Manual

of
Methods of Analysis-
Microbiological Examination
of Food and Water

1
 Table of Contents

Chapter TITLE Page No.

No.
PART- A
General Requirements for Microbiological Laboratories
1. Equipment 6 - 34
2. Glassware/Plasticware 35 – 39
3. Media/ Reagents and Reference cultures 40 - 73
4. Quality Assurance: Assuring Quality of Test Results in Microbiology 74 - 84
Laboratory
5. Good Microbiology Laboratory Practices including Biosafety and 85 - 100
Biohazard Management
Part - B
Methods of Analysis for foods
S. No. METHOD NO. METHOD Page No.
1. FSSAI 15.001:2023 Method for Enumeration of Aerobic Plate Count 102 –109
(APC)
2. FSSAI 15.002:2023 Method for Enumeration of Bacillus cereus 110 -116
3. FSSAI 15.003:2023 Method for Determination of Campylobacter 117 – 123
spp.
4. FSSAI 15.004:2023 Method for Enumeration of Coliforms 124 – 128
5. FSSAI 15.005:2023 Method for Commercial Sterility Test for 129 – 130
Sterilized/UHT Milk /Flavored Milk /
Evaporated Milk
6. FSSAI 15.006:2023 Method for Commercial Sterility Test for 131 – 132
Sterilized/UHT Cream
7. FSSAI 15.007:2023 Method for Determination of Cronobacter spp. 133 – 137
(Enterobacter sakazakii)
8. FSSAI 15.008:2023 Method for Enumeration of Sulfite Reducing 138 – 141
Clostridia based on ISO : 15213
9. FSSAI 15.009:2023 Methods for Isolation and Identification of 142 – 148
Clostridium Perfringens and Clostridium
Botulinum and Enumeration of Clostridium
Perfringensbased on IS 5887-Part 4
10. FSSAI 15.010:2023 Method for Enumeration of Enterobacteriacae 149 – 155
11. FSSAI 15.011:2023 Method for Enumeration of Escherichia 156 – 162
colibased onISO : 16649-2
12. FSSAI 15.012:2023 Methods for Isolation, Identification and 163 – 168
Enumeration of Escherichia Colibased on IS
5887-Part 1

2
 13. FSSAI 15.013:2023 Detection, Isolation and Identification of 169 – 185
Pathogenic E. Coli in Food based on IS 14397
14. FSSAI 15.014:2023 Method for Detection and Confirmation of 186 – 194
Listeria monocytogenes based on IS: 14988-Part
1
15. FSSAI 15.015:2023 Method for Detection and Confirmation of 195 – 202
Listeria monocytogenes based on ISO 11290-1
16. FSSAI 15.016:2023 Detection and Confirmation of Salmonella based 203 – 209
on IS: 5887 Part3
17. FSSAI 15.017:2023 Detection and Confirmation of Salmonella based 210 – 218
on ISO:6579-I
18. FSSAI 15.018:2023 Method for Detection and Confirmation of 219 – 229
Shigella based on ISO: 21567
19. FSSAI 15.019:2023 Method for Detection and Confirmation of 230 – 238
Shigella based on IS 5887-Part 7
20. FSSAI 15.020:2023 Method for Enumeration of Coagulase Positive 239 – 244
Staphylococci based on ISO 6888-1/AMD 1 and
IS 5887- Part 8 (Sec 1)
21. FSSAI 15.021:2023 Method for Enumeration of Coagulase Positive 245 – 248
Staphylococci based on ISO 6888-2 and IS
5887- Part 8 (Sec 2)
22. FSSAI 15.022:2023 Method for Determination of Vibrio cholera and 249 – 258
Vibrio parahaemolyticus
23. FSSAI 15.023:2023 Method for Enumeration of Yeast and Mould 259 - 263
Part C
Methods of Analysis Water / Test methods for Water
S. No. METHOD NO. METHOD Page No.
1. FSSAI 15.024:2023 Method for Enumeration of Aerobic Microbial 265 – 267
count/Standard Plate Count
2. FSSAI 15.025:2023 Method for Enumeration of Escherichia coli and 268 – 269
Coliform
3. FSSAI 15.026:2023 Method for Enumeration of Coliform 270 – 271
(Alternative method)
4. FSSAI 15.027:2023 Method for Determination of Enterococci 272 – 273
(Faecal Streptococci)
5. FSSAI 15.028:2023 Method for Detection of Enterococci (Faecal 274 – 275
Streptococci) (Alternative method)
6. FSSAI 15.029:2023 Method Detection of Salmonella 276 – 279

7. FSSAI 15.030:2023 Method for Detection of Shigella 280 – 286

8. FSSAI 15.031:2023 Method for Detection of Pseudomonas 287 – 290
aeruginosa

3
 9. FSSAI 15.032:2023 Method for Detection of Yeast & Mould 291 – 292

10. FSSAI 15.033:2023 Method for Detection of Sulphite-Reducing 293 – 294

anaerobes (Clostridia)
11. FSSAI 15.034:2023 Method for Detection of Vibrio 295 – 298
parahaemolyticus
12. FSSAI 15.035:2023 Method for Detection of Vibrio cholerae 300 – 304

13. FSSAI 15.036:2023 Method for Detection of Staphylococcus aureus 305 – 307

14. FSSAI 15.037:2023 Method for Detection of Viruses 308 – 312

15. FSSAI 15.038:2023 Method for Detection of MS2 Phage in Water by 313 – 317
Enrichment Spot Assay Technique
16. FSSAI 15.039:2023 Method for Detection of Giardia and 318 – 339
Cryptosporidium - Method A
17. FSSAI 15.040:2023 Isolation and Identification of Giardia and 340 - 347
Cryptosporidium - Method B
Appendix: MPN Table 348 - 350
Note: The test methods given in the manuals are validated/ standardized test methods. However, it
would be the responsibility of the respective testing laboratory to confirm that the above methods are
verified in their laboratory and gives proper result in their laboratory.

4
 ABBREVIATIONS

Sr. No. Abbreviation Expanded Form

1. % Percentage
2. µm Micro meter
0
3. C Degree Celsius
4. Hr Hour
5. L Liter
6. min Minutes
7. mL Milliliter
8. mm millimeter
9. µL Microliter
10. IS Indian Standard
11. ppb Parts per billion
12. ppm Parts per million
13. ISO International Organization for Standardization
14. gm Gram
15. mg Milligram
16. Cfu Colony forming unit
17. sp. species
18. µg Microgram
19. UV Ultra Violet

5
 Part – B
Methods of Analysis for Foods

101
 Method for Enumeration of Aerobic Plate Count (APC)

Method No. FSSAI 15.001:2023 Revision No. & Date 0.0

Introduction The Total Aerobic Mesophilic Plate Count, usually called Aerobic
Plate Count (APC) or Standard Plate Count, is the most commonly
used general indicator of bacterial populations in foods. This method
does not differentiate types of bacteria, and is only used to obtain
general information on the sanitary quality of products, manufacturing
practices, raw materials, processing conditions, handling practices and
shelf life.
Scope This method is applicable to those food product categories and their
sub-categories as mentioned in the Appendix B tables of Food Safety
and Standards (Food Products Standards and Food Additives)
Regulations, 2011 and amendments (Gazette notifications) issued from
time to time.
Caution Precautions should be taken while dealing with suspected food
outbreak samples.
Principle A specified quantity of homogenised food sample is either dispensed
into an empty Petri dish, mixed with a specified molten agar medium or
surface plated on a solid agar culture medium. Other plates are
prepared under the same conditions using decimal dilutions of the test
sample or of the initial suspension. The plates are incubated under
aerobic conditions at 30 °C for 72 h. The number of microorganisms
per gram or millilitre of sample is calculated from the number of
colonies obtained on the plates containing fewer than 300 colonies for
90-100mm plates and 730 colonies for 140mm plates.
Equipment 1. Laminar airflow
2. Hot air oven
3. Autoclave
4. pH meter with measuring accuracy ±0.1

102
 5. Incubator (at 30 ± 1°C)
6. Water bath (at 44 °C to 47 °C)
7. Refrigerator (at 2 °C – 8 °C)
8. Petri dishes (Glass or plastic of 90-100mm diameter or 140mm)
9. Graduated pipettes (0.1 ml divisions) of capacity 1 ml (Class A)
10. Micropipette with tips
11. Tubes and glass bottles
12. Vortex
13. Mechanical stirrer
14. pH meter with measuring accuracy ±0.1
15. Colony counter (optional)
16. Spreader (Sterile glass or plastic)
17. Spiral plater/rotator
Culture Media and Diluent
Reagents The following diluents can be used for preparation of initial
suspension and subsequent serial dilution:

Purpose Diluent
General Use Peptone salt solution
Buffered peptone water
Special Purpose
Highly acidic products of pH ≥ 3.5 Double-strength buffered
to pH ˂ 4.5 peptone water
Gelatine Phosphate buffered diluent
 Plate Count Agar (PCA)
For dairy products, add skimmed milk powder (free from inhibitory
substances) at a level of 1.0g/litre to Plate Count Agar.
For pour plate technique. In case the steriled medium to be used
immediately, cool it to 44 °C to 47 °C in a water bath before use.
If the stored sterilized medium to be used, before starting the
microbiological examination, completely melt the medium, then cool it
to 44 °C to 47 °C in a water bath. The final pH of the media to be
maintained at 7.0 For surface plating technique, pour 15-20 ml of the

103
 medium into sterile petri dishes and solidify. Dry the plates either in a
laminar flow or in a drying cabinet/incubator. While drying in laminar
flow, dry the plates with agar surface facing upwards (at room
temperature) for 30-60 min or overnight at room temperature with the
lids in place. While drying in drying cabinet/incubator, keep the plates
with agar surface facing downwards with half opened lids at 25-50 °C
till the disappearance of water droplets from the surface of the lids.
Reference Cultures Bacillus subtilis subsp. spizizenii (WDCM 00003a)
Escherichia coli (WDCM 00012a or WDCM 00013)
Staphylococcus aureus (WDCM 00032 or WDCM 00034)
Sample Preparation Prepare the test sample in accordance with Chapter 2.

Procedure Two different procedures can be followed for determination of APC in

food matrix.
Procedure Applicability
Pour Plate Technique  Products that require lower LOD (<
102/g or ml for liquid samples and
<103/g for solid samples)
 Products expected to contain
spreading colonies (e.g. Bacillus
spp.; Proteus spp.)
Surface Plating  Chilled and frozen foods, dried
Technique foods, other foods that may contain
(Spread Plate/Spiral heat-sensitive organisms;
Plate technique)  Products likely to contain significant
proportion of obligate aerobic
bacteria
 (e.g. Pseudomonas spp.)
 Products containing small particulate
matter which from are difficult to
distinguish from colonies in pour
plate
 Products with strong colour that

104
 prevents recognition of colonies in
pour plate

Test portion, initial suspension and dilutions

Weigh or measure the test portion, to a tolerance of ±5 %, into a sterile
container or plastic bag. A mass of m g or a volume of V ml (minimum
10 g or 10 ml, unless otherwise stated) representative of the laboratory
sample shall be used. Add a quantity of diluent equal to 9 × m g or 9 ×
V ml to prepare a primary decimal dilution. Homogenize the sample
with a peristaltic blender or rotary homogenizer or vibrational mixer as
mentioned in Chapter 1. This corresponds to 10−1 dilution.

For further decimal dilution, transfer, using a pipette, 1 ml ± 0.02 ml of

the initial suspension into a tube containing 9 ml ± 0.2 ml of sterile
diluent. Mix thoroughly, preferably by using a mechanical stirrer for 5 s
to 10 s, to obtain a 10−2 dilution. If necessary, repeat these steps using
the 10−2 and subsequent dilutions and a new sterile pipette or tip for
each operation, to obtain sufficient (10−3, 10−4 etc.) dilutions to
enumerate the appropriate number of microorganisms.
Inoculation and incubation
Pour Plate Technique
Transfer 1 ml of test sample if liquid, or 1 ml of the initial suspension
(10−1 dilution) in the case of other products to sterile petri dishes in
duplicate. If more than one dilution is to be plated, the number of
dishes can be reduced to one. Similarly transfer 1ml of suspension from
subsequent dilutions (10−2, 10−3 or higher) to separate sterile petri
dishes using a new sterile pipette for each decimal dilution.

If appropriate and possible, select only the critical dilutions steps (at
least two consecutive decimal dilutions) for the inoculation of the Petri
dishes that will give colony counts of between 10 and 300 colonies per
plate.

105
Pour about 12 ml to 15 ml of the plate count agar (PCA) at 44 °C to 47
°C into each Petri dish. The time elapsed between sample preparation
and plating shall not exceed 45 min. Carefully mix the inoculum with
the medium by rotating the Petri dishes horizontally and allow the
mixture to solidify by leaving the Petri dishes standing on a cool
horizontal surface. In case there is possibility that the product contains
microorganisms which will overgrow or swarm on the surface of the
plate, pour an overlay of 4-5 ml of PCA and allow to solidify.

Incubate the plates (in inverted condition) in incubator at 30 ± 1 °C for

72 ± 3 h.

Surface Plating Technique

PCA plates
Pour about 15 ml to 20 ml of the plate count agar (PCA) at 44 °C to 47
°C into each Petri dish and allow it to solidify.
The plates may be stored at 5 °C ± 3 °C for upto 4 weeks.
Immediately before use, these agar plates should be dried.
Label all petri plates with the sample number, dilution, date and any
other desired information.

Inoculation and Incubation

Using a sterile pipette, transfer 0.1 ml of test sample if liquid, or 0.1 ml
of the initial suspension (10−1 dilution) in the case of other products to
the centre of each two agar plates. If more than one dilution is to be
plated, the number of dishes can be reduced to one. Similarly transfer
0.1ml of suspension from subsequent dilutions (10−2, 10−3 or higher) to
separate agar plates using a new sterile pipette/ sterile micro pipette tip
for each decimal dilution.

For counting low number of microorganisms, 1.0 ml of suspension can

be transferred to surface of large agar plate (140 mm diameter) or three
small agar plates (90 mm diameter). In both cases, use duplicate plates
for surface plating (i.e. 2 large plates or 6 small plates).

106
 Spread the inoculum uniformly and as quickly as possible over the
surface of the agar plate, without touching the sides of the petri dish
with a spreader (glass, metallic or disposable type). Same spreader can
be used for all the dilutions of one sample, provided they are used
progressively from highest to lowest dilution (containing greatest
amount of test material). Allow the inoculum to be absorbed for 15
minutes.

The time elapsed between sample preparation and plating shall not
exceed 45 min. Incubate the plates (in inverted condition) in incubator
at (30 ± 1) °C for (72 ± 3) h.
Counting of colonies
After the specified incubation period of (72 ± 3) h, select the agar
plates with, if possible, fewer than 300 colonies. If plates of 140mm
diameter have been used, select plates up to 730 colonies. Count the
colonies with visually or with a colony counter. Special care has to be
taken to distinguish pinpoint colonies from food particles.
Plates with spreading colonies may be avoided if the swarming colony
occupies more than 1/4th of plate area.

Expression of Results Calculate the number N of microorganisms present in the test sample as
a weighted mean from two successive dilutions using the following
formula:

where
is the sum of the colonies counted on the two dishes retained
from two successive dilutions, at least one of which contains a
minimum of 10 colonies;
V is the volume of inoculum placed in each dish, in millilitres;
d is the dilution corresponding to the first dilution retained [d = 1
when the undiluted liquid product (test sample) is tested].

107
Round off the calculated result to two significant figures. When doing
this, if the third figure is less than 5, do not modify the preceding
figure; if the third figure is greater than or equal to 5, increase the
preceding figure by one unit.

Express the result preferably as a number between 1.0 and 9.9

multiplied by the appropriate power of 10, or a whole number with two
significant figures.
Report the result as the number N of microorganisms per millilitre
(liquid products) or per gram (other products).
Special Cases
i. When one dish (test sample or initial suspension or first dilution)
contains less than 10 colonies
If the plate contains less than 10 colonies, but at least four, calculate the
result using the formula:

Report it as the estimated number NE of microorganisms per millilitre

(liquid products) or per gram (other products).
If the total is from 3 to 1, the precision of the result is too low and the
result shall be reported as: “Microorganisms are present but less than
4/Vd per gram or per ml”
ii. When the dish (test sample or initial suspension or first dilution)
contains no colonies
Report the result as follows:
“less than 1/Vd microorganisms per millilitre” (liquid products) or
“less than 1/Vd microorganisms per gram” (other products)
where
d is the dilution factor of the initial suspension or of the first
dilution inoculated or retained
V is the volume of the inoculum used in each dish, in millilitres
iii. Where the counting of colonies for each one of the dishes for all
inoculated dilutions produces a number greater than 300 (or 730 for

108
 140 mm diameter plates), report the result as follows:
“more than 300/Vd”
where
d is the dilution of the last inoculated dilution;
V is the volume of the inoculum used in each dish, in milliliters

Reference 1) ISO 4833-1 (2013): Microbiology of the food chain —Horizontal

method for the enumeration of microorganisms —Part 1: Colony
count at 30 °C by the pour plate technique
2) ISO 4833-2 (2013) (corrigendum 2014): Microbiology of the food
chain —Horizontal method for the enumeration of microorganisms
—Part 2: Colony count at 30°C by the surface plating technique.
3) IS 5402 (2012): Microbiology of Food and Animal Feeding Stuffs
— Horizontal Method for the Enumeration of Microorganisms —
Colony-Count Technique at 30°C (Second Revision)
4) ISO 6887-1 (2017): Microbiology of the food chain — Preparation
of test samples, initial suspension and decimal dilutions for
microbiological examination — Part 1: General rules for the
preparation of the initial suspension and decimal dilutions
Approved by Scientific Panel on Methods of Sampling and Analysis

109
 Method for Enumeration of Bacillus cereus

Method No. FSSAI 15.002:2023 Revision No. & Date 0.0

Introduction Bacillus cereus is a pathogenic bacterium, which causes foodborne
diseases classified by the International Commission on
Microbiological Specifications for Foods (ICMSF, 2002) in Risk
Group III: “diseases of moderate hazard usually not life
threatening, normally of short duration without substantial
sequelae, causing symptoms that are self-limiting but can cause
severe discomfort”. They are Gram-positive rods, spore forming,
facultative anaerobic and each species is differentiated from B.
cereus by, basically, for one single characteristic.
Scope This method is applicable to those food product categories and
their sub-categories as mentioned in the Appendix B tables of
Food Safety and Standards (Food Products Standards and Food
Additives) Regulations, 2011 and amendments (Gazette
notifications) issued from time to time.
Caution Precautions should be taken while dealing with suspected food
outbreak samples.
Principle A specified quantity of homogenised food sample (liquid/initial
suspension) is surface plated on a selective culture medium on to a
petri dish. Other plates are prepared under the same conditions
using decimal dilutions of the test sample or of the initial
suspension. The plates are incubated under aerobic conditions at
30 °C for 18 to 48 h. The number of microorganisms per gram or
millilitre of sample is calculated from the number of colonies
confirmed on plates at dilution levels and further confirmed
according to the test specified.
Equipment 1. Laminar airflow
2. Biosafety cabinet
3. Hot air oven

110
 4. Autoclave
5. Incubator (at 30 ± 1°C, 37 ± 1°C and 55 ± 1°C)
6. Water bath (at 44 °C to 47 °C)
7. Refrigerator (at 2°C – 8 °C)
8. pH meter with measuring accuracy ±0.1
9. Microscope
10. Petri dishes (Glass or plastic of 90-100mm diameter or
140mm)
11. Graduated pipettes (0.1 ml divisions) of capacity 1 ml (Class
A)
12. Micropipette with tips
13. Tubes and glass bottles
14. Vortex
15. Mechanical stirrer
16. pH meter with measuring accuracy ±0.1
17. Colony-counter (optional)
18. Spreader (glass or plastic)
19. Inoculation loops and straight wire
20. Spiral plater/rotator
21. Drying cabinet or incubator at 37 ± 1°C and 55 ± 1°C
Culture Media and i) Dilution fluid
Reagents ii) Complete medium (MYP agar) having Basal Medium
iii) Polymyxin B solution
iv) Egg yolk emulsion
v) Sheep blood agar
Details of preparation given in Chapter 3
Reference Cultures Bacillus cereus ATCC 14579, ATCC 10876, ATCC 11778
Further details of culturing given in Chapter 3
Sample Preparation Sample preparation based on the product categories are given in
Chapter 2
Procedure Test portion, initial suspension and dilutions
Make a 1:10 dilution of the well mixed sample, by aseptically
transferring sample to the desired volume of diluent. Aseptically

111
weigh 10 gm of solid or semi-solid sample into a sterile blender
jar or into a stomacher bag. Add 90 mL of sterile diluent. Blend for
2 min at low speed (approximately 8000 rpm) or mix in the
Stomacher for 30-60 sec. Powdered samples may be weighed and
directly mixed with the diluent. Shake vigorously. In most of the
samples particulate matter floats in the dilution water. In such
cases allow the particles to settle for two to three minutes and then
draw the diluent from that portion of dilution where food particles
are minimum and proceed.

Dilution (decimal dilution)

Prepare decimal dilutions from initial suspension as follows. Shake
each dilution 25 times in 30 cm arc. For each dilution use fresh
sterile micropipette tips. Alternately use auto pipette. Pipette 1 ml
of food homogenate (Initial suspension) into a tube containing 9
ml of the diluent and mix properly by using vortex/Cyclomixer.
From the second dilution transfer 1mL tube containing 9 ml of the
diluents and mix properly by using vortex/Cyclomixer.

Repeat the above to prepare the further dilutions using different

diluent tubes, until the desired dilution is obtained.

Inoculation and Incubation

MYP Agar plates
Pour about 15 ml to 20 ml of the plate count agar (MYPA) at 44
°C to 47 °C into each Petri dish and allow it to solidify.
The plates may be stored at 5 °C ± 3 °C for upto 4 weeks.
Immediately before use, these agar plates should be dried
Label all Petri plates with the sample number, dilution, date and
any other desired information.
1. Pipette 0.1 ml of the test sample (if product is liquid), or of
the initial suspension (in case of other products), of such
dilutions which have been selected, for plating (spread

112
 plate technique) on MYP Agar, in duplicate.
2. In some samples where it is desirable to estimate low
numbers of B. cereus, the limits of detection may be raised
by a factor of 10 by examining 1.0 ml of the test sample if
the initial product is liquid, or 1.0 ml of the initial
suspension for the other products. Pipette 1 ml of inoculum
either over the surface of three small dishes (90 mm) or on
the surface of a large Petri dish (140 mm). In both the
cases, prepare duplicates by using two large plates or six
small plates.
3. Spread the inoculum over surface of agar plate, using a
sterile spreader. Keep the plates in upright position until
inoculum is absorbed by the agar.
4. Invert the inoculated plates and incubate them for 18 h to
24 ±2 h at 30± 1 °C. Reincubate the plates for an additional
24 h, if colonies are not clearly visible, prior counting.

Enumeration
After completion of incubation period, select only those plates
(preferably at two successive dilutions) that contain less than 150
colonies (typical and/or atypical colonies) for enumeration.
If there are less than 15 characteristic colonies present on plates
inoculated with the liquid product or the lowest dilution of other
products, it is possible to make an estimated count as described in
the expression of results below.

The presumptive colonies are large, pink (indicating absence of

mannitol fermentation) and generally surrounded by a zone of
precipitation (indicating the production of lecithinase).
 Numerous mannitol fermenting microbes lead to the
production of acid, then the characteristic pink colour of B.
cereus colonies may be reduced or disappear entirely.
 Some strains of B. cereus produce little or no lecithinase.

113
 Colonies of these strains will not be surrounded by a
precipitation zone. These colonies should also be subjected
to confirmation tests.

Confirmation
Select 5 presumptive colonies from each plate for confirmation. If
the plates are overcrowded, streak 5 presumptive colonies on MYP
plates. After incubation at 30 °C for 18 h to 24 h select the well
isolated colony for confirmation.

Haemolysis test on sheep blood agar

Streak, stab or spot the selected colonies onto the surface of sheep
blood agar and incubate at 30 °C for 24 h ±2 h. Interpret the
haemolysis reaction.

Biochemical interpretation
Test Result confirming presumptive Bacillus cereus
 MYP agar - Formation of pink colonies surrounded by
precipitate
 Haemolysis - Positive reaction
Expression of Results When the method used requires identification, a given number A
(generally 5) of presumptive colonies is identified from each of the
dishes retained for the colony counting. After identification,
calculate, for each of the dishes, the number of colonies complying
with identification criteria, using Equation:
a= XC

where
b is the number of colonies complying with identification criteria
among the identified colonies A;
C is the total number of presumptive colonies counted on the dish.
Calculate the number N of identified microorganisms present in
the test sample by replacing ΣC by Σa in the equation,

114
 Where
∑a is the sum of the colonies confirming presumptive Bacillus
cereus identified o all the dishes selected,
V is the volume of inoculum placed in each dish, in millilitres;
n1 is the number of dishes selected at the first dilution;
n2 is the number of dishes selected at the second dilution; is the
dilution rate corresponding to the first dilution selected (the initial
suspension is a dilution).
d is the dilution corresponding to the first dilution retained [d = 1
when the undiluted liquid product (test sample) is retained.

EXAMPLE:
A count of a product after inoculation with 0.1 ml of product gave
the following results:
- for the first dilution selected (10-2): 65 typical colonies and 85
typical colonies and no atypical colonies
- for the second dilution selected (10-3): 3 typical colonies and 7
typical colonies and no atypical colonies.
The following numbers were stabbed:
- from 65 colonies, 5 colonies were stabbed and all 5 proved to be
biochemical confirmed, giving a = 65;
- from 85 colonies, 5 colonies were stabbed, 3 of which proved to
be biochemical confirmed, giving a = 51;
- from 3 colonies, all 3 were stabbed and proved to be biochemical
confirmed, giving a = 3;
- from 7 colonies, 5 colonies were stabbed and all 5 proved to be
biochemical confirmed, giving a = 7.
= 57272

The result, after rounding off is 5.7 X 104 cfu/gm or ml

115
 No. of presumptive Bacillus cereus is expressed in cfu/g or ml

Reference 1) ISO 7932:2004-Microbiology of food and animal feeding

stuffs — Horizontal method for the enumeration of
presumptive Bacillus cereus — Colony-count technique at
30˚C.
2) IS 5887-6 - 2012 - Microbiology of Food and Animal Feeding
Stuffs – Horizontal Method for the Enumeration of
Presumptive Bacillus cereus, Part 6 Colony-count Technique at
30°C.
3) ISO 6887-1 (2017): Microbiology of the food chain —
Preparation of test samples, initial suspension and decimal
dilutions for microbiological examination — Part 1: General
rules for the preparation of the initial suspension and decimal
dilutions.
Approved by Scientific Panel on Methods of Sampling and Analysis

116
 Method for Determination of Campylobacter spp.

Method No. FSSAI 15.003:2023 Revision No. & Date 0.0

Introduction Campylobacter is one of the major causes of diarrhoea in
humans and C. jejuni subsp. jejuni and C. coli are the species
most frequently associated with acute foodborne gastroenteritis.
Food-borne diseases caused by C. jejuni subsp. Jejuni include
gastroenteritis, septicemia, meningitis, abortion and the Guillain-
Barré Syndrome (GBS). GBS is classified by the International
Commission on Microbiological Specifications for Foods
(ICMSF, 2002) into risk group IB: “diseases of severe hazard for
restricted population; life threatening or resulting in substantial
chronic sequelae or presenting effects of long duration”.
Scope This method is applicable to those food product categories and
their sub-categories as mentioned in the Appendix B tables of
Food Safety and Standards (Food Products Standards and Food
Additives) Regulations, 2011 and amendments (Gazette
notifications) issued from time to time.
Caution In order to safeguard the health of laboratory personnel, it is
essential that tests for detecting Camplylobacter are only
undertaken in properly equipped laboratories, under the control
of a skilled microbiologist, and that great care is taken in the
disposal of all incubated materials. It is also the responsibility of
the user to establish appropriate safety and health practices.
Principle The detection of Campylobacter species involves enrichment in a
selective liquid medium that is incubated at 37°C for 4-6 h
followed by 41.5°C for 44 h. The enrichment culture is inoculated
onto a blood free selective agar medium, which are incubated
microaerobically at 41.5°C for24 h and examined for characteristic
colonies.
Confirmation of suspect colonies of Campylobacter species is by

117
 means of biochemical, morphological and physiological tests.
Equipment 1. Laminar airflow
2. Biosafety cabinet
3. Hot air oven
4. Autoclave
5. Incubator (Operating at 25 ± 1 °C, 37 ± 1 °C and 41.5 ±
1°C)
6. CO2 incubator
7. Water bath (at 44 °C to 47 °C)
8. pH meter with measuring accuracy ±0.1
9. Microscope
10. Refrigerator (at 2°C – 8°C)
11. Petri dishes (Glass or plastic of 90-100mm diameter or
140mm)
12. Graduated pipettes (0.1 ml divisions) of capacity 1 ml
(Class A)
13. Micropipette with tips
14. Tubes and glass bottles
15. Vortex
16. Mechanical stirrer
17. pH meter with measuring accuracy ±0.1
18. Spreader (glass or plastic)
19. Inoculation loops and straight wire
20. Spiral plater/rotator
21. Apparatus suitable for creating microaerophilic
conditions
Culture Media and Obligatory
Reagents  Bolton Broth
 Modified Charcoal Cefoperazone Deoxycholate Agar (mCCDA)
 Columbia Blood Agar (CBA)
 Oxidase Kovacs Reagent
Optional
 Mueller Hinton Blood Agar

118
  3% Hydrogen Peroxide
 Nalidixic acid (30 μg) discs
 Cephalothin (30 μg) discs
 Sodium Hippurate Solution
 Ninhydrin Solution
 Indoxyl acetate discs (2.5 to 5.0 mg)
 Bolton broth
 Sterile lysed horse blood
 Antibiotic solution 1
 Antibiotic solution 2
 Antibiotic solution 3
 Preston broth
 Modified charcoal cefoperazonedeoxychlolate agar (mCCD
agar)
 Colombia blood agar
 Sterile sheep or horse blood
 Reagent for oxidase activity
 Reagent for catalase activity
 Reagent for hydrolysis of hippurate
 Indoxyl acetate discs

Details of preparation given in Chapter 3

Reference Cultures Details given in Chapter 3
Campylobacter jejuni WDCM 00005 (positive control and
Escherichia coli WDCM 00013 (negative control).

Sample Preparation Details given in Chapter 2

Procedure Depending on the type of sample and the purpose of the test, one
or more of three different detection procedures is/are used:
1. Detection procedure A
For samples with low numbers of campylobacters and low levels
of background microflora and/or with stressed campylobacters

119
(e.g., cooked or frozen products), homogenize 10 g or 10 mL of
sample with 90 mL of Bolton broth. Incubate at 37°C for 4 to 6
hours, then at 41.5 °C for 44 ± 4 hours, in a microaerobic
atmosphere (oxygen content of 5 ± 2%, carbon dioxide 10 ± 3%,
optional hydrogen 10%, with the balance Nitrogen).

2. Detection procedure B
For samples with low numbers of campylobacters and high level
of background microflora (e.g. raw meats, including poultry):
Homogenize 10 g or 10 mL of sample with 90 mL of Preston
broth. Incubate at 41.5 ± 1°C for 24 ± 2 hours, in a microaerobic
atmosphere.

3.Detection procedure C
For samples with high numbers of campylobacters, enrichment is
not done.

Isolation (Selective-differential plating)

In general, an amount of test portion is mixed with enrichment
media to yield a ten-fold dilution.

1. Detection procedure A
Using the enrichment culture obtained in Bolten Broth after
Procedure A, two selective solid media are inoculated: 1. —
modified Charcoal Cefoperozone Deoxycholate agar (mCCD
agar); 2. — any other solid selective Campylobacter medium
using different selective principles from those in mCCD agar.
2. Detection procedure B
Using the enrichment culture obtained after Procedure B, the
selective mCCD agar is inoculated.
3. Detection procedure C
Using the enrichment culture obtained after Procedure C, the test
portion is plated directly or after suspending in an appropriate

120
amount of liquid onto the selective mCCD agar.

Incubate the plates at 41.5 ± 1 °C/44 ± 4 h in a microaerobic

atmosphere. Incubate the second selective isolation medium
plates according to the manufacturers’ instructions.

After the incubation period, examine the plates for typical

colonies of Campylobacter.
The typical colonies on mCCDA are grayish, often with a
metallic sheen, with a tendency to spread. Other forms of
colonies may occur.
Follow the manufacturers’ instructions to select typical colonies
on the second isolation medium.

As Campylobacter rapidly loses culturability in air, follow the

procedure of confirmation without delay.
For a clear distinction between positive and negative
confirmation reactions, it is helpful to verify this with well
characterized positive and negative control strains.
Campylobacter jejuni WDCM 00005 (positive control) and
Escherichia coli WDCM 00013 (negative control).
In addition, to the confirmation and identification tests described
in here other tests (PCR tests, serological methods, matrix-
assisted laser desorption/ionization time-of-flight mass
spectrometer (MALDI-TOF-MS) analysis, etc.) can be used,
providing the suitability of the alternative procedure is verified
(see ISO 7218).
Confirmation:
For confirmation, take a typical colony from each plate and a
further four colonies if the first is negative. Store the isolated
plates at 5°C preferably at micraerophilic conditions for
confirmation
Streak each colony onto a Columbia Blood Agar (CBA) plate in

121
order to allow the development of well-isolated colonies and
incubate the plates in a microaerobic atmosphere at 41.5 ± 1 °C
for 24–48 h. Use the pure cultures obtained on CBA for
examination of morphology, motility using microscope, oxidase
activity, microaerobic growth at 25°C, aerobic growth at 41.5°C.
NOTE The suspect colony could be previewed for characteristic
morphology and motility before streaking on CBA.

 Morphology and motility: From the Columbia Blood Agar

(CBA) plate examine the fresh colony for morphology and
motility using a microscope. Cultures showing curved bacilli
with a spiralling “corkscrew” motility should be retained for
the confirmatory tests below.
 Growth at 25°C (microaerobic): Inoculate the culture from
the CBA plate onto the surface of a new CBA plate. Incubate
the plate at 25 ± 1°C for 44 ± 4 h in a microaerobic
atmosphere and examine for growth of Campylobacter
colonies.
 Growth at 41.5°C (aerobic): Inoculate the culture from the
CBA plate onto the surface of a new CBA plate. Incubate the
plate at 41.5 ± 1°C for 44 ± 4 h in an aerobic atmosphere and
examine for growth of Campylobacter colonies.
 Oxidase test: Using a platinum/iridium loop or glass rod,
take a portion of a well-isolated colony from each individual
CBA plate and streak it onto a filter paper moistened with the
Oxidase Kovacs Reagent. The appearance of a mauve, violet
or deep blue color within 10 s indicates a positive reaction. If
a commercially available oxidase test kit (must be approved
by FSSAI under RAFT scheme) is used, follow the
manufacturer’s instructions.
Interpretation: Campylobacter spp. the cultures exhibiting the
following characteristics: small curved bacilli with a spiraling
“corkscrew” motility, microaerobic growth at 25°C negative,

122
 aerobic growth at 41.5°C negative, oxidase positive.
Expression of results Based on the observations and interpretation of the results report
presence or absence of Campylobacter spp. in test portion
specifying the mass in grams or mililitres of the sample taken.
Campylobacter spp.= present or absent/ gm or mL.

Reference ISO 10272-1:217-Microbiology of food chain — Horizontal

method for detection and enumeration of Campylobacter spp. -
Part 1: Detection method.
Approved by Scientific Panel on Methods of Sampling and Analysis

123
 Method for Enumeration of Coliforms

Method No. FSSAI 15.004:2023 Revision No. & Date 0.0

Introduction Coliforms are a broad group of aerobic or facultative anaerobic,
rod-shaped, gram-negative non-spore forming bacteria which can
ferment lactose with the production of acid and gas when
incubated at 32–37°C. Coliform are members of
Enterobacteriacae family. Most common genera are E. coli,
Citrobacter, Enterobacter, Klebsiella and Hafnia.
Scope This method is applicable to those food product categories and
their sub-categories as mentioned in the Appendix B tables of
Food Safety and Standards (Food Products Standards and Food
Additives) Regulations, 2011 and amendments (Gazette
notifications) issued from time to time.
Caution Carry out the test under the control of skilled microbiologist and
great care shall be taken in the disposal of all the incubated
material. Follow safe and good laboratory practices to avoid cross
contamination.
Principle Violet red bile lactose agar (VRBL) medium is used for
enumeration of coliforms. VRBL contains selective inhibitors -
inhibit the accompanying gram-positive and unrelated flora.
Coliforms rapidly ferment lactose and produce red colonies
surrounded by red purple halo. Lactose non-fermenters and late
lactose fermenters produce pale colonies. Confirmation is carried
out in brilliant green bile broth medium, which is also having
selective components for the growth of coliforms.
Equipment Refer Chapter 1 for general equipment.
1. Laminar airflow
2. Biosafety cabinet
3. Hot air oven
4. Autoclave

124
 5. Incubator (Operating at 30 ± 1°C or 37± 1°C)
6. Water bath (at 44 °C to 47 °C or at 100 °C)
7. pH meter with measuring accuracy ±0.1
8. Microscope
9. Refrigerator (at 2°C – 8 °C)
10. Petri dishes (Glass or plastic of 90-100mm diameter or
140mm)
11. Graduated pipettes (0.1 ml divisions) of capacity 1 ml (Class
A)
12. Micropipette with tips
13. Tubes and glass bottles
14. Durham tubes
15. Vortex
16. Mechanical stirrer
17. pH meter with measuring accuracy ±0.1at 25°C
18. Spreader (glass or plastic)
19. Inoculation loops and straight wire
20. Spiral plater/rotator
21. Colony counter (optional)
Culture Media and Primary Diluent
Reagents Violet Red Bile Lactose Agar (VRBL)
Brilliant Green Lactose Bile Broth (BGBB) ISO 4832:2006

For media preparation and other details refer Chapter 3

Reference Cultures Specific strains or equivalent member of coliform group (E. coli
etc.)
Sample Preparation Refer Chapter 2
Procedure Test portion, initial suspension and dilution
Weigh or measure the test portion, to a tolerance of ±5 %, into a
sterile container or plastic bag. A mass of m g or a volume of V ml
(minimum 10 g or 10 ml, unless otherwise stated) representative of
the laboratory sample shall be used. Add a quantity of diluent
equal to 9 × m g or 9 × V ml to prepare a primary decimal

125
dilution. Homogenize the sample with a peristaltic blender or
rotary homogenizer or vibrational mixer as mentioned in Chapter
1. This corresponds to 10−1 dilution.

For further decimal dilution, transfer, using a pipette, 1 ml ± 0.02

ml of the initial suspension into a tube containing 9 ml ± 0.2 ml of
sterile diluent. Mix thoroughly, preferably by using a mechanical
stirrer for 5 s to 10 s, to obtain a 10−2 dilution. If necessary, repeat
these steps using the 10−2 and subsequent dilutions and a new
sterile pipette or tip for each operation, to obtain sufficient (10−3,
10−4, etc.) dilutions to enumerate the appropriate number of
microorganisms.

Inoculation and Incubation

Label all Petri dishes with the sample code, dilution, date and any
other information.
1. Pipette 1ml of the test sample (if the product is liquid), or 1 ml
of primary suspension (if prepared) to the centre of each petri
dishes. Similarly prepare plates from subsequent dilution as
required.
2. Pour approximately 15 ml of the molten VRBL agar, (cooled
at 44 °C to 47 °C) into each petri dishes. Time elapse between
inoculation and addition of agar into plates shall not exceed 15
mins.
3. Carefully mix the inoculum with the medium and allow the
medium to solidify.
Also prepare a control plate with 15 ml of the medium for
checking its sterility.
4. After complete solidification, pour about 4 ml of molten
VRBL agar (cooled at 44 °C to 47 °C) onto the surface of
inoculated medium and allow to solidify. Invert the inoculated
plates and incubate them at 30 °C or 37 °C for 24 ± 2 h.

126
 Enumeration
After completion of incubation period, count purplish red colonies
with a diameter of at least 0.5 mm (sometimes surrounded by a
reddish zone of precipitated bile). Consider all these as typical
colonies of coliform and do not require further confirmation.
Count other atypical colonies (smaller size) also and all colonies
derived from milk products that contain sugar other than lactose,
immediately after the incubation and confirm.
Confirmation
Select 5 colonies of each atypical types and inoculate into tubes of
brilliant green lactose bile broth and incubate at 30 °C or 37 °C for
24 ± 2 h. Consider all colonies as coliforms that show gas
formation in Durham tubes. Take the results into account in the
calculation.
Calculation 1. Select petri dishes having 10 to 150 colonies for enumeration.

2. Use the following formula for calculation

N is Number of colonies per ml or g of product

∑ C is the sum of colonies counted on all the dishes retained
n1 is the no. of dishes retained in the first dilution
n2 is the no of dishes retained in the second dilution
d is the dilution factor corresponding to first dilution

1. In case petri dishes have total/typical colonies less than 10,

calculate the results using the following formula:
N= C/(v × d)
N is Number of colonies per ml or g of product
C is the average of colonies on the petri dishes retained
v is the volume of inoculums used in each dish
d is the dilution corresponding to the dilution retained.

127
Expression of Results1. Results shall be expressed as a number between 1.0 and 9.9
multiplied by 10 x, where x is power of 10.
2. If plates from all dilutions have no colonies, the result is expressed
as less than 1 cfu/ml or 10 cfu/g or mL (if primary suspension
prepared)
Reference 1) ISO 4832:2006: Microbiological of food and animal feeding
stuffs- Horizontal method for the enumeration of coliforms –
Colony count technique
2) IS 5401(Part 1): 2012: Microbiology of food and animal
feeding stuffs - Horizontal method for the detection and
enumeration of coliforms: Part 1 colony count technique
(Second Revision)
3) ISO 6887-1 (2017): Microbiology of the food chain —
Preparation of test samples, initial suspension and decimal
dilutions for microbiological examination — Part 1: General
rules for the preparation of the initial suspension and decimal
dilutions
Approved by Scientific Panel on Methods of Sampling and Analysis

128
 Method for Commercial Sterility Test for Sterilized/UHT
Milk /Flavored Milk / Evaporated Milk

Method No. FSSAI 15.005:2023 Revision No. & Date 0.0

Sterilization and ultra-high temperature treatment are used in the
production of milk, flavoured milk, and evaporated milk to protect
them from pathogens and spoilage bacteria while maintaining the
product's organoleptic and nutritional quality. Because absolute
sterility is impossible to achieve without severely compromising
Introduction
food integrity, thermal processed products must meet the
commercial sterility criterion before they can be sold. Commercial
sterility is defined as the absence of microorganisms capable of
growing in food under normal non-refrigerated conditions under
which the food is likely to be held during distribution and storage.
This method is applicable to those food product categories and
their sub-categories as mentioned in the Appendix B tables of
Scope Food Safety and Standards (Food Products Standards and Food
Additives) Regulations, 2011 and amendments (Gazette
notifications) issued from time to time.
Caution --
UHT processing and aseptic filling are intended to achieve
commercial sterility, microbial defects can occur at any stage of
production due to insufficient heat treatment or high
contamination of raw materials, resulting in food spoilage by
spore-forming microorganisms. In commercial testing, the
products are incubated in their final packaging for seven to
Principle
thirteen days at 30 °C to allow surviving spores or contaminating
microorganisms to recover and grow to detectable levels. When
the products are intended to be stored at higher temperatures (> 40
°C), additional incubation at 55 °C for five to seven days is also
used. A second step focuses on detecting viable microorganisms
that grew in the product using pH and acidity measurement.

129
Equipment Refer Chapter 1 for general equipment
Dilute rosaniline acetate solution
Chemicals and Phenolphthalein solution
Reagents Sodium hydroxide solution
pH indicator strips
Reference Cultures --
Sample Preparation
a) pH
1. pH test is performed to assess the variation in pH levels
during the incubation period of 0 and 7 days incubation.
2. pH variation can be an indicator of microbial activity which
may be leading to acid production.
3. The determination of pH shall be done as per IS 1479 (Part 1)
using indicator strips.
4. Sample which does not show any physical alteration during
incubation at 55±1 °C for 7 days and where the pH does not
show a difference of more than 0.3 unit from the initial pH, is
considered sterile.
Procedure b) Titratable acidity
1. Titratable acidity test is performed to assess the variation in
developed acidity levels during the incubation period of initial 0th
day and final after 7 days incubation.
2. Excessive variation can be an indicator of microbial activity
which may be leading to acid production.
3. The determination of titratable acidity shall be done as per IS
1479 (Part 1)
4. Sample which does not show any physical alteration during
incubation at 55 ± 1 °C for 7 days and where the acidity does not
show a difference of more than 0.02 percent from the initial
acidity is considered sterile.
Calculation --
a) Based on observation made on pH strip where pH difference
Expression of Results
is not more than 0.3 from the initial pH is regarded as sterile

130
 b) Based on observation made on titratable acidity wherein the
difference in TA not more than 0.02% lactic acid is
considered as sterile
1) IS4238: 2020: Sterilized and Ultra High Temperature
Reference Sterilized Milk —Specification
2) IS 1479 (Part 1): 2016 (RA 2021): Methods of Test for Dairy
Industry Part 1 Rapid Examination of Milk (First Revision)
Approved by Scientific Panel on Methods of Sampling and Analysis

131
 Method for Commercial Sterility Test for Sterilized/UHT
Cream

Method No. FSSAI 15.006:2023 Revision No. & Date 0.0

Introduction Sterilization and ultra-high temperature treatment are used in the
production of cream to protect them from pathogens and spoilage
bacteria while maintaining the product's organoleptic and
nutritional quality. Because absolute sterility is impossible to
achieve without severely compromising food integrity, thermal
processed products must meet the commercial sterility criterion
before they can be sold. Commercial sterility is defined as the
absence of microorganisms capable of growing in food under
normal non-refrigerated conditions under which the food is likely
to be held during distribution and storage
Scope This method is applicable to those food product categories and
their sub-categories as mentioned in the Appendix B tables of
Food Safety and Standards (Food Products Standards and Food
Additives) Regulations, 2011 and amendments (Gazette
notifications) issued from time to time.
Caution --
Principle UHT processing and aseptic filling are intended to achieve
commercial sterility, microbial defects can occur at any stage of
production due to insufficient heat treatment or high
contamination of raw materials, resulting in food spoilage by
spore-forming microorganisms. In commercial testing, the
products are incubated in their final packaging for seven to
thirteen days at 30 °C to allow surviving spores or contaminating
microorganisms to recover and grow to detectable levels. When
the products are intended to be stored at higher temperatures (> 40
°C), additional incubation at 55 °C for five to seven days is also
used. A second step focuses on detecting viable microorganisms
that grew in the product using acidity measurement and incubation

132
 test by storing the pack at 38oC for 14 days to check bulging of
cans and other product characteristics like curdling, sliminess, etc.
Equipment Incubator, 2 white porcelain basins,l0-ml burette fitted with a
soda-lime guard tube, porcelain basins
Culture Media and Dilute rosaniline acetate solution
Reagents Phenolphthalein solution
Sodium hydroxide solution
Reference Cultures --
Sample Preparation --
Procedure A) Titratable acidity
a. Weigh 10.0 g cream into each of two white porcelain basins
of approximately 60-ml capacity; add to both, 10 ml of water
and stir to disperse the cream.
b. Prepare from one dilution a colour control by adding and
stirring 2 ml dilute rosaniline acetate solution.
c. Stir 2 ml phenolphthalein solution into the other dilution and,
while stirring, vigorously, add as rapidly as possible sodium
hydroxide solution, from a 10-ml burette fitted with a soda-
lime guard tube, until the colour matches the pink colour of
the control.
d. The titration shall be preferably done in north daylight or
under illumination from a daylight lamp.

B) Incubation test
Incubate the cans/retort pouches at a temperature of 38 oC for 14
days.
The samples shall pass the test if:
a) the cans do not show any bulge due to positive pressure within,
and
b) the product inside the can has not curdled or thinned and is free
from any objectionable taste or odour, sliminess, etc.
Calculation --

133
Expression of Results a. Based on observation made on titratable acidity wherein the
titratable acidity is not more than 0.15% lactic acid is
considered as satisfactory for the assigned test.
b. After 14 days of incubation, the cans do not show bulging of
cans, not curdling or thinned and free from objectionable
odour, or odour and free from objectionable taste or odour,
sliminess, etc is considered as it meets the sterility
requirement of sterilized cream.
Reference 1. IS4884: 2021 Sterilized/UHT Sterilized Cream — Specification
Approved by Scientific Panel on Methods of Sampling and Analysis

134
 Method for Determination of Cronobacter spp.

Method No. FSSAI 15.007:2023 Revision No. & Date 0.0

Introduction Cronobacter spp. (Enterobacter sakazakii) causes a foodborne
disease classified by the International Commission on
Microbiological Specifications for Foods (ICMSF, 2002) in Risk
Group IB: “diseases of severe hazard for restricted population; life
threatening or resulting in substantial chronic sequelae or
presenting effects of long duration”.
Scope This method is applicable to those food product categories and
their sub-categories as mentioned in the Appendix B tables of
Food Safety and Standards (Food Products Standards and Food
Additives) Regulations, 2011 and amendments (Gazette
notifications) issued from time to time.
Caution In order to safeguard the health of laboratory personnel, it is
essential that tests for detecting Cronobacter spp. are only
undertaken in properly equipped laboratories, under the control of
a skilled microbiologist, and that great care is taken in the disposal
of all incubated materials. It is also the responsibility of the user to
establish appropriate safety and health practices.
Principle A test portion is inoculated into BPW, then incubated between 34
°C and 38 °C for 18 h ± 2 h. The selective enrichment medium is
inoculated with the culture and incubated at 41.5 °C ± 1 °C for 24
h ± 2 h. The chromogenic agar is streaked for isolation with the
enrichment culture and incubated at 41.5 °C ± 1 °C for 24 h ± 2 h.
Typical colonies are selected from the chromogenic agar, purified
on a non-selective agar such as TSA and biochemically
characterized.
Equipment 1. Laminar airflow
2. Biosafety cabinet
3. Hot air oven

135
 4. Autoclave
5. Incubator (Operating at 34 °C to 38 °C, 37°C ± 1°C and 41.5
± 1°C)
6. Water bath (at 47°C and 50°C and 37°C ± 1°C)
7. pH meter with measuring accuracy ±0.1
8. Microscope
9. Refrigerator (at 2 °C– 8 °C)
10. Petri dishes (Glass or plastic of 90-100mm diameter or
140mm)
11. Graduated pipettes (0.1 ml divisions) of capacity 1 ml
(ClassA)
12. Micropipette with tips
13. Tubes and glass bottles
14. Vortex
15. Homogenizer
16. pH meter with measuring accuracy ± 0.1
17. Spreader (glass or plastic)
18. Inoculation loops and straight wire
19. Spiral plater/rotator
20. Inoculation loops and straight wire
21. Spectrophotometer
Culture Media and  Buffered peptone water
Reagents  Cronobacter selective broth (CSB)
 Chromogenic Cronobacter isolation (CCI) agar
 Tryptone soya agar (TSA)
 Media and reagents for biochemical characterization
Details of preparation given in Chapter 3
Reference Cultures Details given in Chapter 3
Sample Preparation Details given in Chapter 2
Procedure Pre enrichment
Add 10 g or 10 ml of the test sample to 90 ml of pre warmed
(room temperature) pre-enrichment medium (BPW), to yield a
tenfold dilution. For specific products, follow the procedures

136
specified in chapter 3.
For preparing quantities larger than 10 g, BPW should be pre-
warmed between 34 °C - 38 °C before inoculated with the test
portion.
Pre enrichment
Incubate the inoculated pre-enrichment medium set temperature
between 34 °C and 38 °C for 18 h ± 2 h.

Enrichment
After incubation, mix the inoculated pre-enrichment medium and
transfer 0.1 ml of the obtained culture above into 10 ml of CSB
and mix well. Incubate at 41.5 °C for 24 h ± 2 h.

Isolation of presumptive Cronobacter spp.

From enrichment culture, mix well and with the help of a streak
onto the surface of the CCI agar (Brought to room temperature if
they are stored at a lower temperature) to obtain well-separated
colonies.
Incubate the plate at 41.5 °C for 24 h ± 2 h.
After incubation, examine the chromogenic plate for the presence
of typical colonies of presumptive Cronobacter.
Typical presumptive Cronobacter colonies on CCI are small to
medium-sized (1 mm to 3 mm) and blue to blue-green in colour.
Non-Cronobacter colonies are often white or white with a green
centre, grey or black, yellow or red.

Confirmation
Purification of colonies: Select 5 typical colonies from the CCI
medium. In case colonies are not well separated, streak a typical
colony onto the CCI agar again.
If on the dish there are fewer than five typical colonies, take all the
marked colonies for confirmation.
Streak the selected colonies onto the non-selective agar (e.g.:

137
TSA) to gain well-isolated colonies.
Invert and incubate the plates between 34 °C to 38 °C for 21 h ± 3
h.
If the cultures on the non-selective agar are mixed, sub-culture the
suspect colony onto a plate of the non-selective agar further and
incubate between 34 °C to 38 °C for 21 h ± 3 h to obtain a pure
culture.
If positive, subject to biochemical confirmation tests. If negative,
progress through the other selected colonies until either all are
negative or a positive is found.
Strains can be kept on the non-selective agar at 5 °C, but cannot be
stored for more than 7 days.

Biochemical confirmation
Fresh subcultures of the colonies should be obtained before
performing confirmation tests.

Table showing confirmation tests for Cronobacter spp.

Oxidase Acid from:

Hydrolysis of 4-Nitrophenyl α- D-Arabitol

D-glucopyranoside substrate
L-Lysine decarboxylase D-Sorbitol
L-ornithine decarboxylase D-Sucrose
Methyl Red (Optional) A-Methyl-D-glucoside
Voges-Proskauer (Optional) (optional)
Select one yellow pigmented colony from each TSA plate for
further biochemical characterization below. Miniaturized
biochemical identification kits (must be approved by FSSAI under
RAFT scheme) may be used.

Oxidase test: Using a inoculation loop or glass pasteur pipette,

place a portion of the isolated colony on a filter paper moistened

138
with the Oxidase Kovac’s Reagent. The appearance of a mauve,
violet or deep blue color within 10 s indicates a positive reaction.
If a commercially available oxidase test kit (must be approved by
FSSAI under RAFT scheme) is used, follow the manufacturer’s
instructions.

Citrate test: Streak the selected colonies onto the slant surface of
Simmons Citrate Agar. Incubate the tubes at 30 ± 1°C for 24 ± 2 h.
Positive test is indicated by presence of growth and color change
from green to blue. Negative test is indicated by no growth or very
little growth and no color change.

Arginine dihydrolase and lysine/ornithine decarboxylase tests:

Inoculate the culture into tubes of Decarboxylation Medium (with
0.5% L-lysine, L-ornithine or L-arginine). Incubate the tubes at 30
± 1°C/24 ± 2 h. A violet color after incubation indicates a positive
reaction. A yellow color indicates a negative reaction.

Hydrolysis of 4 Nitrophenyl (PNP) α-D-glucopyranoside

substrate
Using a loop or wire, suspend an individual colony grown on the
non-selective agar such as TSA in 2ml of physiological salt
solution. 0.85% NaCl. Add 2ml of the α-Glucosidase enzymatic
assay solution. Incubate in a water bath at 37˚C for 4h and
measure the formation of yellow colouration in a
spectrophotometer at 405 nm. A minimal absorption of 0.3 at 405
nm after 4h, equivalent to 16 mM PNP, can be considered
positive.

Carbohydrate fermentation tests: Inoculate the culture into

tubes of Carbohydrate Fermentation Medium (with 1% D-sorbitol,
L-rhamnose, D-sucrose, D-melibiose or amygdaline). Incubate the

139
 tubes at 30 ± 1°C/ 24 ± 2 h. A yellow color after incubation
indicates a positive reaction. A red color indicates a negative
reaction.

Methyl Red (Optional)

Inoculate the prepared glucose peptone medium and incubate at
37°C for 24-48h. Add 2 drops of methyl red solution prepared by
dissolving 0.04g of methyl red in 40 ml of absolute ethanol and
dilute with water to make up to 100 ml. A positive reaction is
indicated by red colour and a negative reaction by yellow colour.

Voges-Proskauer (VP) (Optional)

Inoculate the prepared glucose peptone medium and incubate at
37°C for 24-48 h. To 1ml of the growth, add 0.6 ml of α-naphthol
solution prepared as 5% solution in ethanol, shake and add 0.2 ml
of 40% aqueous potassium hydroxide. Shake and slope the tube
and observe for up to 4h for appearance of a pink colour which
indicates a positive reaction.

Interpretation Based on the observations and interpretation of the results, report

Expression of results: presence or absence of Cronobacter spp. in test portion specifying
the mass in grams or mililitres of the sample Cronobacterspp.=
present or absent/ 10 gm or ml.
Reference ISO 22964:2017 -Microbiology of food chain — Horizontal
method for the detection of Cronobacter spp.
Approved by Scientific Panel on Methods of Sampling and Analysis

140
 Method for Enumeration of Sulfite Reducing Clostridia based on
ISO 15213

Method No. FSSAI 15.008:2023 Revision No. & Date 0.0

Introduction Sulphite-reducing clostridia are Gram-positive anaerobic spore-
forming rod-shaped bacteria which have ability of reducing sulphite to
sulphide under anaerobic conditions within 24-48 hours. Anaerobic
sulfite-reducing bacteria are generally considered as indicators of
Clostridia contamination in food products. Clostridium is
a genus of Gram-positive bacteria, which includes several significant
human pathogens, including Clostridium botulinum, Clostridium
perfringens, Clostridium difficile, etc.
Scope This method is applicable to those food product categories and their
sub-categories as mentioned in the Appendix B tables of Food Safety
and Standards (Food Products Standards and Food Additives)
Regulations, 2011 and amendments (Gazette notifications) issued from
time to time.
Caution Carry out the test under the control of skilled microbiologist and great
care shall be taken in the disposal of all the incubated material. Follow
safe and good laboratory practices to avoid cross contamination.
Principle Iron Sulfite Agar is used for sulfite reducing clostridia. The medium
contain disodium sulfite and Iron ammonium citrate. Disodium sulfite
is reduced by sulfite reductase enzyme released by bacteria. So when
H2S is produced from sulfite reduction, the colony becomes black due
to the formation of iron sulfide from citrate. As incubation is done
under anaerobic conditions, only anaerobic bacteria having ability of
sulfite reduction are isolated and enumerated. Clostridia colonies are
confirmed by doing respiratory and spore forming test.
Equipment 1. Laminar airflow
2. Biosafety cabinet
3. Hot air oven
4. Autoclave
5. Anerobic Jar for creating anerobic atmosphere and a system to
check the anerobic condition

141
 6. Incubator (Operating at 37 °C ± 1°C and 50 °C ± 1°C)
7. Water bath (at 44 °C to 47 °C)
8. pH meter with measuring accuracy ±0.1
9. Microscope
10. Refrigerator (at 2 °C– 8 °C)Petri dishes (Glass or plastic of 90-
100mm diameter or 140mm)Graduated pipettes (0.1 ml divisions)
of capacity 1 ml (Class A)
11. Micropipette with tips
12. Test tubes (16 x 160 mm) and flasks or bottles of capacity 500 ml.
13. Vortex
14. Mechanical stirrer
15. pH meter with measuring accuracy ±0.1
16. Spreader (glass or plastic)
17. Inoculation loops and straight wire
18. Spiral plater/rotator
19. Inoculation loops and straight wire

Culture Media and  Saline Peptone Diluent/ or buffered peptone water (BPW)
Reagents  ron Sulfite Agar
 Ellner's Medium
For media preparation and other details refer Chapter 3
Reference Cultures Clostridium spp (Clostridium sporogenes)
Sample Preparation Refer Chapter 2
Procedure Preparation of the samples and serial dilutions.
a). Following the procedures described in Chapter 2
Heat treatment of the initial suspension is necessary to eliminate
vegetative forms of spore forming bacteria. Temperature and heating
time may vary accrding to actual need from combinations producing
definite pasteurization effect a moderate heat acivaion effect (e.g 75 C
for 20 min o boiling to several minues)., Homogenize 10g of sample
with 90 mL of saline peptone water (SPW) or buffered peptone water
(BPW) (10-1 dilution). From this first dilution prepare serial decimal
dilutions,

142
b) Inoculation.

Take two sterile Petri dishes. Using a sterile pipette/ micropipette tip,
transfer to the dish 1 ml of the test sample if the product is liquid, or 1
ml of the initial suspension in case of other products. Repeat the
procedure described with the further dilutions, if necessary, using a
fresh sterile pipette/ micropipette tip for each dilution.

Pour Approx. 18 to 20 ml molten iron sulfite agar, previously cooled to

440C to 470C in water bath.
Time elapse between inoculation and addition of agar into plates
should not exceed 15 mins. The time between the end of the
preparation of the initial suspension and pour plating shall not exceed
45 min.
Carefully mix the inoculums into medium by horizontal movements
and allow the medium to solidify.
After solidification, add 5-10 ml of iron sulfite agar into dishes as
overlay.
c) Incubation
1. Incubate all the petri dishes in anaerobic jar at 370C±10C
for 24 to 48h.
2. If thermophillic bacteria are suspected, prepare a second
set of petri dishes and incubate this set at 500C±10C.
 Observation
1. Read the results after 24 to 48 h, depending on the
bacterial growth. Count all black colonies, possibly
surrounded by a black zone as sulfite reducing bacteria.
 Confirmation
1. For confirmation of clostridia, pick 5 characteristics
colonies colonies from each dish and proceed the
confirmatory test i.e respiratory test and spore forming
test.
2. In respiratory test allow to grow characteristic colonies
aerobically to check their growth in presence of oxygen.

143
 3. Spore forming test can be done by inoculating spore
inducing medium i.e. Ellner's medium and observe the
spore production.
Calculation 1. Select petri dishes having less than 300 total colonies and
less than 150 typical colonies for calculation.
2. Use the following formula for calculation

N is Number of colonies per ml or g of product

∑ C is the sum of colonies counted on all the dishes
retained
n1 is the no. of dishes retained in the first dilution
n2 is the no of dishes retained in the second dilution
d is the dilution factor corresponding to first dilution
3. In case petri dishes have total/typical colonies less than 10, calculate
the results using the following formula:
N= C/ (v × d)
N is Number of colonies per ml or g of product
C is the average of colonies on the petri dishes retained
v is the volume of inoculums used in each dish
d is the dilution corresponding to the dilution retained.
Expression of Results  Results shall be expressed as a number between 1.0 and 9.9
multiplied by 10x, where x is power of 10.
 If plates from all dilutions have no colonies, the result is
expressed as less than 1 cfu/ml or 10 cfu/g or mL (if primary
suspension prepared)
Reference ISO 15213 (2003): Microbiology of food and animal feeding stuffs —
Horizontal method for the enumeration of sulfite-reducing bacteria
growing under anaerobic conditions.
Approved by Scientific Panel on Methods of Sampling and Analysis

144
 Methods Isolation and Identification of Clostridium perfringens and
Clostridum botulinum and enumeration of Clostridium perfringens
based on IS 5887-Part 4
Method No. FSSAI 15.009:2023 Revision No. & Date 0.0
Introduction Several micro-organisms contaminating food give rise to clinical
symptoms, such as abdominal pain, nausea, vomitting, diarrhoea and
sometimes pyrexia. A well-known exception is that of botulism where
the symptoms are those of difficulty in swallowing, diplopia, aphonia
and difficulty in respiration. Poisoning through food is characterized by
symptoms of explosive nature which occur in otherwise healthy
individuals. Such explosive nature of food poisoning helps in
differentiating conditions from those of out-breaks of food-borne
infectious diseases which generally spread over a period of several days.
This part of the standard covers the method for isolation and
identification of some Clostridium species responsible for food
poisoning.
Scope This method is applicable to those food product categories and their sub-
categories as mentioned in the Appendix B tables of Food Safety and
Standards (Food Products Standards and Food Additives) Regulations,
2011 and amendments (Gazette notifications) issued from time to time.
Caution The test must be carried out under the control of skilled microbiologist
and great care shall be taken in the disposal of all the incubated material.
Follow safe and good laboratory practices to avoid cross contamination.
Utmost biosafety precautions to be taken while performing the test for
C. botulinum.
Principle Clostridium perfringens: The method of identification of food-
poisoning strains of Clostridium perfringens is based on colonial
characters, morphology and the Nagler reaction. The frequency of spore-
bearing Clostridium perfringens is low and reduces the diagnostic values
of this criterion.
Clostridium perfringens: The method is based on growth on blood agar
medium associated with haemolysis and on egg-yolk medium, C.

145
 botulinum colonies produce opalescence and a pearly layer and are
lactose negative. Followed by demonstration of toxin by in vivo test in
guinea pigs/mice.
Equipment 1. Laminar airflow
2. Biosafety cabinet
3. Hot air oven
4. Autoclave
5. Incubator (Operating at 30 °C ± 1°C and 37°C ± 1°C)
6. Anerobic jar
7. Water bath (at 44 °C to 100 °C)
8. pH meter with measuring accuracy ±0.1
9. Microscope
10. Refrigerator (at 2 °C– 8 °C)
11. Petri dishes (Glass or plastic of 90-100mm diameter or 140mm)
12. Graduated pipettes (0.1 ml divisions) of capacity 1 ml (Class A)
13. Micropipette with tips
14. Tubes and glass bottles
15. Vortex
16. Mechanical stirrer
17. pH meter with measuring accuracy ±0.1
18. Spreader (glass or plastic)
19. Inoculation loops and straight wire
20. Spiral plater/rotator
21. Inoculation loops and straight wire
22. Colony counter
Medium i) Cooked meat medium
ii) Willis and Hobb’s medium with neomycin (Egg yolk Medium)
iii) Medium for C. botulinum type E
iv) Nutrient broth
v) Nutrient agar
vi) Spore Inducing Medium (Ellner’s Medium)
vii) Animals
viii) Mice

146
 ix) Guinea Pig
x) Blood agar with neomycin
xi) C. perferingens antitoxin
xii) C. botulinum antitoxin
Procedure Isolation of C. perfringens
The sample is blended in a sterile blender/jar for 2 minutes
using approximately 200 ml of diluting fluid per approximately 25 g of
the sample. The diluting fluid should be 0.1 percent peptone in water
sterilized at 120°C for 20 min, final pH6.8 ±0.1or 3.4 percent of
potassium dihydrogen phosphate (KH2PO4) in water, pH adjusted to 7.2
and sterilized at 120°C for 20 min.
An aliquot of the specimen is inoculated into cooked meat medium and
the inoculated tube heated in a steamer at 100°C for one hour and
incubated over- night at 37°C.
An aliquot of the specimen is also inoculated directly on to blood agar
edium and the egg-yolk medium and incubated in an anaerobic jar at
37°C overnight.
Subcultures are made from the growth in medium on to the two solid
media (i.e Blood agar with Neomycin and Willis and Hobb’s Medium
with Neomycin) and incubated in an anaerobic jar at 37°C overnight.

Isolation of C. botulinum
Preheat the sample at 80°C for 30 min and inoculate into cooked
meat medium and the two solid media (Blood agar with neomycin
and Wilfis and Hobb’s medium with neomycin). The solid media are
incubated anaerobically and all the three inoculated media are
incubated at 37°C for 5 to 10 days.

Isolation of C. botulinum type E strains:

C. botulinumtype E strains exhibit low thermal resistance and are missed
in specimens which have been heated prior to inoculation. Inoculate the
specimen in duplicate tubes of the medium (Medium for C. botulinum
type E) and incubate at 30°C for 3 days

147
1) In sterile test tubes take aliquots of 2 ml samples of growth and mix
with equal volume of absolute ethanol. Let stand at 25°C for one
hour with occasional mixing.
2) Streak onto Willis and Hobb’s medium with neomycin and inoculate
into medium for C. botulinum type E. Incubate overnight at 37°C the
solid medium being incubated in an anaerobic jar.
3) Examine the solid medium for presence of colonies with opalescence
zones indicating growth of C .botulinum Type E. If such colonies are
present, carry out test for toxin using the growth in medium for C.
botulinum type E inoculated with ethanol treated culture.

Identification of C. perfringens
1) Grams stain: Test from liquid culture and solid media - Gram-
positive rods, large and stout withstraight sides and rounded ends.
Spores are oval, central or subterminal and distend the bacillary
body.
2) Colonial Characters: On blood agar medium growth is associated
with haemolysis which may not be larger than the colony. On Willis
and Hobb’s medium with neomycin, colonies produce opalescence
and a pearly layer and are lactose negative.
3) Spore prduction
Inoculate growth from any of the media as in Blood agar with
Neomycin and Willis and Hobb’s medium with neomycin into spore
inducing medium. The growth in cooked meat medium may also be
inoculated into medium.
NOTE — Some workers have noted reduced heat resistance of
spores when cultures are grown in spore inducing medium.

4) Nagler reactin – In vitro Test for alpha Toxin

One half of a plate of Willis and Hobb’s Medium with Neomycin is
spread over with two or three drops of standard Clostridium
perfringens antitoxin and dried. The area is demarcated. The two
halves of the plate are inoculated with the suspect strain and

148
 incubated at 37°C anaerobically. Lecithinase activity is shown by
precipitates around colonies in the half without antitoxin and this
reaction is inhibited in the other half with specific antitoxin. The
production of the enzyme lecithinase C, as demonstrated in the
Nagler reaction by all types of C. perfringens is used to distinguish
C. perfringens from other species of Clostridia. However, C.
bifermentes also produce lecithinase and maybe differentiated from
C. perfringens by C. bifermentes showing proteolytic activity, ready
sporulation and non-fermentation of lactose. Lactose fermentation is
carried out in 1 percent peptone water sugar medium incubated
anaerobically at 37°C.

Identification of C. botulinum
1) Gram's Stain

2) Colonial Characters
By growth on blood agar medium and egg-yolk medium, as
described and for Type E strains.
3) In vivo Test for Toxin
Grow suspect strain in cooked meat medium for to 10 days. Obtain
filtrate and divide into two portions, one of which is heated at 100°C
for 10 min. Use three guinea pigs for intraperitonal injection with
filtrate as follows:

a) One animal is protected with polyvalent botulinum antitoxin and

injected with 2 ml of unheated filtrate;
b) One animal as injected with 2 ml of unheated filtrate and is
unprotected; and
c) One animal is injected with 2 ml of heated filtrate.
Death with paralytic symptoms of the unprotected animal receiving
unheated filtrate and survival of the other two animals diagnose
the presence of botulinum toxin.

149
Demonstration of Toxin of C. botulinum Type E
The procedure as in In vivo Test for Toxin may fail to demonstrate toxin
of C. botulinumType E. For such strains the procedure shall be as
follows.
1) To filtrate from growth in medium as obtained after procedure
described above trypsin is added to a final concentration of 0.1
percent. Incubate at 37°C for 60 min.
2) Dilute specific type E antitoxin 1 in 5 with 0.1 M phosphate buffer
of pH 6.5 containing 0.2 percent gelatin.
3) To 1.5 ml of diluted antitoxin, add equal volume of trypsinized
filtrate mix and keep at room temperature for 30 min.
4) Inject 1 ml of the mixture intra-peritoneally into a pair of white
mice. Also inject a pair of mice with 0.5 ml of the filtrate heated at
100°C for 10 min and another pair of mice with 0.5 ml of unheated
trypsinized filtrate.
Observe the mice up to 96 h. Death of the unprotected mice and
survival of the mice receiving neutralized toxin and the heated toxin
diagnose toxin of C. botulinumType E.
Since bacteriological diagnosis of food-poisoning due to C. botulinum is
based on the demonstration of the toxin in the food or intestinal content,
the presence of the toxin in such materials need to be demonstrated.

Procedure
The material is soaked overnight in equal volume of sterile normal
saline. The suspension is centrifuged and the supernatant sterilized
by filtration. This is then directly used to note the presence of
botulinum toxin by animal inoculation as with culture filtrate
described in vivo test and demonstration of C. botulinum Type E
toxin.
SEROTYPING
Food poisoning strains of Clostridium perfringens may be serotyped by
slide agglutination using colonies from blood agar and testing with
specific agglutinating sera, if these are available.

150
 ENUMERATION
Clostridium perfringens 25 to 50 g of the sample is taken in a sterile
blender/jar and to this is added diluting fluid to have dilution of 10-1.
Blend at 8,000 to 10,000 rev/min for 2 min. Make serial ten-fold
dilutions with the diluting fluid in duplicate series up to 10-7. Streak 0.1
ml from each tube evenly on to blood agar medium and also on to egg-
yolk medium .Incubate in an anaerobic jar at 37°C for 18 to 24 h. It is
useful to incubate aerobically duplicate plates similarly inoculated for
comparison. The suspect colonies of Clostridium perfringens are
counted and the number of viable colonies per gram of sample
determined by multiplying by the dilution factor(s) and dividing by the
mass of the sample.

Reference IS 5887 (Part 4): Methods for Detection of Bacteria Responsible for
Food Poisoning, Part 4: Isolation and Identification of Clostridium
Perfringens (Clostridium Welchii) and Clostridium Botulinum and
Enumeration of Clostridium Perfringens (Second Revision)
Approved by Scientific Panel on Methods of Sampling and Analysis

151
 Method for Enumeration of Enterobacteriacae

Method No. FSSAI 15.010:2023 Revision No. & Date 0.0

Introduction Enterobacteriaceae are a large family of gram - negative non
spore forming rod shape bacteria, with the ability to ferment
glucose. Members of the Enterobacteriaceae are classified into
coliform and non coliform group based on ability to ferment
lactose. Common food borne genera of
Enterobacteriaceae family are Salmonella, Yersinia,
Escherichia coli, Shigella, Citrobacter, Ervinia, Hafnia,
Klebsiella, Proteus, Providencia, Morganella, Serratia etc.
Scope This method is applicable to those food product categories and
their sub-categories as mentioned in the Appendix B tables of
Food Safety and Standards (Food Products Standards and Food
Additives) Regulations, 2011 and amendments (Gazette
notifications) issued from time to time.
Caution Carry out the test under the control of skilled microbiologist and
great care shall be taken in the disposal of all the incubated
material. Follow safe and good laboratory practices to avoid
cross contamination.
Principle The quantification of Enterobacteriaceae can be achieved by the
standard plate count method usingviolet red bile glucose
(VRBG) agar as the culturemedium. Characteristic
Enterobacteriaceae colonies on VRBG are glucose fermenting
are show oxidae negative reaction.

Equipment 1. Laminar airflow

2. Biosafety cabinet
3. Hot air oven
4. Autoclave
5. Incubator (Operating at 30 °C ± 1°C)
6. Water bath (at 44 °C to 47 °C)

152
 7. pH meter with measuring accuracy ±0.1
8. Microscope
9. Refrigerator (at 2 °C– 8 °C)
10. Petri dishes (Glass or plastic of 90-100mm diameter or
140mm)
11. Graduated pipettes (0.1 ml divisions) of capacity 1 ml (Class
A)
12. Micropipette with tips
13. Tubes and glass bottles
14. Vortex
15. Mechanical stirrer
16. pH meter with measuring accuracy ±0.1
17. Spreader (glass or plastic)
18. Inoculation loops and straight wire
19. Spiral plater/rotator
20. Colony counter (optional)

Culture Media and For Colony count technoque

Reagents Diluent: Saline peptone water (SPW) or buffered
peptone water (BPW)
(Refer Chapter 3 to check on special cases in which either the
type or volume of diluent vary as a function of the sampleto be
examined).
Violet Red Bile Glucose Agar (VRBG)
Nutrient Agar
Glucose OF medium
oxidase reagent (N,N,N’,N’- Tetramethyl-p-phenylenediamine
dihydrochloride) / Buffered brilliant green bile glucose broth (SS
and double stenghth for MPN Method)
For media preparation and other details refer Chapter 3
Reference Cultures Member of Enterobacteriacae (E.coli, etc)
Sample Preparation Refer Chapter 2
Procedure Preparation of the samples and serial dilutions:

153
Following the procedures described in Chapter 2, homogenize 10
g of sample with 90 mL of saline peptone water (SPW) or
buffered peptone water (BPW) (10-1 dilution). From this first
dilution prepare serial decimal dilutions.
Most probable number (MPN) technique

I. This technique is recommended when the number sought

is expected to be in the range 1 to 100 per millitre or per
gram of the test sampleTake three tubes of double-
strength medium. Transfer to each of these tubes, using a
pipette, 10 ml of the test sample if the product is liquid,
or 10 ml of the initialsuspension in the case of other
products.
II. Take three tubes of single-strength medium. Transfer to
each of these tubes, using another pipette 1 ml of the test
sample if the product is liquid, or 1 ml of the initial
suspension in the case of other products.
III. Take three more tubes of single-strength medium.
Transfer to each of these tubes, using another pipette 1
ml of the first decimal dilution (10-1) (of the test sample
if the product is liquid, or 1 ml of the first decimal
dilution of the initial suspension (10-2) in case of other
products.
Incubate these nine tubes at 35 °C or 37°C for 24 h.

Isolation
Streak a loopful from each of the nine incubated cultures on the
violet red bile gucose agar and incubate the plates at 35 or 37 °C
for 24 h.
Selection of colonies for confirmation
From each of the plates incubated on which typical pink to red
colonies (with or without precipitation haloes or coloufless,
mucoid colonies have developed, select at random five such
colonies for biochemical confirmation and subculture on nutrient

154
agar paltes. Keep these plates at 35°C or 37°C for 24 h. Select a
well isolated colon for biochemical confirmation from the
number of confirmed positive tubes, calculation of the most
probable number of Enterobacteriaceae per ml or per gm of Test
sample using the MPN table (Appendix A).

Colony Count Techniques;

Inoculation and Incubation:
Label all petri dishes with the sample code, dilution, date and
any other information.
1. Take two pert dishes. Pipette 1ml of the test sample (if the
product is liquid), or 1 ml of primary suspension (if
prepared) to the centre of each petri dish. Similarly prepare
plates from subsequent dilution as required
2. Pour approximately 15 ml of the molten VRBG agar, (cooled
at 44 °C) into each petri dish. Time elapse between
inoculation and addition of agar into plates shall not exceed
15 min.
3. Carefully mix the inoculum with the medium and allow to
solidify.
4. After complete solidification, pour about 5 ml to 10 ml of
molten VRBG agar (over lay) onto the surface of inoculated
medium and allow to solidify.
5. Incubate the plates in an inverted position at 35°C or 37 ±1
°C for 24 ±2 hours.

Enumeration and selection of colonies

After incubation, select the plates that contain less than 150
characteristic colonies for enumeration.
Characteristic colonies are pink to red or purple in color with or
without precipitation haloes.

Confirmation

155
 Select 5 colonies from each dish. If there are less than 5 colonies
on the plate, take all presumptive colonies present for
confirmation. When there is no characteristic colony present,
take five whitish colonies for confirmation. Streak the selected
colonies onto the non selective agar medium (e.g. nutrient agar)
and incubate these plates at 35 or 37 °C for 24 ±2 hours.
Select isolated colonies for biochemical confirmation.

Biochemical confirmation Tests

1. Oxidase Test:
Using an inoculation loop or glass rod, take a portion of well
isolated colony and streak onto a filter paper moistened with
the oxidase reagent.
Consider the test negative, when the colour of the filter paper
does not turn dark blue purple within 10s.
2. Fermentation Test:

Using an inoculation loop or glass rod, stab the same

colonies selected in oxidase test, into a tube of Glucose OF
medium and overlay the surface with minimum of 1 cm of
sterile mineral oil. Incubate at 37 °C for 24 ± 2 h. If yellow
colour developed in the tube throughout, consider as glucose
fermentation positive.

Interpretation of biochemical tests

The colonies that are oxidase negative and glucose positive are
confirmed as Enterobacteriaceae.

Calculation Calculation of the most probable number (MPN)

I. Count the number of tubes giving a positive reaction for

each dilution.
II. One of the selected typical colonies of a subculture is
oxidase-negative and glucose-positive; the tube from
which the subculture is derived shall be regarded as being

156
 positive.
III. Using the MPN table (see Appendix A), determine from
the number of positive tubes in the different dilutions, the
most probable number (MPN) index.
IV. In the case of liquid products, the number of
Enterobacteriaceae per millilitre is calculated by
dividing the MPN index by 10. In the case of other
products for which initial suspensions are prepared, the
number per gram is equal to the MPN.
1518
Colony count Method
4. Select petri dishes having 10 to 150 characteristics colonies for
enumeration.
5. Spreading colony shall be considered as single colony.
6. Use the following formula for calculation

N is Number of colonies per ml or g of product

∑ C is the sum of colonies counted on all the dishes
retained
n1 is the no. of dishes retained in the first dilution
n2 is the no of dishes retained in the second dilution
d is the dilution factor corresponding to first
dilution
In case petri dishes have total/typical colonies less than 10,
calculate the results using the following formula:
N= C/(v × d)
N is Number of colonies per ml or g of product
C is the average of colonies on the petri dishes
retained
v is the volume of inoculums used in each dish
d is the dilution corresponding to the dilution
retained.

157
Expression of Results Results shall be expressed as a number between 1.0 and 9.9
multiplied by 10 x, where x is power of 10.
 If plates from all dilutions have no colonies, the result is
expressed as less than 1 cfu/ml or 10 cfu/g or mL (if primary
suspension prepared).
Reference 1) ISO 21528-2:2017 Microbiologyof the food chain —
Horizontal method for the detection and enumeration of
Enterobacteriaceae — Part 2: Colony-count technique
2) IS 7402: Microbiology - General Guidance for the
Enumeration of Enterobacteriaceae without Resuscitation
MPN Technique and Colony-count Technique
Approved by Scientific Panel on Methods of Sampling and Analysis

158
 Method for Enumeration of Escherichia coli based on ISO : 16649-
2

Method No. FSSAI 15.011:2023 Revision No. & Date 0.0

Introduction E. coli is included both in the group of total coliforms as in that of
thermo tolerant coliforms. Its natural habitat is the intestinal tract of
hot-blooded animals, although it may also be introduced into foods via
non faecal sources.
Scope This method is applicable to those food product categories and their
sub-categories as mentioned in the Appendix B tables of Food Safety
and Standards (Food Products Standards and Food Additives)
Regulations, 2011 and amendments (Gazette notifications) issued from
time to time.
This method specifies a horizontal method for the enumeration of β-
glucuronidase-positive Escherichia coli in products intended for human
consumption. It uses a colony-count technique at 44 °C on a solid
medium containing a chromogenic ingredient for detection of the
enzyme β-glucuronidase.
Caution Precautions should be taken while dealing with suspected food
outbreak samples.
Principle Duplicate plates of tryptone-bile-glucouronic medium (TBX) are
inoculated with the specified quantity of the test sample/initial
suspension. Under the same conditions, using decimal dilutions of the
test sample/initial suspension, two plates per dilution are inoculated.
The dishes are incubated then examined to detect the presence of
colonies which, from their characteristics, are considered to be β-
glucuronidase-positive Escherichia coli. The number of colony-
forming units (CFU) of β-glucuronidase-positive Escherichia coli per
gram or per milliliter of sample is calculated.

Equipment 1. Laminar airflow

2. Biosafety cabinet
3. Hot air oven

159
 4. Autoclave
5. Incubator (Operating at 44 °C ± 1°C)
6. Water bath (at 44 °C to 47 °C)
7. pH meter with measuring accuracy ±0.1
8. Microscope
9. Refrigerator (at 2 °C– 8 °C)
10. Petri dishes (Glass or plastic of 90-100mm diameter or 140mm)
11. Graduated pipettes (0.1 ml divisions) of capacity 1 ml (Class A)
12. Micropipette with tips
13. Tubes and glass bottles
14. Vortex
15. Mechanical stirrer
16. pH meter with measuring accuracy ±0.1
17. Inoculation loops and straight wire
18. Spiral plater/rotator
19. Inoculation loops and straight wire
Culture Media and Diluent
Reagents Culture medium: Tryptone-bile-glucuronic medium (TBX)
Details of preparation given in Chapter 3
Reference Cultures Further details of culturing given in Chapter 3

Sample Preparation Sample preparation based on the product categories are given in
Chapter 2
Procedure Test portion, initial suspension and dilution
Aseptically weigh 10 gm of solid or semi-solid sample into a sterile
blender jar or into a homogenizer bag. Add 90 mL of sterile diluent.
Blend for 2 minutes at low speed (approximately 8000 rpm) or mix in
the homogenizer for 30-60 seconds. Powdered samples may be
weighed and directly mixed with the diluent with vigorous shaking (50
times through 30 cm arc). In most of the samples particulate matter
floats in the diluent. In such cases allow the particles to settle for two to
three minutes and then draw the portion of dilution where food
particles are minimum and proceed.

160
 Inoculation
Using a sterilepipetteor a micropipette, transfer to a Petri dish, 1 ml of
the test sample (if liquid), or 1 ml of the initial dilution (10–1) in the
case of other products. Inoculate two plates per dilution. Repeat the
procedure with the further decimal dilutions, if necessary, using a new
sterile pipette / micropipette tip for each dilution. Pour into each Petri
dish approximately 15 - 20 ml of the TBX medium (kept at 44 °C to 47
°C). Carefully mix the inoculum with the medium and allow the
mixture to solidify, with the Petri dishes standing on a cool horizontal
surface. Time elapse between inoculation and addition of agar into
plates shall not exceed 15 min.

Incubation
Incubate the plates in an inverted position at 44 °C for 18 h to 24 h.
The total incubation time shall not be longer than 24 h.

Enumeration
After incubation count the typical blue-green colonies of β-
glucuronidase-positive Escherichia coli in each dish containing less
than 150 typical CFU and less than 300 total CFU (typical and non
typical).
Expression of Results The calculation given below takes into account those cases most
frequently encountered when conducting tests in accordance with good
laboratory practice. Some special, fairly improbable, cases can arise
(e.g. very different CFU numbers between the two dishes from the
same dilution, or very different proportions from that of the dilution
factor between the dishes from two successive dilutions). It is then
necessary that the counting results be examined, interpreted and
possibly rejected by a competent microbiologist.

For a valid result, in general it is considered that it is necessary to count

the CFU on at least one dish containing minimum 15 blue CFU.

161
Calculate N, the number of CFU of β-glucuronidase-positive
Escherichia coli present in the test sample per milliliter or per gram, as
the weighted mean from two successive dilutions using the following
equation:

Where
∑a -the sum of the CFU counted on all the dishes retained from two
successive dilutions, at least one of which contains a minimum 15 blue
CFU.V -Volume of inoculum placed in each dish, in millilitres;
n1 - Number of dishes considred inthe first dilution;
n2 - Number of dishes considred in the second dilution;
d -Dilution factor corresponding to the first dilution retained [d = 1
when the undiluted liquid product (test sample) is retained].

Round off the results to two significant figures. Express the result as
the number of β-glucuronidase-positive Escherichia coli per milliliter
(liquid products) or per gram (other products) as a whole number to
two significant figures (if below 100) or as a number between 1.0 and
9.9 multiplied by the appropriate power of 10.

Estimation of low numbers

If the two dishes [at the level of the test sample (liquid products) or of
the initial suspension (other products) or of the first inoculated or
retained dilution] contain less than 15 blue CFU, calculate NE, the
number of CFU of β-glucuronidase-positive Escherichia coli present in
the test sample, as the arithmetical mean from two parallel plates using
the following equation:

NE where

- Sum of the blue CFU counted on the two dishes;

V -Volume of the inoculum, in millilitres, applied to each dish;

162
n -Number of dishes retained (n = 2 in this case);
d -Dilution factor corresponding to the initial suspension [d = 1 in the
case of liquid products where the directly inoculated test sample
isconsidered].

Round off the results to two significant figures

Express the result as follows:

Estimated number of β-glucuronidase-positive Escherichia coli per
millilitre (liquid products) or per gram (other products): NE = Y.

If the two dishes at the level of the test sample (liquid products) or the
initial suspension (other products) of the first inoculated or retained
dilution do not contain any blue CFU, express the result as follows:
- less than 1/d of β-glucuronidase-positive Escherichia coli per
milliliter (liquid products) or per gram (other products), where d is the
dilution factor of the initial suspension or the first inoculated or
retained dilution [d = 1 in the case (liquid products) where the directly
inoculated test sample is retained].

If for the two dishes from the first dilution d1 the total number of blue
and non-typical CFU is higher than 300 with visible blue CFU, and if
for the two dishes from the subsequent dilution d2 containing less than
300 colonies, no blue CFU can be counted, express the result as
follows:
- less than 1/d2 and more than 1/d1 β-glucuronidase positive
Escherichia coli per milliliter (liquid products) or per gram (other
products), where d1 and d2 are the dilution factors corresponding to
dilutions d1 and d2.

If for the two dishes from the first dilution d1 the total number of
typical CFU and non-typical CFU is higher than 300 without visible
blue CFU, and if for the two dishes from the subsequent dilution d2

163
containing less than 300 colonies, no blue CFU can be counted, express
the result as follows:
- less than 1/d2 CFU of β-glucuronidase-positive Escherichia coli per
millilitre (liquid products) or per gram (other products),
where d2 is the dilution factor corresponding to dilution d2.

Method of calculation: Special cases

In the case where the number of blue CFU is higher than 150 for the
two dishes from the first dilution d1, with a number of blue CFU below
15 for the two dishes from the subsequent dilution d2:
-if the number of blue CFU on each of the two dishes from dilution d1
is within the range 167 to 150 (upper part of the confidence interval of
a weighted mean equal to 150), use the calculation method for the
general case;
-if the number of blue CFU on each of the two dishes from dilution d1
is higher than 167 (upper limit of the confidence interval of a weighted
mean equal to 150 CFU), only take into account the result of the counts
of dilution d2 and carry out a low number count.
- In the case where counting the blue CFU on each of the dishes from
all the inoculated dilutions gives a number higher than 150, express the
result as follows:
more than 150/d -glucuronidase-positive Escherichia coli per
milliliter (liquid products) or per gram (other products), where d is
the dilution factor of the last inoculated dilution.
- In the case where only the two dishes from the lowest dilution
(highest concentration) contain less than150 typical CFU, calculate the
number N' of -glucuronidase-positive Escherichia coli present in the
test sample as the arithmetical mean of the colonies counted on the two
dishes, using the following equation:

164
 where
is the sum of the blue CFU counted on the two dishes;
V is the volume of the inoculum, in millilitres, applied to each dish;
n is the number of dishes retained (n = 2 in this case);
d is the dilution factor to the initial suspension or the first inoculated or
retained dilution [d = 1 in the case (liquid products) where the directly
inoculated test sample is retained].
Round off the results to two significant figures
Reference ISO: 16649-2- Microbiology of food and animal feeding stuffs-
Horizontal method for the enumeration of β –glucuronidase-positive
Escherichia coli- Part 2 Colony-count technique at 44°C using 5-brom-
4 chloro-3-indolyl β-D-glucuronide.
Approved by Scientific Panel on Methods of Sampling and Analysis

165
 Method for Enumeration of Escherichia coli based on IS 5887-Part
1

Method No. FSSAI 15.012:2023 Revision No. & Date 0.0

Introduction E. coli is included both in the group of total coliforms as in that of
thermo tolerant coliforms. Its natural habitat is the intestinal tract of
hot-blooded animals, although it may also be introduced into foods via
non faecal sources.
Scope This method is applicable to those food product categories and their
sub-categories as mentioned in the Appendix B tables of Food Safety
and Standards (Food Products Standards and Food Additives)
Regulations, 2011 and amendments (Gazette notifications) issued from
time to time.
Caution Precautions should be taken while dealing with suspected food
outbreak samples.
Principle A: Spread Plate Method: A specified quantity of test sample
(homogenised or serially diluted) is surface plated on a solid agar
culture medium. After incubation period typical or suspected colonies
are identified, enumerated and reported after confirmation.

 B: MPN Method: The test sample to be tested is serially diluted and

inoculated in MacConkey Broth Medium tubes in 3 replicates for each
dilution. E. coli present in the sample, ferment lactose in the medium
to produce acid and gas. The presence of acid is shown by a change in
the medium’s colour, whilst the presence of gas is indicated by the
collection of gas bubbles in an inverted Durham tube present in the
media. The pattern of positive tests (growth) in the replicates and
statistical probability tables are used to determine the count (most
probable number) of E.coli and reported after confirmation.
Equipment 1. Laminar airflow
2. Biosafety cabinet
3. Hot air oven
4. Autoclave

166
 5. Incubator (Operating at 37 °C and 44 °C ± 1°C)
6. Water bath (at 44 °C to 47 °C)
7. pH meter with measuring accuracy ±0.1
8. Microscope
9. Refrigerator (at 2 °C– 8 °C)
10. Petri dishes (Glass or plastic of 90-100mm diameter or 140mm)
11. Graduated pipettes (0.1 ml divisions) of capacity 1 ml (Class A)
12. Micropipette with tips
13. Tubes and glass bottles
14. Vortex
15. Mechanical stirrer
16. pH meter with measuring accuracy ±0.1
17. Inoculation loops and straight wire
18. Test Tube Racks
Culture Media and  Diluent (0.1 % Peptone and final pH adjusted 6.8 ± 0·1, or 3·4 percent
Reagents of potassium diyhdrogen phosphate (KH2PO4) in water, pH adjusted to
7.2)
 Culture medium: Tergitol -7 agar
 Nutrient Broth
 Nutrient Agar
 MacConkey Broth Medium
 Single strength
 Double strength
 Eosin Methylene Blue Lactose Agar Medium
 TSI Medium
 Medium for carbohydrate fermentation test
 Simmon, Citarate Agar
 Medium for Indole production
 Medium for Urease Test
 Medium for Motility Test
 Slides
 Gram Stain

167
 Methyl Red and Voges-Proskauer Test. –
Details of preparation given in Chapter 3
Reference Cultures Further details of culturing given in Chapter 3

Sample Preparation Sample preparation based on the product categories are given in
Chapter 2
Procedure Test portion, initial suspension and dilution
Preparation of Sample –
Take 25 g of the sample in a sterile blender jar and to this add 200 ml
of diluting fluid (sterile 0.1 % Peptone and final pH adjusted 6.8 ± 0.1,
or 3·4 percent of potassium diyhdrogen phosphate (KH2PO4) in water,
pH adjusted to 7.2).
Homogenise the sample properly. Make serial ten-fold dilutions with
the diluting fluid (0.1% peptone water), in duplicate series, up to 10-6.

Method of Isolation
1. Inoculate 1 ml of the homogenised sample into 10 ml of single
strength MacConkey broth medium.
2. If the numbers of organisms are assumed to be very small, then
inoculate 10 ml of double strength MacConkey broth medium.
3. Also streak loopfuls on to MacConkey agar medium and to
eosin methylene blue lactose agar and if available Tergitol-7
agar.
4. Incubate all the inoculated media at 37°C overnight.
5. If there is growth with fermentation of lactose in' the
MacConkey broth medium, then streak out a loopful on to each
of the solid media and incubate at 37°C overnight.

TESTS FOR IDENTIFICATION

Take as many suspect colonies from the solid media as possible, but
not less than 5, to investigate. The suspect colonies are smooth and are
lactose fermenting on MacConkey agar and on eosin methylene blue
lactose agar and are yellow colonies surrounded by yellow zones on

168
Tergitol-7 agar medium
Escherichia coli - Suspect when conforming to the characters
mentioned below and tested
1. Gram Stain: Gram negative rod shape bacteria
2. Motility test: Motile
3. Indole: Positive
4. H2S production in TSI medium: Negative
5. MR: Positive
6. VP: Negative
7. Citrate: Not Utilized
8. Urease: Negative
9. Salicin: Acid and gas production variable
10. Sucrose: Acid and gas production variable
11. Acid and gas formation in MacConkey broth: Positive
with acid and gas at 44°Cfor 2 days

Confirmation Test
1. Gram Stain
Perform gram staining with 24 hr growth culture from Nutrient agar
and observe the presence of gram negative rod shape bacteria

2. Motility Test
Inoculate by stabbing with a straight wire into the top of the motility
test medium with the colonies to be tested, inside the glass tubing to a
depth of about 5 mm. Take care that inoculation is not made on to the
surface of the medium outside the glass tubing. Incubate at 37°C for 18
to 24 hours. Motile strains shall be found to show growth on the
surface of the medium outside the ‘inner glass tube’ having travelled
through the entire medium inside this inner tube. If negative on the first
day, keep the inoculated tube at room temperature for a further 4 to 6
days to see if evidence of motility is present.

3. Test for Indole Production

169
Inoculate tubes of tryptone water with loopful of 24 hr growth culture
of typical/suspected colonies or MPN positive tubes (from Nutrient
Agar). Incubate the inoculated tubes at 37°C for 24 h. Add 0.5 m1 of
the Kovac's reagent to the tubes. Mix well and shake tubes gently. The
appearance of red color indicates the presence of indole. The
appearance of yellow color is a negative reaction.

4. Test for H2S Production in TSI Agar

With help of inoculating wire or a needle inoculate the TSI agar
(streaking slant and stabbing butt) with the culture obtained from the
nutrient agar plates and incubate at 37°C for upto 7 days. The absence
of blackening in the butt of TSI indicates a negative reaction.

5.Test for Methyl Red (MR)

Inoculate the MR-VP medium and incubate at 37°C for 2 days. Add 2
drops of methyl red solution prepared by dissolving 0.04 g of methyl
red in 40 ml of absolute ethanol and diluting with water to make up to
100 ml. A positive reaction is indicated by red colour and a negative
reaction by yellow colour.

6. Test for Voges-Proskauer Reaction (VP)

Inoculate the MR-VP medium and incubate at 37°C for 2 days. To 1 ml
of the growth add 0.6 ml of alpha-naphthol solution prepared as 5
percent solution in ethanol. Shake and add 0.2 ml of 40 percent
aqueous solution of potassium hydroxide. Shake and slope the tube and
observe for up to 4 hours for the appearance of a pink colour which
indicates a positive reaction.

7. Test for Citrate Utilization

Inoculate the strain on to SCA medium with a young nutrient agar slant
culture using a straight wire. Incubate at 37°C for up to 4 days for
growth of the organism. No medium color change indicates a negative
reaction and medium color change to blue indicate a positive reaction.

170
8. Urease Test
Inoculate the organisms from the 24hour incubated nutrient broth
culture heavily over the entire slope of urea slant and incubate at 37°C
for 18 to 24 hours. A positive urease is shown by the medium
becoming, pink or red on incubation. If negative, continue incubation
for at least 4 days. Proteus species gives a positive result and may be
used as ‘positive control’.

9. Test for Carbohydrate fermentation

Inoculate the Andrade Peptone water medium and the carbohydrates i.e
sucrose or salicin @ 1 % and incubate at 37°C for 18 hours. Record the
presence of acid from pink colour and that of gas in the Durham’s
Tube. Alternatively, readymade carbohydrate disc may be used.

Enumeration of Escherichia coli

Preparation of Sample –
Take 25 to 50 g of the sample in a, sterile blender jar and to this add
diluting fluid to have dilution of 10-1. Blend at 8000 to 10000 rev/min
for 2 minutes. Make serial ten-fold dilutions with the diluting fluid
(0.1% peptone water), in duplicate series, up to 10-6.

A) Plate Count
Spread out 0.1 ml from each dilutin tube, evenly n Tergitol-7 agar, and
incubate at 37°C for 24 hours. Enumerae the colonies of E. coli which
are of yellow clour surrounded by yellow zone and confirm these as
being E. coli by the tests specified in this method. The number of
viable colonies of E. coli per gram of sample shall be determined by
multiplying by the dilution factor(s) and dividing by the mass of
sample. Tergitol-7 agar plates/MacConkey agar plates or eosin
methylene blue lactose agar plates may be used

B) Determination'of the Most Probable Number of E. coli

171
 I. Obtain serial dilutions of the sample with a fresh sterile pipette,
a measured volume of 1 ml of the homogenized mixture and of
the five following serial-dilutions of both dilution series in
triplicate to the tubes of 10 ml of single strength MacConkey
broth containing Durham's tube for collection of gas.
II. Start with highest dilution and proceed to the lowest, filling and
emptying the pipette three times before transferring the 1 ml
portions to the tubes of medium.
III. When the number of E. coli is assumed to be very small, start
by transferring 1.0 ml of the homogenized mixture in triplicate
to 10-1 ml of double strength MacConkey broth medium
containing Durham's tube for collection of gas, using a sterile
10 ml pipette.
IV. Incubate in a water-bath at 44°C for 48 hours.
V. Examine the tubes showing production of acid and gas, and
using Table, obtain the most probable number (MPN) of E. coli
per gram of the sample.
VI. Use for the calculation the results from three dilutions, selecting
the highest dilution showing three positive tubes below which
no sets with a smaller number of positive tubes occur, and the
two following higher dilutions.
VII. The number obtained from Table 1 of Appendix A has to be
multiplied by the lowest dilution factor, namely that of the first
set of tubes, to obtain the most probable number of E. coli per
gram of the sample.
For example, when dilution 100 (= 10 ml of macerate), 10-1 and
10-2 are found to give the following numbers of positive tubes:
2, 2, 1, the MPN is 2.8 bacteria per gram, and when the
dilutions 100, 10-1, 10-2, 10-3, 10-4 and 10-5 are found to give the
following numbers of positive tubes: 3, 3, 3, 2, 0, 0, the MPN is
9.3 (3, 2, 0), multiplied by the dilution factor 102, that is, 9.3 X
102 bacteria per gram. The MPN is reported as the average of
the results obtained from each of the duplicate dilution series.

172
 Refer APPENDIX A for MPN calculations
Expression of Results Plate count Method
The number of viable colonies of E. coli per gram of sample shall be
determined by multiplying by the dilution factor(s) and dividing by the
mass of sample.
MPN
The MPN is reported as the average of the results obtained from each
of the duplicate dilution series.

Reference IS 5887 (Part 1): Methods for Detection of Bacteria Responsible for
Food Poisoning, Part 1: Isolation, Identification and Enumeration of
Escherichia Coli
Approved by Scientific Panel on Methods of Sampling and Analysis

173
 Method for Detection, Isolation and Identification of Pathogenic E.
coli in Food based on IS 14397

Method No. FSSAI 15.013:2023 Revision No. & Date 0.0

Introduction Apart from serving as indicator of faecal contamination, E. coli is also
known to cause gastrointestinal disturbances especially in infants and
children in addition to traveller's diarrhoea. Hence, presence of these
organisms in food assumes greater importance. These pathogenic E.
coli are classified into five groups, namely, Enteropathogenic E. coli
serotypes, Enterotoxigenic E. coli serotypes, Enteroinvasive E. coli
serotypes, Enterohaemorrhagic E. coli and Enteroaggregate
(Enteroadhesive) E. coli.
Scope This method is applicable to those food product categories and their
sub-categories as mentioned in the Appendix B tables of Food Safety
and Standards (Food Products Standards and Food Additives)
Regulations, 2011 and amendments (Gazette notifications) issued from
time to time.
Caution Precautions should be taken while dealing with suspected food
outbreak samples.
Principle The scheme for detection, isolation and identification of EPEC, ETEC,
EIEC, EHEC and EAEC involves following steps: a) Presumptive
coliform test; b) Test for identification of typical coliform bacilli (E.
coli or faecal coli); c) Serological identification of EPEC, ETEC,
EIEC; and d) Confirmation of various strains.

Equipment 1. Laminar airflow

2. Biosafety cabinet
3. Hot air oven
4. Autoclave
5. Incubator (Operating at 44°C ± 1°C)
6. Water bath (at 44 °C to 47 °C)
7. pH meter with measuring accuracy ±0.1
8. Microscope

174
 9. Refrigerator (at 2 °C– 8 °C)
10. Petri dishes (Glass or plastic of 90-100mm diameter or 140mm)
11. Graduated pipettes (0.1 ml divisions) of capacity 1 ml (Class A)
12. Micropipette with tips
13. Tubes and glass bottles
14. Vortex
15. Mechanical stirrer
16. pH meter with measuring accuracy ±0.1
17. Spreader (glass or plastic)
18. Inoculation loops and straight wire
19. Spiral plater/rotator
20. Inoculation loops and straight wire
Culture Media and  Diluent
Reagents  Culture medium: Tryptone-bile-glucuronic medium (TBX)
 Nutrient Broth
 Nutrient Agar
 MacConkey Broth Medium
 Single strength
 Double strength
 Tryptone Water
 Kovac’s Reagent
 Brilliant Green Lactose Bile Broth
 Details of preparation given in Chapter 3
Reference Cultures Further details of culturing given in Chapter 3

Sample Preparation Sample preparation based on the product categories are given in
Chapter 2
Procedure ISOLATION
Presumptive Coliform Test
Prepare serial dilutions of the sample; The choice of dilutions depends
upon the type of samples to be tested. Transfer 1 ml of the sample and
its dilutions (1:10 and 1:100) into Mac Conkey's broth tube in
triplicate.

175
Production of Acid and Gas
Incubate the tubes for 24 h at 37 °C and observe for the production of
acid and gas. The production of acid is indicated by change of color of
the medium. Production of gas is observed in the Durham's tubes
which may be partially or completely filled with gas. If no change is
observed incubate for another period of 24 h and record the
observation.

TEST FOR IDENTIFICATION OF TYPICAL COLIFORM

BACTERIA (FAECAL OR E. COLI)
Elikman's Test
Inoculate BGLB broth tubes with a loopful of the culture from a
positive presumptive coliform tube in triplicate. Incubate the tubes for
24-48 h at 44°C and observe for the production of acid and gas. Only
faecal E. coli strains are capable of producing acid and gas in BG LB
broth.

Test for Indole Production

Incubate 3 tubes of tryptone water eachwith loopful of culture from
positive presumptive coliform test tube.
Incubate the inoculated tubes at 44°C for 24 h. Add 0.5 m1 of the
Kovac's reagent to the tubes. Mix well and examine after 1 min. The
appearance of red color indicates the presence of indole. The
appearance of yellow color is a negative reaction.

SEROLOGICAL IDENTIFICATION OF E. COLI STRAINS

Serological identification of E. coli strains depends upon the detection
of O, H and K antigens, though most often only O antigen is detected.

Determination of O antigen of Test Strains

This needs inactivation of K antigen (which cause O inagglutinability).

176
Suspend the growth from an agar slant culture in saline to give fairly
light suspension (about 7.5 x 108 organisms/ml). Heat the suspension at
121°C for 2 h 30 min to inactivate K antigens (where K antigens are of
B type, heating at 100°C for 1 h will be sufficient). Transfer loopfuls of
the antigen suspension on to a clean glass slide. Add loopful of
different pool of O, K sera (O sera if available) to the drops of antigen
on the slide. Mix and rock gently. Observe for agglutination which
should be strong and clearly visible within one min. Weak and late
agglutination reactions should not be taken into account. Always a
saline control should be used in carrying out slide agglutination
reactions.

If the agglutination is seen with any one of the pools of O-K antisera
(or O antisera), then the same antigen should be checked with all the
individual factor sera that constitute the pool to arrive at the ‘O’
antigen of the strain in question.
The results of slide agglutination should be confirmed by tube
agglutination.

Tube Agglutination
Make serial dilutions of the antiserum (identified as above) in 0.5 ml
volumes in saline from 1:10 to 1:640 (round bottomed glass tubes of
approximately 9 mm x 85 mm size are suitable). To each, add 0.5 ml of
the antigen suspension. This doubles the dilution of the serum. A
control tube should be set up containing only the antigen and saline
(0.5 ml each). Shake the tubes and incubate in water bath (50 °C)
overnight. Examine for agglutination. Ag- agglutination titres 1 in 20
are insignificant. Titres at or near the stated titre of the serum are
significant.

CONFIRMATION OF ENTEROPATHOGENICITY OF EPEC

STRAINS
As the mode of pathogenicity of EPEC strain is not clearly understood,

177
no standard methods are available for determination of
enteropathogenicity except serotyping.

CONFIRMATION OF ENTEROTOXIGENCITY OF ETEC

STRAINS
ETEC are known to produce two types of toxins; (i) heat labile toxin
(LT), and (ii) heat stable toxin (ST). Heat labile toxin (LT) is detected
by both in-vitro methods (Biken test and tissue culture methods) and
in-vivo method (Rabbit ileal loop assay method).
Biken Test
This test holds promise of being the simplest and most practical for
laboratories with limited facilities while at the same time being
reliable.

Principle
Thespecific antitoxin reacts with toxin liberated by an actively growing
organism on a special medium and produces a line of precipitation at
the sites where they meet in optimal proportion.
Apparatus
The Biken test kit:
Media and Reagents:
Biken agar No. 2
Composition (for 100 ml):
Casamino acid: 2.0g
Yeast extract: 1.0 g
NaCl: 0.25 g
K2HP04: 1.5 g
Glucose: 0.5 g
Trace Salts Solution (5% MgSO4, 2% CoC16.6H20 and 0.05 ml 0.5%
FeCl3)
Noble agar or agarose: pH to 7.5 with 1 M NaOH. Sterilize by
autoclaving at 121°C for 15 ml and distribute 15 ml in test tubes for
each plate.

178
Lincomycin: 2.7 mg/ml (it increases toxin production).
Polymixin B disc: 6 mm discs containing about SOC IU (it helps toxin
release).
Anti-LT serum: The optimal dilution of the antitoxin has to be
predetermined by testing two-fold dilutions (for example 1:2 to 1:32)
of the antitoxin against the toxin antigen by the gel-diffusion technique.
This is carried out in a Petri-dish using a media containing 1.5 g of
Noble agar in 100ml of phosphate buffer solution (0.01 M) with 0.25
percent sodium azide. Punch one central well for the toxin antigen and
other well around it at an equal distance for different dilutions of
antitoxin. The optimal dilution lies between the two highest dilutions
showing lines of precipitation after overnight incubation, for example,
if the highest dilutions showing precipitation are 1:16 and 1:8, the
optimal dilution for working purposes is 1:12.
Pure LT for determining the optimal working dilution of antitoxin.
Gel puncher: 4 mm diameter.
Template for inoculation of strains.

Procedure
Transfer 0.5 ml of lincomycin solution to a sterile Petri-dish 90 mm in
diameter.
Dissolve Biken agar No. 2 on a boiling water bath. Cool the medium to
about 50 to 60°C, and pour into the Petri-dish containing lincomycin
solution. Mix well by rotating the plate at least 10 times. Prepared
plates can be stored at 4°C for 3-5 weeks if kept in a plastic bag to
prevent drying. Before using freshly prepared media, dry the agar
surface.

Inoculation of test cultures

Spot inoculate the test strains to ensure a fairly large area of confluent
growth around the site where the central well will be punched. Allow
for a distance of about 4 mm between the inner edge of the growth and
the margin of the site of the central well and take care that the strains

179
do not touch each other when they grow during the incubation period.
Four test cultures, or three test and one positive control culture, may be
inoculated around each central well.
After 48 h of incubation, put a polymyxin B disc on top of the growth
of each strain. Incubate for 6 h.
Punch a well (4 mm in diameter) in the centre of the area so that the
distance between the well and the edges of the growth is about 4 mm.
Put 20 µl of the optimal dilution of the antiserum against LT into the
central well. Incubate again for 20-24 h.

Examine for lines of precipitation in the zone between the growth and
the central well. The lines may not always be very distinct at this time,
but are seen better after further incubation for 15-20 h, or when the
plates are placed on a light box with black background. The
precipitation bands developed can be stained with commassie blue (0.1
percent) to make them more prominent.

Rabbit ligated Ileal Loop Test

Reagents and Apparatus
In addition to the usual microbiological apparatus, the following are
required.
Animal operating board
Clippers
Hypodermic syringe with 25-gauge needle
Stainless steel wound clips, 0.9 cm.
Surgical thread
Sodium pentabarbitone, 3 percent.
Ether
Normal saline
Han’s F-JO Nutrient mixture

Procedure
Select three albino rabbits weighing about 2 kg each. Supply them with

180
water but no food for 24-48 h before surgery. Restrain rabbits on their
back using a small animal operating board. Remove abdominal fur with
clippers. Using the clean technique and employing general anaesthesia
(1 ml of 3 percent sodium pentabarbitone/kg body weight followed by
ether inhalation), make a midline incision, identify and gently lift out
the small bowel from the abdominal cavity, taking care to keep it moist
with sterile saline. Tie a single cotton suture around the proximal end
of the small bowel 5 cm below the stomach.
Inject 10 ml of sterile saline into the upper small bowel using a 25-
gauge needle. Gently manipulate the saline between two fingers down
the bowel and into the caecum. Place another tie above the ileo-caecal
junction.

Tie off a series of 5 cm segments, starting proximally, with 2 cm

segments separating each 5 cm segment. As many as 10-15 of the 5 cm
segments can be isolated in a 2 kg rabbit. Do not injure blood vessels
or interrupt the blood supply to the intestine. Inject positive control
filtrates, saline controls, and test supernates or filtrates, 1 ml per 5 cm
segments, using a sterile tuberculin syringe. and 25-gauge needle [or
each injection. Whole live cultures can be used as a screening test for
enterotoxin; however, positive results must be confirmed using
filtrates. Inject the culture filtrates in triplicate into three rabbits.
Randomize the injection pattern so that test filtrates and controls are
present in proximal middle, and distal regions of the small bowel.
Injection should be made into the anti-mesenteric border of the
intestine near the separating ligature so that the site can be tied off with
another ligature. Close the peritoneum with suture and close the skin
with sutures or 0.9 cm stainless steel wound clips.

After 18 h, sacrifice each rabbit with intravenous phenobarbital or by 5

ml of air rapidly injected by the same route. Open the abdomen and
excise the small bowel with ligated segments intact. Positive loops
appear angry and distended with clear to brownish, or rarely,

181
haemorrhagic fluid, and the negative loops remain collapsed. Measure
the volume of fluid in each positive segment by aspirating into a
syringe and dispensing into a graduated cylinder. Measure the length of
the empty segment in cm.

Interpretation of the results

Determine the ratio of fluid accumulation (ml) to the length (cm) of the
loop. An average value for the three rabbits is calculated. A value of
1.0 or greater is considered positive in the 18 h test. Exclude results in
a rabbit if the saline-control segment contains; measurable fluid with a
ml/cm ratio of LO at 18 h.

Y-1 Adrenal Cell Tissue Culture Assay

The Y-l adrenal cells are maintained in monolayer culture in Ham's F-
I0 nutrient mixture (HAM). Prepare the medium from a dry powder or
from a 10X concentrate. In either case, each litre of medium is
supplemented with 150 ml horse serum, 25 ml foetal calf serum, 40 mg
of gentamicin, and 100 000 units of penicillin. Adjust the pH with
NaHCO2 to 7.2. Sterilize by pressure filtration through a 0.22 µ
membrane filter. Aliquot the medium using sterile technique and freeze
at – 20 °C until use. Once thawad it may be stored at 4°C for up to 1
week, if kept sterile bacterial quality control should be done by adding
0.2 ml of medium to Mueller-Hinton broth and incubating for two
weeks. Observe for turbidity indicating contamination.
Routinely, tissue culture flasks with a 75 cm2 growth area are filled
with 25 ml of medium and incubated at 37°C in a 5 percent CO2
humidified atmosphere. All cell manipulations are done in a laminar
flow hood, and cells are examined using a sub stage phase microscope.

Weekly Procedures for Tissue Culture Assay and Maintenance

Day 1
Aseptically suction the F-10 medium from the confluent monolayer
(one week of growth in flask). Wash the cell monolayer with 5 ml

182
sterile phosphate-buffered saline (PBS) and remove with suction.
Add 1.5 ml of 0.2 percent trypsin to the flask and leave the trypsin
covered monolayer at room temperature until cells begin to loosen
from the plastic surface (5-10 min).
Add 5 ml F-10 medium to the flask to neutralize the trypsin (The
Ham's F-10 is stored at - 20°C and should be thawed and brought to
37°C in a water bath prior to all medium changes and cell sub
culturing). If any monolayer remains, scrape it from the flask surface
with a sterile rubber scrubber or suitably aspirate and flush with
medium.
Transfer the suspended cells (approximately 6.5 ml) to a sterile control
centrifuge tube and centrifuge at 500 to 1,000 g for 5 min.
Suction off the supernatant leaving a sediment of Y-1 adrenal cells in
the centrifuge tube. Re-suspend the cells in 5 ml fresh F-10 medium
with a Pasteur pipette. Using a Pasteur pipette dispensed six drops of
the cell suspension into each flask to which has been added 25 ml fresh
F-10 medium. As a general rule 2 flasks are carried.
For the toxin assay, which is run in flat-bottomed 96 well micro titre
plates, make a 1:10 to 1:100 dilution of the cell suspension. The
suspension is dispensed, approximately 0.15 ml per well. Therefore,
one flask usually can fill 12 to 25 plates. Stack the' plates and cover the
top plate to prevent contamination and evaporation. Place flask (with
loose caps) and/or micro titre plates in the CO2 incubator.

Day 2
Check flasks and/or wells under the microscope for evidence of cell
growth.

Day 3
Change F-10 medium in the stock flasks by suctioning off the old
medium and replacing it with 25 ml fresh F-10. Do not change the
medium in the assay plates.

183
In the Late Afternoon of Third Day - Using a Pasteur pipette, inoculate
each well of the micro titre plates containing a monolayer ofY-1
adrenal cells with 2 drops of the toxin supernatants or whole live broth
cultures (including controls). Be sure the micro titre plates and
appropriate record sheets are coded before the actual transfer. After 15
to 30 min, suction the medium from each well of the plate, being
careful to touch only the corner of the well so as not to damage the
monolayer. Wash once with medium and remove with suction. Finally,
add 0.15 ml of medium to each well. During the washing procedure
wash only half of the plate at one time to prevent drying of the
monolayer. Restack micro titre plates and incubate in CO2 at 37°C
overnight.

Day 4 (morning) read assay

L T causes rounding of Y -1 adrenal cells.

Chinese Hamster Ovary (CHO) Cell Assay

Preparation of Medium
The CHO cells are grown in Eagle's minimum essential medium
(MEM). To prepare the medium, follow manufacturer's instructions for
dissolving powdered MEM. Add 100 µg/ml streptomycin and 100
units/ml penicillin G per litre. Mix well. Filter through a 0.22µ
Millipore membrane filter. Aseptically, aliquot two 90 ml amounts into
280 ml screw cap flasks. Add 10 ml of sterile calf serum (CS) to 1 flask
and 10 ml of CS and 1 ml of sterile foetal 1 calf serum (FCS) to the
other. The two flasks containing 10 percent CS and 10 percent CS plus
1 percent FCS and the remaining MEM with antibiotic can be stored at
4°C for not more than 3 weeks.

Maintenance of Tissue Culture

Obtain Chinese hamster ovary cell culture from a laboratory that is
routinely conducting this assay. Cells must be frozen in dry ice during

184
shipment. Using Pasteur pipette, add a drop of cell suspensions to 4.5
ml of the MEM medium with 10 percent calf serum (CS) and
antibiotics in a 25 cm'" tissue culture flask. Two or more flasks are
routinely carried. Maintenance of the tissue culture requires weekly
passage to fresh medium. To determine the time for passage, observe
cells for confluence in the monolayer using a sub stage phase
microscope.
Passage of cells
Cells are passaged at least once a week and 2 flasks are routinely
maintained. Fresh MEM with 10 percent CS and antibiotics should be
added the day before passage. Simply decant the old medium and add
4.5 ml of the fresh medium.
Decant MEM with 10 percent CS and antibiotics from confluent cells.
Add 2 ml of 0.25 percent trypsin, wash over cells for about 5 min and
decant. Incubate at 37°C in 6% CO2 for 20 min with the monolayer
side of the flask up. Add 2 ml MEM with 10 percent S and antibiotics
to stop trypsin digestion and agitate to suspend all cells and break up
any clumping. Take the cell suspension into a sterile centrifuge tube
and spin down at 800 rpm for 5 min at room temperature
Add desired quantity of cell suspension to a new tissue culture flask
with fresh MEM with 10 percent CS and antibiotics. The amount added
may be adjusted to achieve new monolayer cell confluency at a
specified time point Usually one drop of the cell suspension from a
Pasteur pipette into 4.5 ml of medium should reach confluency in 3
days. Incubate at 37°C in 6% CO2.
Assay for Enterotoxin
Two to four old cultures are preferred for LT assay. Fresh MEM with
10 percent CS plus 1 percent FCS and antibiotics should be added the
day before the assay. Simply decant the old medium and add 4.5 ml of
the fresh medium. Decant MEM with 10 percent CS 1 percent FCS and
antibiotics from confluent cells. Add 2 ml of 0.25 percent trypsin, wash
over cells, and decant quickly. Incubate at 37°C in 6 percent CO2 for
20 min with the monolayer side of the flask up.

185
Add 2 ml MEM with 10 percent CS plus 1 percent FCS and antibiotics
to stop trypsin digestion and agitate to suspend all cells and break up
any clumping. Dilute the cell suspension further in MEM with 10
percent CS plus 1 percent FCS and antibiotics to a concentration of
approximately 2x104 cells/ml Dispense 0.20 ml of cells into each Lab.
Tek chamber (eight chamber slide), Add proximately 0.02 ml toxin or
E. Coli culture supernatants to the Lab Tek Chamber. Each test should
include filtrates from both positive and negative control cultures,
incubate in 6 percent CO2 at 37°C.
Read assay at 20-24 h and not later than 24 h, after pouring off all fluid
and fixing the cells in 10 percent buffered formalin or 100 percent
methanol for 5 min; some prefer to stain with giemsa (1:40) for 20 min.
Lab Tek chamber slides are dissembled and treated as slides that is
stained in Coplin jars.

Result
The number of elongated cells in each 100 cells may be counted and
recorded, or a ‘+ ‘or ‘-’ interpretation may be made when the reaction
is not questionable. Elongation is defined as bipolar and three times
longer than wide. Greater than 10 percent elongated cells is considered
positive.
Infant Mouse Test for Detection of Heat distinguished and identified
biochemically from Stable (ST) Enterotoxin
Select 2-3 days old infant suckling mice. Inject 0.1 ml of the test
inoculum mixed with 50 µl of 0.5 percent Evans blue dye intragastrally
into each mouse separately, the control being injected by the same
amount of peptone water. Sacrifice the animals after 4-5 h and examine
the gastrointestinal tract. Record the distension of G.I. Tract if any.
Determine fluid accumulation ratio by dividing weight of the G.I. Tract
with the total body weight of the mouse. A ratio of 0.09 or more is
considered as a positive reaction. The E. coli strain producing this
value is considered enterotoxigenic.

186
CONFIRMATION OF EIEC STRAINS (SERENY TEST)
A few strains of E. coli cause dysenteric symptoms like shigellosis
frequently with blood and mucus in the stool. These E. coli strains are
usually non-motile, lysine negative, slow in fermenting lactose and
produce little or no gas. Invasive E. coli found so far belongs to a
limited number of serotypes, but as the antisera are not generally -
available, such strains are identified by the Sereny test, which is very
reliable.

Procedure
Inoculate the test strain into a heart infusion agar (HIA) plate and
streak without flaming the loop between quadrants on the plate so that
maximum growth will be obtained. Incubate the plate at 37°C for 18-
24 h. Remove the growth with a cotton swab and suspend it in 1 ml of
physiological saline. Inoculate a drop (50µl) of this suspension into a
guinea pig’s eye, using a sterile Pasteur pipette. Be sure not to
traumatize the eye. Inoculate only one eye. The other eye may serve as
negative control. Known positive shigella strains may be used in a
separate guinea pig as a positive control. Observe the guinea pig’s eye
daily for 72 h for development of keratoconjuctivitis. Guinea pigs with
redness and swelling of the eye are considered positive. If the test is
positive, report: invasive E. coli.

NOTE - Positive guinea pigs cannot be reused. They are infectious and
their carcasses should be disinfected by autoclaving or by other means
before they are discarded. Guinea pigs may be reused, however, if they
were negative in previous tests, using the eye not used previously

ISOLATION OF ENTEROHAEMORRHAGIC E. COLI (EHEC)

Apart from the ordinary characteristic of E. coli, Enterohaemorrhagic
E. coli O157:H7 can be distinguished and identified biochemically
from E. coli as it is 100% sorbitol non-fermenter and 100% ducitol and
raffinose fermenter. These characters are used as screening tests for

187
identifying the organism.
Non fermentation of sorbitol is observed on Sorbitol Mac Conkey Agar
Medium (SMAC) and on Sorbitol Liquid Broth.

Preparation of Sorbitol MacConkey Agar Medium (SMAC)

The SMAC media is prepared with MacConkey agar base 40.0 g
(without lactose) and D-sorbitol 10.0 g.
MacConkey Agar Base Contains
Peptone - 2.0 percent
Sodium taurocholate - 0.5 percent
Agar - 3.0 percent
Distilled Water - 100 ml.

Procedure
At first peptone and taurocholate is dissolved in the water by shaking
and heating. Agar is added in it and is dissolved in the steamer or
autoclaved. If necessary, clear by filtration, pH is adjusted to 75. Then
40.0 g of MacConkey agar base, 1 percent D-sorbitol and neutral red is
added and mixed properly in 1,000 ml of distilled water. Heat in the
autoclave with free steam (100 °C) for 1 h then at 115°C for 15 min.
Pour in the plate and preserve the plate in refrigerator till use.
Sorbitol MacConkey Agar Medium is used, as it is a differential and
selective medium, for Enterohaemorrhagic E. coli O157:H7. Here
instead of lactose, sorbitol is used because EHEC O157 H7 do not
ferment sorbitol that is EHEC O157:H7 is non-sorbitol fermenter
(NSF). In contrast, other E. coli ferment sorbitol. Sorbitol Mac Conkey
agar plate is incubated at 37 °C for overnight after the primary
inoculation. Non-sorbitol fermenting E. coli colonies are picked up and
subcultured to obtain the pure NSF growth.
Characterisation of Enterohaemorrhagic E. coli (EHEC)
Colony Characteristics
On primary isolation, colonies are translucent, large, thick, moist,
smooth. The colonies are similar in appearance to non- lactose

188
fermenting colony on Mac Conkey agar that is colourless and
contrasted well with bright pink colonies of sorbitol positive organism
of the faecal flora. EHEC colonies therefore are easily recognisable on
SMAC medium culture where as they are indistinguishable from faecal
flora in Mac Conkey agar culture medium.

Preparation of Sorbitol Liquid Media

At first base media is prepared by adding the following ingredients:
Peptone – 1%
Sodium chloride - 0.5%
Distilled water - 100 ml
Above ingredients are mixed and autoclaved at 121°C, for 15 min. Add
1 percent Andrade’s indicator and adjust the pH to 7.6. Then 1% D-
sorbitol is added in the base media which is again steamed in the
autoclave under free steam for one hour after dispensing in small tubes
with Durham’s tube.
Procedure of the Sorbitol Fermentation -Test
Inoculate the media with the organism and incubate at 37°C overnight
to observe the fermentation. EHEC is sorbitol non-fermenter.
Preparation of Dulcitol and Raffinose Liquid Media
Here base media is prepared as in the case of sorbitol liquid media.
After adjusting the pH and addition of Andrade’s indicator add 1
percent raffinose and 1% Dulcitol separately to the base media. The
media is again sterilised by free steaming after dispensing in small
tubes with Durham’s tube.
Procedure of the Dulcitol and Raffinose Fermentation Test
Inoculate both the media with the organism and incubate at 37°C
overnight and observe for fermentation. EHEC E. coli O157:H7 is 100
percent positive to Dulcitol and Raffinose fermentation test.
Slide Agglutination Test for Confirmation of EHEC
Now for confirmation of the organism as Enterohaemorrhagic E. Coli
the slide agglutination test may be performed by O157 and H7
antiserum, respectively.

189
Procedure of the Slide Agglutination Test
E. coli colonies are emulsified in saline on a grease free, dry
microscopic slide. This is kept at the left side of the slide as control.
Another emulsion is prepared in the same way and is kept on the right
side of the slide. One loopful of the O157 antisera, is added to the
emulsion to be tested, end is observed for agglutination.
Procedure of the slide agglutination test with H7 antiserum is same as
previous one.
IDENTIFICATION OF ENTEROADHERENT E. COLI (EAEC)
Some EPEC tend to adhere to intestinal mucosa in vivo. Majority of
EPEC strains isolated from out breaks showed mannose resistant
adhesion to HEp2, whereas normal flora of E. Coli rarely adhered.
Two distinct pattern of EPEC adherence is noted
a) Localised adhesion (LA), and
b) Diffuse adhesion (DA).
LA pattern is characterised by organisms attaching to one or two small
areas of the cell surface in micro colonies, whereas DA pattern attaches
in hybridization with the probe correlates well with scattered pattern to
the whole of cell surface.
Cell Lines used - HEp-2
Hela
It is thought that identical results are obtained in adhesion assays.
However, some workers report that some isolates adhere to Hela and
not to Hep-2 cells.
The adhesion property is thought to be plasmid mediated (size 50-70 M
Da) and known as EPEC adhesive factor (EAF).
A DNA probe composed of 1 K Da portion of EAF plasmid has been
developed by isolating plasmid fragment of adhesion. This probe has
been used to detect mid fragment of adhesion. This probe has been
used to detect EPEC in epidemiological studies and hybridization with
the probe correlates well with production of LA.
The ability of EPEC to adhere to HEp-2 cells, in localised manner and
to hybridize with EAF was most commonly found amongst EPEC

190
 serogroups 055, 0111, 0119, 0127, 0128 and 0142 (called Class-I
EPEC). These strains are most commonly associated with outbreaks of
infantile diarrhoea.
Strains showing DA and no adhesion to HEp-2 cells belong to
serogroups less commonly incriminated in out breaks of diarrhoea and
belong to EPEC SWEROGROUPS O44, O86, O144 (CLASS II EPEC).

Expression of Pathogenic E.coli Present or absent

Results

Reference IS 14397: Detection, Isolation and Identification of Pathogenic E. Coli

In Food
Approved by Scientific Panel on Methods of Sampling and Analysis

191
 Method for Detection and confirmation of Listeria monocytogenes in
Food based on IS: 14988-Part 1

Method No. FSSAI 15.014:2023 Revision No. & Date 0.0

Introduction Listeria monocytogenes is an intracellular, foodborne pathogen
potentially lethal for humans and animals.
Scope This method is applicable to those food product categories and their sub-
categories as mentioned in the Appendix B tables of Food Safety and
Standards (Food Products Standards and Food Additives) Regulations,
2011 and amendments (Gazette notifications) issued from time to time.
Caution While testing L. monocytogenes it is recommended that a properly
equipped laboratory under supervision of skilled Microbiologist is done.
The material used during testing is carefully disposed off after
sterilization. Pregnant personnel may be asked to avoid handling of L.
monocytogenes cultures and undertaking the tests.
Principle Listeria monocytogenes may be present in small numbers and are often
accompanied by considerably larger numbers of other microorganisms,
therefore selective enrichment is necessary. It is also necessary to detect
injured and stressed Listeria monocytogenes and the primary selective
enrichment medium, with reduced inhibitor concentration, fulfils at least
part of this function.

The detection of Listeria monocytogenes requires 4 successive stages

viz. primary enrichment, secondary enrichment, identification and
confirmation.

Primary enrichment in a selective liquid enrichment medium with

reduced concentration of selective agents (half-Fraser broth):

Inoculation of a selective primary enrichment medium containing half

the concentrations of acriflavine and nalidixic acid (half-Fraser broth
and incubation of the initial suspension at 30 °C for 24 h to 26 h.

Secondary enrichment with a selective liquid enrichment medium

192
 with full concentration of selective agents (Fraser broth):

Inoculation of full-strength secondary liquid enrichment medium (Fraser

broth) with a culture obtained from 3.1. Incubation of the Fraser broth at
37 °C for 24 h.

Plating out and identification:

From the cultures obtained from Primery and secondary enrichment,

plating out on the two selective solid media:
 Oxford Agar

 PALCAM Agar

Incubation of the paltes at 30°C, 35°C or at 37 °C for a total of 48 h for

presence of characteristic colonies.

Confirmation

Subculturing of the colonies of presumptive L. monocytogenes plated

out, and confirmation by means of appropriate morphological and
biochemical tests.

Equipment 1. Laminar airflow

2. Biosafety cabinet
3. Hot air oven
4. Autoclave
5. Incubator (Operating at 25°C ± 1°C, 30 °C ± 1°C and 35°C to 37°C
± 1°C)
6. Anerobic jar or anerobic workstation
7. Gas Mixure for microaerobic incubation (5% -12 % CO2, 5% -15 %
O2 and 75 % N2
8. Water bath (at 47 °C ± 2°C)
9. pH meter with measuring accuracy ±0.1
10. Microscope
11. Refrigerator (at 2 °C– 8 °C)
12. Petri dishes (Glass or plastic of 90-100mm diameter or 140mm)
13. Graduated pipettes (0.1 ml divisions) of capacity 1 ml (Class A)

193
 14. Micropipette with tips
15. Tubes and glass bottles
16. Vortex
17. Mechanical stirrer
18. pH meter with measuring accuracy ±0.1
19. Spreader (glass or plastic)
20. Inoculation loops and straight wire
21. Spiral plater/rotator
22. Inoculation loops and straight wire
23. Colony counter
24. Equipment for Henry Illumination Test
Culture media i) Phosphate buffered peptone water
and reagents ii) Listeria enrichment broth
iii) Half Frazer broth
iv) Frazer broth
v) Modified Oxford Agar
vi) PALCAM Agar
vii) Tryptone Soya Yeast Extract Agar
viii) Tryptone Soya Yeast Extract Broth
ix) Sheep Blood Agar
x) Carbohydrate utilization broth (Rhamnose and Xylose)
xi) Motility Agar
xii) CAMP Medium and test organisms
xiii) Hydrogen peroxide solution

194
 Preparation of sample and primary enrichment
Procedure
Aseptically open the sample container and weigh x gm sample into a
sterile mixture bag/ empty wide mouth container with screw cap or
suitable closure or take x ml of liquid sample. Add x ml of sterile half-
Fraser broth to obtain a ration of test portion to selective primary
medium of 1/10 (mass to volume or volume to volume). Make a uniform
suspension by blending if necessary and incubate at 30 °C for 24 ±2 h.

Secondary enrichment:
Inoculate 0.1 ml of the culture from Half Fraser Broth onto 10 ml tubes
of Fraser Broth. Incubate the tubes at 35 °C or 37 °C for 48 h

Selective differential plating:

From the culture obtained in the primary enrichment (Half Fraser Broth
after 24 ± 2 h at 30 °C) inoculate (streaking) the surface of the selective
isolation medium, Oxford agar and PALCAMplates.

From the culture obtained in the secondary enrichment (Fraser Broth

after 48 h ± 2 h at 35°C or37 °C) inoculate (streaking) the surface of the
selective PALCAM and Oxford agar plates.
Incubate the plates at 30 °C, 35 °C or 37 °C and observe for typical
colonies after 24 h. PALCAM agar plates are incubated
microaerobically.
Incubation of Oxford agar at 30 °C is suitable for food stuffs only lightly
contaminated by a supplementary flora for products heavily
contaminated by supplementary flora, incubate Oxford agar plates at 37
°C. If no suspect colonies are evident or if the growth is poor after 24 h,
re-incubate the plates for an additional 24 h and read again.

Appearance of After the incubation period, examine the plates for typical Listeria
colonies monocytogenes colonies.
On PALCAM agar and Oxford agar - gray to black colonies surrounded
by a black halo.

195
Confirmation Select five typical colonies from one plate of each medium. If presumed
of Listeria colonies are less than five on a plate, take all of them.
species
Purify each culture by streaking the selected colonies from each plate on
to the surface of a well dried Tryptic Soya Yeast Extract Agar (TSYEA)
for obtaining well separated colonies. Invert the plates and incubate at 35
°C or 37 °C for 18 to 24 hr or until the growth is satisfactory.

Typical colonies are 1 mm to 2 mm in diameter, convex, colorless and

opaque with an entire edge. Carry out the following tests from colonies
of a pure culture on the TSA YE.

Catalase reaction:
With the help of loop pick up an isolated colony and place it in H2O2
solution on a glass slide. Immediate production of gas bubbles indicates
catalase positive reaction.

Gram staining:
Perform Gram staining on a colony, Listeria are Gram positive slim
short rods.

Motility Test:
Take colony from TSYEA plate and suspend it TSYE broth. Incubate at
25°C for 8 to 24 hr until cloudy medium is observed. Take a drop o f
culture and place it on a glass slide. Cover the top with a cover slip and
observe under a microscope. Listeria is seen as slim rods with a
tumbling motility (cultures grown above 25°C fail to show this motion.
Compare them with a known culture – cocci or large rods with rapid
motility are not Listeria.

As an alternative stab motility agar tube with an isolated colony from

TSYEA and incubate at 25 °C for 48 h Typical umbrella like appearance
around the stab indicate motility positive culture. If growth is not

196
 positive incubate up to five days and observe for the stab again.

Confirmation Heamolysis test:

of Listeria Take a colony from TSA YE and stab it on a well dried surface of sheep
monocytogenes blood agar plate. Simultaneously stab positive (L. monocytogenes) and
negative (L. innocua) control cultures. Invert the plates and incubate at
35 °C or 37 °C for 24 ± 2 h. Examine the plates.
L. monocytogenes show clear light zones of beta haemolysis. L. innocua
does not show any haemolysis. Examine the plates in a bright light to
compare test cultures with the controls.

Carbohydrate utilization:
Inoculate each of the carbohydrate utilization broths (rhamnose and
xylose) with a culture from TSYE broth and incubate at 35°C or 37°C
for upto 5 days. Appearance of yellow color indicates a positive reaction
within24 to 48 hr.

CAMP test
On a well dried surface of sheep blood agar or CAMP Medium streak
each of the Staphylococcus aureus and Rhodococcus equi cultures in
single lines and parallel to each other and diametrically opposite, a thin
even innoculum is required.
Streak the test strain separated in a similar manner at right angles to
these cultures as that the test strain and S. aureus and R.equi cultures do
not touch but their closest are about 1 mm or 2 mm apart. Several test
strains can be streaked on the same plate. Simultaneously streak control
cultures of L. monocytogenes, L. innocua and L. ivanovii. Incubate blood
agar plates at 35 to 37°C for 18 to 24 hr and CMP medium for 35 or
37°C for 16 to 18 hr.
Observe plates against bright light. In L. monocytogenes case there is
enhanced zone of beta haemolysis at the intersectiono of S. aureus.
L. innocua does not show any enhanced zone of haemolysis with S.
aureus or R. equi.
In case of L. ivanovii enhamced beta zone of haemolysis is seen on R.

197
equi side.

Inoculate thin agar plates as in diaram. Vertical lines represent streaks of

S. aureus (S) and R. equi (R). Horizontal lines represent sraks of test
cultures. Hatched area indicates the location of enhanced haemolysis.
The dotted area indicates the zone of influence of the S. aureus culture.
Figure 1 Inoculation and Interpretation of CMP test plates

Henry oblique transmitted illumination (Optional)

Examine TSAYE plates using a source of beamed white light, striking
the bottom of the palte at a 45° angle. When examined under this
transmitted illumination from directly above the plates Listeria colonies
are bluish in color with granular surface.

198
 Figure 2 Examination of plates for suspect colonies (Henry
illumination test)
Interpretation All Listeria species are small, Gram positive rods that demonstrate
of results motility and catalase positive reaction. L. monocytogenes are
distinguished from other species by the characteristics listed in table
given below.

Species Haemolysis Production Production CAMP Test

of acid with of acid
Rhamnose with S. aureus R. equi
Xylose
L. + + - + -
monocytogenes
L. innocua - V - - -
L. ivanovii + _ + - +
L. seeligeri (+) - + (+) -
L. welshmeri - V + - -
L. grayi - - - - -
subspecies
grayi
L. grayi - V - - -
subspecies
murrayi

V is variable reaction

199
 (+) weak reaction
(-) no reaction
+ is >90% of positive
Expression of Based on the observations and interpretation of the results report
results presence or absence of L. monocytogenes in test portion specifying the
mass in grams or mililitres of the sample taken.
L. monocytogenes =present or absent/ gm or mL or 25/gm or mL.
References IS 14988-1: Microbiology of Food and Feeding Stuffs - Horizontal
method for Detection and Enumeration of Listeria Monocytogenes, Part
1: Detection Method
Approved by Scientific Panel on Methods of Sampling and Analysis

200
 Method for Detection and confirmation of Listeria monocytogenes in
Food based on ISO 11290-1

Method No. FSSAI 15.015:2023 Revision No. & Date 0.0

Introduction Listeria monocytogenes is an intracellular, foodborne pathogen
potentially lethal for humans and animals.
For pregnant women and immunocompromised persons it is classified in
group IB:“diseases of severe hazard for restricted population; life
threatening or resulting in substantial chronic sequelae or presenting eff
ects of long duration.”
Scope This method is applicable to those food product categories and their sub-
categories as mentioned in the Appendix B tables of Food Safety and
Standards (Food Products Standards and Food Additives) Regulations,
2011 and amendments (Gazette notifications) issued from time to time.
Caution While testing L. monocytogenes it is recommended that a properly
equipped laboratory under supervision of skilled Microbiologist is done.
The material used during testing is carefully disposed off after
sterilization. Pregnant personnel may be asked to avoid handling of L.
monocytogenes cultures and undertaking the tests.
Principle Listeria monocytogenes may be present in small numbers and are often
accompanied by considerably larger numbers of other microorganisms,
therefore selective enrichment is necessary. It is also necessary to detect
injured and stressed Listeria monocytogenes and the primary selective
enrichment medium, with reduced inhibitor concentration, fulfils at least
part of this function.

The detection of Listeria monocytogenes requires 4 successive stages

viz. primary enrichment, secondary enrichment, identification and
confirmation.

Primary enrichment in a selective liquid enrichment medium with

reduced concentration of selective agents (half-Fraser broth):

Inoculation of a selective primary enrichment medium containing half

201
 the concentrations of acriflavine and nalidixic acid (half-Fraser broth
and incubation of the initial suspension at 30 °C for 24 h to 26 h.

Secondary enrichment with a selective liquid enrichment medium

with full concentration of selective agents (Fraser broth):

Inoculation of full-strength secondary liquid enrichment medium (Fraser

broth) with a culture obtained from 3.1. Incubation of the Fraser broth at
37 °C for 24 h.

Plating out and identification:

From the cultures obtained from Primery and secondary enrichment,

plating out on the two selective solid media:

 Agar Listeria according to Ottaviani and Agosti (AOAL Agar)

 Any other solid selective Media at the choice of the laboratory

complementary to Listeria agar according to Ottaviani and Agosti.

Incubation of the Agar Listeria according to Ottaviani and Agosti

and secondary solid selective agar at 37 °C for a total of 48 h. If
colonies of presumptive L. monocytogenes are evident at 24 h.The
incubation may be stopped at this stage.

Confirmation

Subculturing of the colonies of presumptive L. monocytogenes plated

out, and confirmation by means of appropriate morphological and
biochemical tests.

Equipment 1) Laminar airflow

2) Biosafety cabinet
3) Hot air oven
4) Autoclave
5) Incubator (Operating at 30 °C ± 1°C and 37°C ± 1°C)
6) Anerobic jar
7) Water bath (at 44 °C to 47 °C)
8) pH meter with measuring accuracy ±0.1

202
 9) Microscope
10) Refrigerator (at 2 °C– 8 °C)
11) Petri dishes (Glass or plastic of 90-100mm diameter or 140mm)
12) Graduated pipettes (0.1 ml divisions) of capacity 1 ml (Class A)
13) Micropipette with tips
14) Tubes and glass bottles
15) Vortex
16) Mechanical stirrer
17) pH meter with measuring accuracy ±0.1
18) Spreader (glass or plastic)
19) Inoculation loops and straight wire
20) Spiral plater/rotator
21) Inoculation loops and straight wire
22) Colony counter
Culture media and 1. Phosphate buffered peptone water
reagents 2. Listeria enrichment broth
3. Half Frazer broth
4. Frazer broth
5. Agar Listeria Ottaviani and Agosti (ALOA)
6. Modified Oxford Agar
7. PALCAM Agar
8. Tryptone Soya Yeast Extract Agar
9. Tryptone Soya Yeast Extract Broth
10. Sheep Blood Agar
11. Carbohydrate utilization broth (Rhamnose and Xylose)
12. Motility Agar
13. CAMP Medium and test organisms
14. Hydrogen peroxide solution

Preparation of test
Preparation of sample and primary enrichment
sample
Aseptically open the sample container and weigh 25 gm sample into a
sterile mixture bag/ empty wide mouth container with screw cap or

203
 suitable closure or take 25 ml of liquid sample. Add 225 ml of sterile
half-Fraser broth (pre warm at room temperature). Make a uniform
suspension by blending if necessary and incubate at 30 °C ± for 25 ± 1
h.

Secondary enrichment: Inoculate 0.1 ml of the Half Fraser Broth into

10 ml tubes of Fraser Broth. Incubate the tubes at 37 °C for 24 h ± 2 h
for Listeria monocytogenes.

Selective differential plating:

From the culture obtained in the primary enrichment (Half Fraser Broth
after 24 ± 2 h at 30 °C) inoculate (streaking) the surface of the selective
isolation medium, Agar Listeria Ottaviani and Agosti (ALOA) and
second selective medium agar plates.
From the culture obtained in the secondary enrichment (Fraser Broth
after 24 h ± 2 h at 37 °C) inoculate (streaking) the surface of the
selective isolation medium, Agar Listeria Ottaviani and Agosti (ALOA)
and second selective medium agar plates of choice.
Incubate the plates at 37 °C and observe for typical colonies after 24 h.
Second selective medium agar plates are incubated as per manufacture
instructions. If no suspect colonies are evident or if the growth is poor
after 24 h, re-incubate the plates for an additional 24 h and read again.
After incubation plates can be kept at 5°C for a maximum of 48 h before
reading.
Appearance of After the incubation period, examine the plates for typicalListeria
colonies monocytogenes colonies.
On Agar Listeria Ottaviani and Agosti (ALOA) - blue-green colonies
surrounded by an opaque halo (typical colonies).
Consider as presumptive Listeria spp all blue green colonies with and
without holo.
Confirmation of Select at least one presumptive typical colony from one plate of each
Listeria species medium.
Purify each culture by streaking the selected colonies from each plate on
to the surface of a well dried TSYEA for obtaining well separated

204
colonies. Invert the plates and incubate at 37 °C for 18 to 24 h or until
the growth is satisfactory.
Typical colonies are 1 mm to 2 mm in diameter, convex, colorless and
opaque with an entire edge. Carry out the following tests from colonies
of a pure culture on the TSAYE.

Catalase reaction:
With the help of loop pick up an isolated colony and place it in H2O2
solution on a glass slide. Immediate production of gas bubbles indicates
catalase positive reaction.

Gram staining:
Perform Gram staining on a colony, Listeria are Gram positive slim
short rods.

Motility Test:
Take colony from TSYEA plate and suspend it TSYE broth. Incubate at
25°C for 8 to 24 hr until cloudy medium is observed. Take a drop of
culture and place it on a glass slide. Cover the top with a cover slip and
observe under a microscope. Listeria is seen as slim rods with a
tumbling motility (cultures grown above 25°C fail to show this motion.
Compare them with a known culture – cocci or large rods with rapid
motility are not Listeria.
As an alternative stab motility agar tube with an isolated colony from
TSA YE and incubate at 25 °C for 48 h Typical umbrella like
appearance around the stab indicate motility positive culture. If growth
is not positive incubate up to five days and observe for the stab again.

Heamolysis test on blood agar:

Take a colony from TSA YE and stab it on a well dried surface of sheep
blood agar plate. Simultaneously stab positive (L. monocytogenes) and
negative (L. innocua) control cultures. Invert the plates and incubate at
35 °C or 37 °C for 24 ± 2 h. Examine the plates.

205
L. monocytogenes show clear light zones of beta haemolysis. L. innocua
does not show any haemolysis. Examine the plates in a bright light to
compare test cultures with the controls.
Heamolysis reaction using red blood corpuscles

CAMP test (optional)

On a well dried surface of sheep blood agar streak each of the
Staphylococcus aureus and Rhodococcus equi cultures in single lines
and parallel to each other and diametrically opposite, a thin even
innoculum is required.
Streak the test strain separated in a similar manner at right angles to
these cultures as that the test strain and S. aureus and R. equi cultures do
not touch but their closest are about 1 mm or 2 mm apart. Several test
strains can be streaked on the same plate. Simultaneously streak control
cultures of L monocytogenes, L innocua and L. ivanovii. Incubate plates
at 35 to 37°C for 18 to 24 hr.
Observe plates against bright light. In L. monocytogenes case there is
enhanced zone of beta haemolysis at the intersectiono of S. aureus.
L. innocua does not show any enhanced zone of haemolysis with S.
aureus or R. equi.
In case of L. ivanovii enhamced beta zone of haemolysis is seen on R.
equi side.

206
 Carbohydrate utilization:
Inoculate each of the carbohydrate utilization broths (rhamnose and
xylose) with a culture from TSYE broth and incubate at 35°C or 37°C
for upto 5 days. Appearance of yellow color indicates a positive reaction
within24 to 48 hr.

Interpretation of All Listeria species are small, Gram positive rods that demonstrate
results motility and catalase positive reaction. L. monocytogenes are
distinguished from other species by the characteristics listed in table
given below.
Species Haemolysis Production Production CAMP Test
of acid with of acid
Rhamnose with S. aureus R equi
Xylose

L.monocytogen + + - + -
es
L. innocua - V - - -
L. ivanovii + _ + - +
L. seeligeri (+) - + (+) -
L welshmeri - V + - -

207
 L. grayi - - - - -
subspecies
grayi
L. grayi - V - - -
subspecies
murrayi
Expression of results Based on the observations and interpretation of the results report
presence or absence of L. monocytogenes in test portion specifying the
mass in grams or mililitres of the sample taken.
L. monocytogenes = present or absent/ gm or mL
Reference ISO 11290-1 (2017): Microbiology of the food chain — Horizontal
method for the detection and enumeration of Listeria monocytogenes
and of Listeria spp. — Part 1: Detection method.
Approved by Scientific Panel on Methods of Sampling and Analysis

208
 Method for Detection and Confirmation of Salmonellabased on IS:
5887 Part3

Method No. FSSAI 15.016:2023 Revision No. & Date 0.0

Introduction Salmonella is a genus belonging to the family Enterobacteriaceae. The
family is defined as rod-shaped, Gram-negative, non-spore-forming,
facultative anaerobic and oxidase-negative bacteria.
Scope This method is applicable to those food product categories and their sub-
categories as mentioned in the Appendix B tables of Food Safety and
Standards (Food Products Standards and Food Additives) Regulations,
2011 and amendments (Gazette notifications) issued from time to time.
Caution In order to safeguard the health of laboratory personnel, it is essential
that tests for detecting Salmonella spp. are only undertaken in properly
equipped laboratories, under the control of a skilled microbiologist and
that great care is taken in the disposal of all incubated materials. It is
also the responsibility of the user to establish appropriate safety and
health practices in the laboratory.
Principle The detection of Salmonella requires 4 successive stages viz. pre-
enrichment, enrichment, differential selective and confirmation
Pre-enrichment in non-selective broth:
Salmonella may be present in small numbers and often accompanied by
larger number of other Enterobacteriaceae members and other bacteria.
Therefore, selective enrichment is necessary, furthermore, pre-
enrichment is necessary to permit detection of Salmonella. The
objectiveof this step is to recover injured cells, which can be obtained by
incubating the sample in non-selective conditions, for at least 16-20
hours. The most commonly used media is buffered peptone water
(BPW).

Enrichment in selective broth:

The objective of this step is to inhibit the multiplication of the
accompanying microbiota and preferentially promote the increase of the

209
 number of Salmonella cells, by incubating the pre-enriched sample in
selective broth, for 24 h. In this step, it is recommended that two
differentenrichment media be used, because the resistance of Salmonella
to selective agents varies from strain to strain. The most recommended
media for this purpose are modified Rappaport-Vassiliadis broth (RV)
and Selenite/cystine medium.

Differential selective plating: The objective of this step is to

preferentially promote the development of Salmonella colonies
exhibiting typical characteristics that distinguish them from competitors,
for subsequent serological and biochemical confirmation. Phenol red or
brilliant green agar and any other second medium as per the choice of
testing lkaboratory uness there is specific international standards relating
to the product to be examined, which specifies the composition of this
second medium.
Confirmation: The objective of this step is to confirm the Salmonella
colonies by means of biochemical and serological assays.
Equipment 1. Laminar airflow
2. Biosaftey cabinet
3. Hot air oven
4. Autoclave
5. Incubator (Operating at 35 °C ± 1°C, 37 °C ± 1°C and 42 °C ± 1
°C)
6. Water bath (at 42 °C, 45 °C, 55 °C and 70 °C)
7. pH meter with measuring accuracy ± 0.1 at 25 °C
8. Microscope
9. Refrigerator (at 2 °C– 8 °C)
10. Petri dishes (Glass or plastic of 90-100 mm diameter or 140 mm)
11. Graduated pipettes (0.1 ml divisions) of capacity 1 ml (Class A)
12. Tubes and glass bottles
13. Vortex
14. Mechanical stirrer
15. Spreader (glass or plastic)

210
 16. Inoculation loops and straight wire
17. Spiral plater/rotator
18. Inoculation loops and straight wire made up of platinum/iridium
or nickel/chromium of dia ≈ 3 mm
Culture Media i) Nutrient Agar
ii) Lactose broth
iii)Reconstituted Non-Fat Dry Milk
iv) 1% aqueous Brilliant Green Dye Solution
v) Rappaport- Vassiliadis magnesium chloride/malachite reen
medium (RV –medium)
vi) Triple Sugar Iron (TSI) Agar
vii) Lysine Iron Agar (LIA)
viii) Urea Agar
ix) Phenol Red Dulcitol Broth
x) Phenol Red Lactose Broth
xi) Tryptone Broth
xii) Buffered Glucose (MR-VP) Medium
xiii) Buffer Peptone Water
xiv) Selenite/cysteine Media
xv) Brillient reen /phenol red gar
xvi) Semi Solid Nutrient agar
xvii) L-Lysine decaroxylation Medium
xviii) Saline Solutin
xix) VP Medium
xx) Kovacs reagent
xxi) Saline solution
xxii) Toluene
xxiii) β galactosidase reaent
xxiv) TSI medium
xxv) Urea agar
xxvi) Salmonella antisra

211
Procedure Preparation of sample and pre-enrichment
Aseptically add 25 g/ml of sample into a sterile empty wide mouth
container with suitable closure. Add 225 ml of sterile Buffer peptone
water for pre-enrichment.
In case prescribed portion of test sample is other than 25, use necessary
quantity of pre-enrichment media to yield approximately 1/10 dilution
(mass to volume)
Make a uniform suspension by blending.
Incubate at 35 °C or 37 °C for 16 to 20 h

Selective enrichment
Mix the incubated sample by gentle shaking and transfer 0. 1 ml to 10
ml of RV broth
Incubation Temperature 42 °C for 24 h
Add additional of 10 ml to 100 ml of selenite or cysteine medium and
incubate at 35 °C to 37 °C for 24 h and further 24 h.

Selective media plating

After incubation (24 h), gently mix and streak loopful of incubated RV
medium and selenite/ cystine broth onto selective media plates of BGA
(first selective media)/ or any other second selective media. Repeat the
same process from selenite/ cystine broth of 48 h incubation.
Incubate the plates at 35 °C or 37 °C for 20 h to 24 h.
Re incubate the plates for a further 18 h to 24 h if no typical colonies of
Salmonella are present.
Use a big petri plates (140 mm) or 2 small petri plates (90 mm) to get
isolated colonies.

Observe plates for typical Salmonella colonies.

Typical colonies of Salmonella rown on phenol red/brilliant green agar
cause the colour of the medium to change from pink to red.
Observe the secondary selective plates for typical Salmonella colony.

Confirmation

212
Treatment of typical or suspected colonies
Take suspected five colonies (if present) from each of the selective
plates and streak onto Nutrient agar plates. Incubate at 35 °C or 37 °C
for 18 to 24 h.
Use this culture for biochemical and serological confirmation.
Biochemical tests
By means of inoculation wire inoculate following medium from cultures
of the colonies selected.
TSI Agar
With help of inoculating wire or a needle inoculate the media TSI
(streaking slant and stabbing butt) with the culture obtained from the
nutrient agar plates

Incubate TSI slants at 37 °C for 24 ±2 hand 48 ±2 h respectively.

Table: Typical Salmonella reactions are:
TSI
Slant Alkaline (red)
Butt Acid (Yellow)
H2S production + or -
(blackening in butt)

A culture is treated as presumptive postive if the reactions are typical on

TSI slants.

Using a sterile inoculating needle or wire inoculate a portion of the

positive culture into the following broths.
Incubate at 35 °C or 37°C for 24 h and read for Salmonella typical
reactions.
Biochemical tests
Broth/ Media Time of Results
incubation
Urea broth 35 °C or 37 °C Negative (no change in yellow
24 ±2 h colour of medium)

213
 L-Lysine 35 °C or 37 °C Positive (Purple color)
decarboxylation for 24 ±2 h
medium
Β-galactosidase 35 °C or 37 °C Negative
for few min
Indole Test 35 °C or 37 °C Negative (Yellow brown
for 24 h reaction)
MR-VP medium 35 °C or 37 °C Negative for VP test but
24±2 h positive for MR test.
(Red for positive)

Serological Tests
Elimination of auto agglutinable strains
Place 1 drop of saline on clean glass slide. Take a part of colony to be
tested to make homogenate suspension. Tilt the slide gently for 1 min.
Observe the result for any clumps which shows presence of auto
agglutinable strain.
Auto-agglutinable strains shall not be submitted for following tests.

Examination of H-antigens
Incubation
Inoculate the colony on NA and incubate at 35 °C or 37 °C for 18 to 24h.
Take a part of colony from NA on glass slide and add 1 drop of anti H
serum on it. Mix well, if agglutination occur, the reaction is positive.

Examination of Vi-antigens
Add 1 drop of culture suspension to clean glass slide. Add a drop of
anti-Vi- serum. If agglutination occurs, the reaction is positive.

Monovalent/Polyvalent somatic (O) test.

Add 1 drop of culture suspension to clean glass slide. Add a drop of anti
O serum. If agglutination occurs, the reaction is positive.

214
 Interpretation of agglutination tests
In place of serum, use normal saline as negative control.

Interpretation of Biochemical and Serologiocal Reactions

Biochemical Auto- Serologiocal Interpretation
Reactions agglutination Reactions
Typical No O-, Vi-, H- antigen Strains
positive considered to
be Salmonella
Typical No All reactions May be
negative Salmonella
Typical Yes Not tested
No Typical No O-, Vi-, H- antigen
Reactions positive
No Typical No All reactions Not considered
Reactions negative to be
Salmonella

In accordance with the results of the interpretation, indicate the presence

and absence of Salmonella in a test portion of x of product.
Definitive Confirmation

Strains which are considered to be Salmonella shall be sent to reconised

Salmonella reference centre for definitive typing.

Calculation NA
Expression of Result Salmonella = Present/Absent per 25 gm or 25 ml
Reference IS 5887-3: Methods for Detection of Bacteria Responsible for Food
Poisoning, Part 3: General Guidance on Methods for the Detection of
Salmonella
Approved by Scientific Panel on Methods of Sampling and Analysis

215
 Method for Detection and Confirmation of Salmonella speciesbased
on ISO:6579-I

Method No. FSSAI 15.017:2023 Revision No. & Date 0.0

Introduction Salmonella is a genus belonging to the family Enterobacteriaceae. The
family is defined as rod-shaped, Gram-negative, non-spore-forming,
facultative anaerobic and oxidase-negative bacteria..
Scope This method is applicable to those food product categories and their sub-
categories as mentioned in the Appendix B tables of Food Safety and
Standards (Food Products Standards and Food Additives) Regulations,
2011 and amendments (Gazette notifications) issued from time to time.
Caution In order to safeguard the health of laboratory personnel, it is essential
that tests for detecting Salmonella spp. are only undertaken in properly
equipped laboratories, under the control of a skilled microbiologist and
that great care is taken in the disposal of all incubated materials. It is
also the responsibility of the user to establish appropriate safety and
health practices in the laboratory.
Principle The detection of Salmonella requires 4 successive stages viz. pre-
enrichment, enrichment, differential selective and confirmation
Pre-enrichment in non-selective broth:
Salmonella may be present in small numbers and often accompanied by
larger number of other Enterobacteriaceae members and other bacteria.
Therefore, selective enrichment is necessary, furthermore, pre-
enrichment is necessary to permit detection of Salmonella.
The objective of this step is to recover injured cells, which can be
obtained by incubating the sample in non-selective conditions, for at
least 16-20 hours. The most commonly used media are buffered peptone
water (BPW).
Enrichment in selective broth:
The objective of this step is to inhibit the multiplication of the
accompanying microbiota and preferentially promote the increase of the
number of Salmonella cells, by incubating the pre-enriched sample in

216
 selective broth, for 24 h. In this step, it is recommended that two
different enrichment media be used, because the resistance of
Salmonella to selective agents varies from strain to strain. The most
recommended media for this purpose are modified semi-solid
Rappaport-Vassiliadis broth (MSRV) or Rappaport Vassiliadis Soya
broth (RVS) and Muller- Kauffmann tetrathionate- novobiocin broth
(MKTTn broth).

Differential selective plating:

The objective of this step is to preferentially promote the development
of Salmonella colonies exhibiting typical characteristics that distinguish
them from competitors, for subsequent serological and biochemical
confirmation. The most commonly used media is xylose lysine
desoxicolate (XLD) agar that differentiate Salmonella through its
incapacity to ferment lactose and the concomitant ability to produce
H2S,
Confirmation: The objective of this step is to confirm the Salmonella
colonies by means of biochemical and serological assays.
Equipment 1. Laminar airflow
2. Biosaftey cabinet
3. Hot air oven
4. Autoclave
5. Incubator (Operating at 34°C to 38 °C ,37 °C ± 1°C and 41.5 °C
± 1 °C)
6. Water bath (at 42 °C to 50 °C and 37 °C ± 1°C)
7. pH meter with measuring accuracy ± 0.1 at 20 °C to 25 °C
8. Microscope
9. Refrigerator (at 2 °C– 8 °C)
10. Petri dishes (Glass or plastic of 90-100 mm diameter or 140 mm)
11. Graduated pipettes (0.1 ml divisions) of capacity 1 ml (Class A)
12. Tubes and glass bottles
13. Vortex
14. Mechanical stirrer

217
 15. Spreader (glass or plastic)
16. Inoculation loops and straight wire
17. Spiral plater/rotator
18. Inoculation loops and straight wire made up of platinum/iridium
or nickel/chromium of dia ≈ 3 mm
Culture Media i) Nutrient Agar
ii) Rappaprt-Vassilliadis medium with Soya (RVS broth)
iii) Modified Semi Solid Rappaprt-Vassilliadis (MSRV agar)
iv) Muller Kauffmann tetrathionate broth (MKTT Broth)
v) Xylose Lysine Deoxycholate (XLD) Agar
vi) Triple Sugar Iron (TSI) Agar
vii) Lysine Iron Agar (LIA)
viii) Urea Agar
ix) Phenol Red Dulcitol Broth
x) Phenol Red Lactose Broth
xi) Tryptone Broth
xii) Buffered Glucose (MR-VP) Medium
xiii) Brain Heart Infusion (BHI) Broth
xiv) Buffer Peptone Water
xv) TSI agar
xvi) Kovacs reagent
xvii) Saline solution
xviii) Toluene
xix) β galactosidase reaent
xx) TSI medium
xxi) Urea agar
xxii) Salmonella antisra

218
Procedure Preparation of sample and pre-enrichment
Aseptically add 25 g/ml of sample into a sterile empty wide mouth
container with suitable closure. Add 225 ml of sterile or Buffer peptone
water for pre-enrichment. Make a uniform suspension by blending.
Incubate at 34 to 38°C for 18 ± 2 h.
After incubation this pre-enrichment can be stored for a maximum of 72
h at 5 °C.

Selective enrichment
General
Allow the pre-enrichment media to came at room temperature.
Gently shake the incubated sample avoiding particulate material from
pre-enrichment medium.
After incubation this enrichment can be stored for a maximum of 72 h at
5 °C.

Procedure for food samples from production area

Transfer 0. 1 ml to 10ml of RVS broth or to the surface of MSRV agar
plate with one to three equally spaced spots.
Add 1 ml of the pre enrichment culture to a tube of 10 ml of MKTTn
broth.
Incubate RVS and MSRV at 41.5 °C for 24 h± 3h.
Do not invert MSRV agar plates.
Incubate MKTTn broth at 37 °C for 24 h ± 3h
Suspect MSRV plates will show grey white, turbid zone extending out
from the inoculated drop.
An additional incubation of 24 h ± 3h may be required for dried milk
products and cheese where Salmonella may be sublethally injured.
An additional incubationtime may also be beneficial for some products,
while investigating outbreaks.

Procedure for food samples from pre production area

Inoculate the MSRV agar plate with 0.1 ml of pre enriched culture as
one to three equally spaced spots.

219
Incubate MSRV at 41.5 °C for 24 h± 3h.
Do not invert MSRV agar plates.
Suspect MSRV plates will show grey white, turbid zone extending out
from the inoculated drop.
In case plates are negative after 24 h, re-incubate for a further 24 h ± 3h.

Selective media plating

Gently mix and streak loopful of incubated selective enriched culture
(RVS broth or MSRV agar and MKTTn broth) onto two selective
isolation agar media.
The first isolation medium is Xylose Lysine Deoxycholate (XLD) agar.
The second isolation medium is chosen by the testing laboratory.
Choose a second selective plating medium which is complementary to
XLD agar (and is based on different diagnostic characteristics to those
of XLD agar to facilitate detection of (for instance, lactose positive or
H2Snegative) Salmonella.
From the incubated RVS broth, inoculate (by means of a 10 µl loop) the
surface of an XLD plate so that well-isolated colonies will be obtained.
Proceed in the same way with the second selective platingout medium.
From the MSRV agar, determine the furthest point of opaque growth
from the inoculation points and dip a 1 µl loop just inside the border of
the opaque growth. Withdraw the loop ensuring that no large lumps of
MSRV agar are extracted.
Inoculate the surface of an XLD plate so that well-isolated colonies will
be obtained. Proceed in the same way with the second selective plating-
out medium.
From the MKTTn broth, inoculate (by means of a 10 µl loop) the
surface of an XLD plate so that well-isolated colonies are obtained.
Proceed in the same way with the second selective plating-out medium.
Invert and incubate the XLD plates at 37 °C for 24 ± 3 h
Incubate the second selective plating-out medium in accordance with the
manufacturer’s instructions.
If the selective enrichment media have been incubated for an additional

220
24 h, follow the same plating-out procedure as described above.
Typical colonies of Salmonella on XLD agar have a black centre and a
lightly transparent zone of reddish colour due to the colour change of the
indicator.
Salmonella H2S neative strains are pink with darker pink centre. Lactose
positive Salmonella grown on XLD are yellow with or without
blackening.
Check the second selective plating medium after appropriate incubation
for the presence of colonies which from their characteristics are found to
be presumptive Salmonella.
Confirmation
Combination of biochemical and serological test results indicate whether
an isolate belons to enus Salmonella. Fr characterization of Salmonella
strains, fully serotypingg is needed.

Treatment of typical or suspected colonies

Mark suspect colonies on each palte. Take at least one suspected colony
for subculture and confirmation, if this is neative take four more suspect
colonies (if present) from each of the different selective isolation
medium and streak onto non selective medium e.g. Nutrient agar plates.
Incubate between 34 °C and 38 °C for 24 h ± 3 h.Use this culture for
biochemical and serological confirmation.

Alternatively, if well-isolated colonies (of a pure culture) are available

on the selective plating media, the biochemical confirmation can be
performed directly on a suspect, well-isolated colony from the selective
plating medium.

Biochemical tests
TSI Agar
With help of inoculating wire or a needle inoculate the TSI agar
(streaking slant and stabbing butt) with the culture obtained from the
nutrient agar plates

221
Incubate TSI slants at 37 °C for 24 ± 3 h
Table: Typical Salmonella reactions are:
TSI
Slant Alkaline (red)
Butt Acid (Yellow)
H2S production + or -
(blackening in butt)

A culture is treated as presumptive postive if the reactions are typical on

TSI slants.

Biochemical tests
Using a sterile inoculating needle or wire inoculate a portion of the
positive culture into the following broths and read for Salmonella typical
reactions.
.
Biochemical tests
Broth/ Media Time of Results
incubation
Urea broth 37 °C for 24 h Negative (no change in yellow
colour of medium)
L-Lysine 37 °C for 24 ± Positive (Purple color)
decarboxylation 3h
medium
Β-galactosidase 37 °C for 24 h Negative

MR-VP medium 35 °C or 37 °C Negative for VP test but

24 ± 2 h positive for MR test.
Indole test 37 °C for 24 Negative (Yellow brown
h± 3 h reaction)

Serological Tests
Elimination of auto agglutinable strains

222
Place 1 drop of saline on clean glass slide. Take a part of colony to be
tested to make homogenate suspension. Tilt the slide gently for 1 min.
Observe the result for any clumps which shows presence of auto
agglutinable strain.
Auto-agglutinable strains shall not be submitted for following tests.
Examination of H-antigens
Take a part of colony on glass slide and add 1 drop of anti H serum on it.
Mix well, if agglutination occurs, the reaction is positive.
Examination of Vi-antigens
Add 1 drop of culture suspension to clean glass slide. Add a drop of
anti-Vi- serum. If agglutination occurs, the reaction is positive.
Monovalent/Polyvalent somatic (O) test.
Add 1 drop of culture suspension to clean glass slide. Add a drop of anti
O serum. If agglutination occurs, the reaction is positive.

Interpretation of agglutination tests

In place of serum, use normal saline as negative control.

Interpretation of Biochemical and Serologiocal Reactions

Biochemical Auto- Serologiocal Interpretation
Reactions agglutination Reactions
Typical No O- and H- antigen Strains
positive(and Vi considered to
positive if tested) be Salmonella
Typical No O- and H- antigen May be
negative Salmonella
Typical Yes Not tested
No Typical _ _ Not considered
Reactions to be
Salmonella

Serotyping:
Strans that are confirmed as Salmonella spp, can be further serotyped.

223
Calculation NA
Expression of Result Salmonella = Present/Absent per 25 gm or 25 ml
Reference ISO 6579-1 (2017): Microbiology of the food chain — Horizontal
method for the detection, enumeration and serotyping of Salmonella —
Part 1: Detection of Salmonella spp.
Approved by Scientific Panel on Methods of Sampling and Analysis

224
 Method for Detection and Confirmation of Shigellabased on ISO:
21567

Method No. FSSAI 15.018:2023 Revision No. & Date 0.0

Introduction Shigellosis is an infectious disease spread most commonly by person to
person transmission. The genus consists of 4 species: Sh. dysenteriae
(Subgroup A), Sh. flexneri (Subgroup B), Sh. boyvdii (Subgroup C) and
Sh. sonnei (Subgroup D). These species are distinguishable by their
biochemical and serological reactions
Scope This method is applicable to those food product categories and their sub-
categories as mentioned in the Appendix B tables of Food Safety and
Standards (Food Products Standards and Food Additives) Regulations,
2011 and amendments (Gazette notifications) issued from time to time.
Caution In order to safeguard the health of laboratory personnel, it is essential
that tests for detecting Shigella spp. are only undertaken in properly
equipped laboratories, under the control of a skilled microbiologist, and
that great care is taken in the disposal of all incubated materials. It is
also the responsibility of the user to establish appropriate safety and
health practices.
Principle The detection of Shigella spp requires 4 successive stages viz. pre-
enrichment, enrichment, biochemical and serological confirmation
Pre-enrichment selective broth:
The objective of selective enrichment is to inhibit the competing
microflora present in the samples, favoring at the same time
multiplication of the target microorganism. This is achieved by using
selective agents (0.5 µg/ml of Novobiocin) and/ or restrictive conditions
(anerobically incubated at 41.5 ± 1 °C)for the growth of the competing
microflora
Differential selective plating: The objective of this step is to
preferentially promote the development of Shigella spp. colonies
exhibiting typical characteristics that distinguish them from competitors,
for subsequent serological and biochemical confirmation.

225
 Confirmation by means of biochemical assays and serological assays:
The objective of this step is to verifywhether the colonies obtained on
the plates are actuallyShigella spp colonies, by means of biochemical
and serologicaassays
Equipment 1. Laminar airflow
2. Biosafety cabinet
3. Hot air oven
4. Autoclave
5. Incubator (Operating at 37 °C ± 1°C and 41°C ± 1°C)
6. Water bath (at 44 °C to 47 °C)
7. pH meter with measuring accuracy ±0.1
8. Microscope
9. Glass slides
10. Refrigerator (at 2 °C– 8 °C)
11. Petri dishes (Glass or plastic of 90-100mm diameter or 140mm)
12. Graduated pipettes (0.1 ml divisions) of capacity 1 ml (Class A)
13. Micropipette with tips
14. Glass tubes, flasks and glass bottles
15. Vortex
16. Modified atmospheric jar /Anaerobic Jar /anerobic incubation
cabinet
17. Mechanical stirrer
18. pH meter with measuring accuracy ±0.1
19. Spreader (glass or plastic)
20. Inoculation loops and straight wire
21. Spiral plater/rotator
22. Inoculation loops and straight wire
23. Colony counter
Culture media (i) Nutrient Broth (NB)
(ii) MacConkey agar medium
(iii) Deoxycholate citrate Agar (DCA) medium
(iv) Triple Sugar Iron (TSI) Agar slants

226
 (v) Urea Broth
(vi) Acetate Agar Slants
(vii) Carbohydrate Fermentation Media
(viii) Tryptone Broth (for Indole test)
(ix) Buffered Glucose (MR-VP) Medium
(x) Koser’s Citrate Broth
(xi) Decarboxylase Test Media with Lysine or Ornithine
(xii) Motility Test Medium
(xiii) Thornley’s Semi-Solid Arginine Medium
Procedure Preparation of sample and enrichment
Pre enrichment
Aseptically open the sample container and weigh x gm sample into a
sterile empty wide mouth container with screw cap or suitable closure or
take x ml of liquid sample. Add x ml of sterile Shigella broth containing
0.5 µg/ml of novobiocin to make 1 in 10 dilution of sample. Make a
uniform suspension by blending if necessary and incubate the Shigella
broth under anaerobic conditions with caps and closures loose, or with
equipment giving an equivalent effect, so that gas exchange can readily
occur without contamination at 41.5 ± 1 °C for 16 h to 20 h.

Plating out and colony selection

Isolation
Using the cultures obtained in pre enrichment, gently mix the contents
and allow the larger particles to settle. Inoculate (streaking), by means of
a loop, the surface of the selective agars to obtain well-isolated colonies
on MacConkey agar (Low selectivity), XLD agar (Moderate selectivity)
and Hektoen enteric agar and incubate the plates at 37 ± °C for between
20 h and 24 h.

Identification
After the incubation period, examine the selective agar plates for typical
colonies on
MacConkey agar: Colourless to pale pink, translucent, lactose negative.

227
XLD agar: Translucent with red/ cerise center, same colour as the agar
Hektoen enteric agar: Green and moist raised colonies.
If no typical colonies are seen, reincubate the plates for further 24 h for
typical Shigella colonies.

Colony selection and purification: Select five typical colonies of

Shigella from each selective agar. If there are fewer than 5 typical or
suspect colonies, then take all the marked colonies for confirmation. For
purification, streak the selected colonies onto the surface of Nutrient
Agar (NA) plates. Incubate the plates at 37 °C ± 1 °C for 24 ± 3 h.

Biochemical confirmation:
Use pure cultures from the NA plates for biochemical and serological
tests. As an alternative to conventional biochemical tests, commercial
biochemical kits (must be approved by FSSAI under RAFT scheme) can
be used.
1. Gram’s Stain:
Take one loop full culture and spread evenly over the drop of normal
saline on a clean glass slide to form a smooth smear. Heat fix the smear
by gently passing the slide over the flame. Add crystal violet for 60
seconds. The stain was poured off and slide was gently washed under
slightly running tap water. Add Gram’s Iodine for 60 seconds. Iodine
solution was decanted and slide was washed under slightly running tap
water. Then slide was de-colourized with alcohol for 1 second and
subsequently washed under slightly running tap water. Add Safranin as
counter stain for 30seconds, then wash with slightly running tap water
and air dry. Examine stained slide under microscope at 100X.

2. Test for Catalase:

Take clean glass slide. Take an isolated colony and suspended it in a
drop of 3 % (w/w) Hydrogen peroxide (H2O2) solution on the slide.
Presence of effervescences, caused by the liberation of free oxygen as
gas bubbles, indicated the presence of catalase and was considered
positive.

228
3. Test for Oxidase:
In freshly grown Nutrient Agar slant of culture, add few drops of freshly
mixed test reagent, 1% solution of α-naphthol in 95% ethanol and equal
amount of 1% solution of para-amino-dimethylaniline hydrochloride in
water. A positive reaction is indicated by the appearance of blue colour
within two minutes.

4. Hugh-Leifson’s test:
The strain from fresh nutrient agar growth is stabbed into two tubes of
Hugh-Leifson’s media, one of which is then layered over with a small
amount of sterile liquid paraffin. Incubate both tubes at 37°C and
observe up to 4 days. Acid formation, yellow colour, in the tube without
paraffin indicates oxidative utilization of glucose. Acid in both tubes
indicates fermentative reaction. Lack of acid in either tube indicates the
strain as not being able to utilize glucose oxidatively or fermentatively.

5. Urease Test
Streak the organisms over agar surface and incubate at 37 °C for 18 to
24 h and examine at intervals.
If urea is hydrolysed, a rose-pink to deep cerise colour develops from
the release of ammonia by the decomposition of the urea with a change
in the colour of the pH indicator. There is no change in colour of the
agar with a negative reaction. Shigella species do not hydrolyse urea.

6. Test for H2S Production TSI reactions:

Inoculate each suspect culture into Triple Sugar Iron Agar (TSI) tubes
by streaking the slant and stabbing the butt. Incubate at 37 ± 1°C/24 ± 3
h. Cap the tubes loosely to maintain aerobic conditions. After the
incubation period, examine the tubes for typical Shigella reactions

Butt: Yellow Glucose fermented: positive. Red or unchanged Glucose

not fermented: negative Black Formation of hydrogen sulfide: positive,

229
Bubbles or cracks Gas formation.

Slant surface: Yellow Lactose and/or sucrose utilized: positive. Red or

unchanged Lactose and sucrose not utilized: negative

Typical Shigella cultures show a yellow butt (acid formation) and no gas
bubbles, there is no change in thecolour of the slant (no utilization of
lactose or sucrose) and no hydrogen sulphide production.

7. L-Lysine decarboxylase medium

Inoculate below the surface of the liquid broth. Incubate at 37 ±1 °C for
24 ± 3h. Turbidity and a purple colour after incubation indicate a
positive reaction; yellow indicates a negative result. Shigella species do
not decarboxylate lysine.

NOTE The use of a paraffin overlay in the tubes can help to ensure
anaerobic conditions.
8. L-Ornithine decarboxylase medium
Inoculate below the surface of the liquid broth. Incubate at 37 ± 1°C for
24 ± 3h. If a purple colour develops, the test is positive; a yellow colour
means a negative result. Shigella sonnei decarboxylates ornithine, but
other Shigella species do not.

9. Detection of indole formation

Inoculate a tube containing 5 ml of tryptone/tryptophan medium (B.9.1)
with the pure culture. Incubate at 37 °C ± 1 °C for 24 ± 3 h.
After incubation, add 1 ml of Kovac’s reagent. The formation of a red
ring within 10 min indicates indole formation, and a yellow/brown
colour indicates a negative reaction. Shigella sonnei is negative whilst
other strains give variable reactions.

10. Detection of β-galactosidase

Suspend a loopful of the purified culture from the nutrient agar into 0.25
ml of saline solution in a screw cap bottle or test tube. Add one drop of

230
toluene and shake to mix well. Put the tube in an incubator set at 37 °C
and leave for several minutes. Add 0.25 ml of the complete reagent and
mix. Replace in the incubator set at 37 °C and leave for 24 h ± 3 h,
examining at intervals.
A yellow colour indicates the formation of β-galactosidase, which can
occur in as little as 20 min.
Shigella sonnei is positive. S. dysenteriae and S. boydii give variable
reactions and S. flexneri is negative.
11. Utilization of carbohydrates
Inoculate each of the prepared carbohydrate broths with a small
inoculum. Incubate at 37 °C for 18 to 48 h
A positive reaction when carbohydrate is utilized gives a change in the
pH indicator from purple to yellow.

Interpretation of biochemical results

Strains within some Shigella species vary in their biochemical reactions
(see Table 1), therefore interpretation based only on biochemical results
is difficult and serotyping is essential to establish identity.

Shigella are Gram-negative bacilli, 2 μm to 4 μm by 0.5 μm in size, but

often show a tendency to shorter cocco-bacillary forms and typically do
not produce gas from glucose.
They are non-motile, do not produce hydrogen sulfide or decarboxylate
lysine, and are lactose negative at 24 h. The other tests described above
give variable reactions or differing reactions according to the species.

Within the genus Shigella, mannitol discriminates Shigella dysenteriae

(negative) from other species andL-ornithine decarboxylase
differentiates Shigella sonnei (positive) from other species.
Table 1

Test Shigella Shigella Shigella Shigella

sonnei flexneri dysenteriae boydii
H2S from TSI - -

231
Gas from glucose (TSI) - -

Motility

Urease

L-Lysine decarboxylase -

L-Ornithine
decarboxylase

Indole formation Vd(61 %) Vd(44 %) Vd(29 %)

β-Galactosidase + (95 %) - Vf(50 %) Vf(11 %)

Acid from:

Dulcitol Vg(9,4 %) Vg(4.5 %) Vg(6,7 %)

Glucose + (100 %) + (100 %) + (100 %) + (100 %)

Lactose -c -a -a

Mannitol + (99 %) + b(94 %) + (98 %)

Melibiose V V V

Raffinose (2,5 %) V (53 %)

Salicin

Sorbitol V (31 %) V (29 %) V (42 %)

Sucrose -c (1,5 %)

Xylose Vh(4,0 %) V (57 %)

a
some strains of S. flexnerri serovars 2a and S. boydii 9 produce acid
b
some strains of S. flexnerri serovars 4 and 6b do not produce acid
c
S. sonnei produces acid after several days of incubation
Additional biochemical differentiation
General
It is recommended to carry out additional biochemical differentiation
tests for a better identification of the
strains: some strains of Escherichia coli and Shigella species are similar.

A) Sodium acetate
Streak the slope of the sodium acetate medium with the pure culture.

232
Use a straight wire to minimize the amount of culture medium
transferred with the inoculum, or use an inoculation needle. Incubate
under aerobic conditions for 2 days at 37 ±1°C.
Examine the green medium for growth: a positive result is found when
the medium turns blue.
Look for the growth, a blue colour indicates a positive reaction. If no
growth occurs, incubate the culture for 2 additional days at 37 ±1°C.
Examine the medium again. Shigella species do not grow or grow very
poorly. Strains of E. coli give blue colonies with the surrounding
medium blue/green.

B) Chrisensen’s Citrate
Inoculae the slant surface of Chrisensens Cirate Medium wih pure culure
using an inoculating needle. Incubae aerobically for 2 days at 37 ±1°C.
Examine to check cream pink growh. In case no growh observed
incubate furher 24 h. Shigella species do not grow.

C) Sodium mucate
Inoculae the test broth and contol broh wih pure culure. Incubate
aerobically for 2 days at 37 ±1°C. Examine to check growh. In case no
growh is observed incubate furher for 24 h. Blue colur indicaes negative
reaction and yellow colour indicaes posiive reaction.
All Shigella sp except Shigella sonnei give negative result.
Table 2 - Additional biochemical testsa to differentiate some strains
of Shigella spp.
Biochemical reactions (growth) for a determined period
after incubation

Sodium acetate Christensen’s Sodium mucate

Species
citrate

+ % at + % at + % at + % at + % at + % at
2 days 2 days 2 days 2 days 2 days 2 days

S. -(0) -(0) -(0) -(0) -(0) -(0)

dysenteriae

233
 S. flexneri -(0) -(0) -(0) -(0) -(0) -(0)

S. boydii -(0) -(0) -(0) -(0) -(0) -(0)

S. sonnie -(0) -(0) -(0) -(0) V(6,4) V(36,7)

E. coli V + V V + +
(83,8) (93,5) (>15,3) (>34,2) (91,6) (93,0)

+ >90% strains positive.

- >90% strains negative
V Variable results with between 10% to 89% of strains positive.
% Percentage of positive strains after determined incubation.
a
From Bacteriological Analytical Manual, 8th Edition (Revised
1997), FDA, USA.

Serological confirmation
Antigenic differentiation
Shigella species are non-motile and therefore do not have flagella
antigens. Differentiation within and between species depends upon the
analysis of distinct somatic group “O” and specific “O” type antigens
(see Table 3).

Growth from a fresh culture on nutrient agar is required. Carry out

agglutination tests on clean glass slides or plates of glass of the
appropriate size.

Table 3 — Antigenic differentiation within the Shigella species

Shigella Antigenic Serovars (specific antigen designation)

species group
S. dysenteriae A 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13

S. flexneri B 1a, 1b, 2a, 2b, 3a, 3b, 3c, 4a, 4b, 5a, 5b, 6,
X, Y

S. boydii C 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,

15, 16, 17, 18

S. sonnei D 1

234
NOTE 1 The group antigens (A, B, C, D) can contain minor antigens
that may cross react with other group antigens; this is avoided by the use
of absorbed antisera and/or its dilution to a stipulated level. Some
species, particularly Shigella dysenteriae, have envelope antigens that
will mask the group and serovar antigens which prevent agglutination
with specific type antisera. The envelope antigen is removed by heating
a suspension at 100 °C for 15 min to 60 min.
NOTE2 The Shigella sonnei group D antigen is present in both the
smooth and rough colony types and has no crossreactivity with the other
Shigella group antigens. Unlike some other Enterobacteriaceae,
therough colony types of
S. sonnei do not necessarily auto-agglutinate. Shigella sonnei has no
envelope antigen.

Agglutination tests
Follow precisely the instructions given by the manufacturer for
preparing antisera and conducting agglutination tests.

Place one drop of the group antiserum and one drop of saline solution
separately on a glass slide. Disperse part of the colony to be tested in the
saline and part of the colony in the antiserum solution so as to obtain a
homogeneous and turbid suspension in each. Rock the slide gently for
30 s to 60 s. Observe the result against a dark background, if necessary
with the aid of a magnifying lens.

If the bacteria in the antiserum have clumped into more or less distinct
particles and there is no agglutination in the saline, the isolate is positive
for the group tested. If there is agglutination in the saline, the strain is
considered to auto-agglutinate, and shall not be used further tests. The
testing of other colonies from the same culture and the other isolates
selected for examination from the original selective agar and giving
biochemical reactions indicative of Shigella should then be tested.

Expression of results:
Based on the observations and interpretation of the results report

235
 presence or absence of Shigella spp. in test portion specifying the mass
in grams or mililitres of the sample taken.
Expression of Results Shigella spp= present or absent/ gm or ml.

Reference ISO 21567 (2004): Microbiology of food and animal feeding stuffs —
Horizontal method for the detection of Shigella spp.
Approved by Scientific Panel on Methods of Sampling and Analysis

236
 Method for Detection and Confirmation of Shigella based on IS
5887-Part 7

Method No. FSSAI 15.019:2023 Revision No. & Date 0.0

Introduction Shigellosis is an infectious disease spread most commonly by
person to person transmission. The genus consists of 4 species:
Shigella dysenteriae (Subgroup A), Shigella flexneri (Subgroup B),
Shigella boydii (Subgroup C) and Shigella sonnei (Subgroup D).
These species are distinguishable by their biochemical and
serological reactions
Scope This method is applicable to those food product categories and their
sub-categories as mentioned in the Appendix B tables of Food
Safety and Standards (Food Products Standards and Food Additives)
Regulations, 2011 and amendments (Gazette notifications) issued
from time to time.
Caution In order to safeguard the health of laboratory personnel, it is
essential that tests for detecting Shigella spp. are only undertaken in
properly equipped laboratories, under the control of a skilled
microbiologist, and that great care is taken in the disposal of all
incubated materials. It is also the responsibility of the user to
establish appropriate safety and health practices.
Principle The detection of Shigella spp requires. pre-enrichment, isolation on
differential selective medium and confirmation.

Pre-enrichment in non-inhibitory medium

Differential selective plating: The objective of this step is to
preferentially promote the development of Shigella spp. colonies
exhibiting typical characteristics that distinguish them from
competitors, for subsequent serological and biochemical
confirmation.
Confirmation:The objective of this step is to verifywhether the
colonies obtained on the plates are actuallyShigella spp colonies, by

237
 means of biochemical and serologicl assays.
Equipment 1. Laminar airflow
2. Biosafety cabinet
3. Hot air oven
4. Autoclave
5. Incubator (Operating at 37 °C ± 1°C and 41°C ± 1°C)
6. Anaerobic chamber
7. Water bath (at 44 °C to 47 °C)
8. pH meter with measuring accuracy ±0.1
9. Microscope
10. Glass slides
11. Refrigerator (at 2 °C– 8 °C)
12. Petri dishes (Glass or plastic of 90-100mm diameter or 140mm)
13. Graduated pipettes (0.1 ml divisions) of capacity 1 ml (Class A)
14. Micropipette with tips
15. Glass tubes, flasks and glass bottles
16. Vortex
17. Modified atmospheric jar /Anaerobic Jar /anerobic incubation
cabinet
18. Mechanical stirrer
19. Spreader (glass or plastic)
20. Inoculation loops and straight wire
21. Spiral plater/rotator
22. Inoculation loops and straight wire
23. Colony counter
Culture media i) Nutrient Broth (NB)
ii) MacConkey agar medium
iii) Deoxycholate citrate Agar (DCA) medium
iv) Triple Sugar Iron (TSI) Agar slants
v) Urea Broth
vi) Acetate Agar Slants
vii) Carbohydrate Fermentation Media
viii) Tryptone Broth (for Indole test)

238
 ix) Buffered Glucose (MR-VP) Medium
x) Koser’s Citrate Broth
xi) Decarboxylase Test Media with Lysine or Ornithine
xii) Motility Test Medium
xiii) Thornley’s Semi-Solid Arginine Medium
Expression of Results Shigella spp= present or absent/ gm or ml.

Reference IS 5887 (Part 7): Methods for Detection of Bacteria Responsible for
Food Poisoning, Part 7: General Guidance on Methods for Isolation
and Identification of Shigella.

Approved by Scientific Panel on Methods of Sampling and Analysis

239
 Method for Enumeration of Coagulase Positive Staphylococci based
on ISO 6888-1/AMD 1 and IS 5887- Part 8 (Sec 1)

Method No. FSSAI 15.020:2023 Revision No. & Date 0.0

Introduction Staphylococci are aerobic Gram-positive bacteria, with diameters of 0.5
– 1.5 µm and characterized by individual cocci, in grape-like clusters.
They are non-motile, non-spore forming facultative anaerobes. Some of
species of Staphylococcus genera have ability to produce coagulase
enzyme i.e Staphylococcus aureus, Staphylococcus hyicus,
Staphylococcus intermedius etc. Coagulase positive
Staphylococciproduce Enterotoxins, which is responsible for food-
poisoning if contaminated food is consumed. S. aureus is the species
with the more pathogenic of the genus Staphylococcus.
Scope This method is applicable to those food product categories and their sub-
categories as mentioned in the Appendix B tables of Food Safety and
Standards (Food Products Standards and Food Additives) Regulations,
2011 and amendments (Gazette notifications) issued from time to time.
Caution Carry out the test under the control of skilled microbiologist and great
care shall be taken in the disposal of all the incubated material. Follow
safe and good laboratory practices to avoid cross contamination.
Principle Enumeration medium BPA, contain Lithium chloride and potassium
tellurite which inhibit most of the contaminating micro flora except
Staphylococci. Staphylococci can reduce tellurite to telluride, which
results in grey to black coloration of the colonies. With the addition of
egg yolk, the medium becomes yellow, slightly opaque. A clear halo
develops around colonies from coagulase positive Staphylococci. Grey-
black colonies and a halo on this medium are presumed to be indicative
of coagulase positive Staphylococci which are confirmed by doing
coagulase test.
Equipment 1. Laminar airflow
2. Biosafety cabinet
3. Hot air oven

240
 4. Autoclave
5. Incubator (Operating at 35°C ± 1°C or 37 °C ± 1°C, 25 °C ± 1°C ,44
°C ± 1°C and 50°C ± 1°C)
6. Water bath (at 44 °C to 47 °C ± 2 °C)
7. pH meter with measuring accuracy ±0.1
8. Microscope
9. Refrigerator (at 2 °C– 8 °C)
10. Petri dishes (Glass or plastic of 90-100mm diameter or 140mm)
11. Graduated pipettes (0.1 ml divisions) of capacity 1 ml (Class A)
12. Micropipette with tips
13. Tubes and glass bottles
14. Vortex
15. Mechanical stirrer
16. Spreader (glass or plastic)
17. Inoculation loops and straight wire
18. Spiral plater/rotator
19. Inoculation loops and straight wire
20. Colony counter

Culture Media and  Primary Diluent saline peptone water (SPW) or buffered peptone
Reagents water (BPW)
 Baired Parker Agar (BPA) Medium
 Potassium tellurite solution
 Egg Yolk Medium
 Sulfamezathine Solution
 Brain Heart Infusion Broth (BHIB)
 Coagulase (Rabbit) Plasma

For media preparation and other details refer Chapter 3

Reference Cultures Staphylococcus aureus
Sample Preparation Refer Chapter 2
Procedure Preparation of the samples and serial dilutions.
a). Following the procedures described in Chapter 2, make a 1:10

241
dilution of the well mixed sample, by aseptically transferring sample to
the desired volume of diluents saline peptone water (SPW) or buffered
peptone water (BPW). Aseptically weigh 10 gm /10ml of solid or semi-
solid sample into a sterile blender jar or into a stomacher bag. Add 90
mL of sterile diluent.
Prepare subsequent serial dilutions as required by pipetting 1 ml of
previous dilution into a subsequent tube containing 9mL of the diluent.
Shake each dilution. Vortex mixing can be performed to prepare
homogenate.
Certain types of product result in viscous or thick initial suspensions
when prepared with the usual 1 in 10 dilution and additional diluent may
be necessary to facilitate further testing. In such cases, the diluent shall
be added in other ratios (e.g. 1 in 20, 1 in 50, 1 in 100) until a
satisfactory initial suspension for further operations is achieved. These
non-standard ratios shall be taken into account in subsequent operations,
particularly in the calculation and expression of results.
Inoculation and Incubation
BPA plates
Pour about 15 ml to 20 ml of the Baird- Parkar Agar Medium (BPA) at
44 °C to 47 °C into each Petri dish and allow it to solidify.
Thepaltes may be stored at 5 °C ± 3 °C for upto 24 h
Immedietletly before use, these agar plates should be dried
Label all petri plates with the sample number, dilution, date and any
other desired information.
Inoculation
 Take two BPA plate prepared previously. Using sterile
pipette/auto pipette, spread 0.1ml of test sample (liquid or
primary suspension if prepared).
 Similarly prepare plates from subsequent dilution as required.
 Limit of detection can be raised by a factor of 10 by spreading 1
ml of liquid sample/primary suspension in case of food sample,
on the surface of 3 petri dishes of 90 mm or using a single 140
mm petri dish.

242
  Carefully spread the inoculums as quickly as possible over the
surface of agar medium, using the spreader, trying not to touch
the sides of agar plate.
 Allow all the plates to dry with their lids on for 15 min at
laboratory temperature.
Incubation
 Invert all petri dishes and incubate at35°C±1°C or 37°C±1°C for
24±2h. Then reincubate for a further 24h±2h.
Selection of plates and Observation
 Observe all plates for presence of typical or atypical colonies.
 Typical colonies appear as 1-2.5 mm diameter shiny black/grey
color and surrounded by clear zone which may be partially
opaque. Atypical colonies may appear as grey colonies free of
clear zone or shiny black colonies with or without narrow white
edge and free from clear zone.
Confirmation
 Select 5 typical and 5 atypical colonies from each plate selected
for enumeration.
 In case of low count obtained (less than 15) on lowest dilution,
select all colonies for confirmation.

Coagulase test
From the surface of each selected colony, transfer an inoculum with
sterile inoculation loop/wire and transfer it to a tube of BHI broth.
Incubate at 37°C±1°C for 24±2h. After that aseptically add 0.1 ml of
this culture to a sterile test tube and add 0.3 ml of the rabbit plasma (or
specified by manufacturer) and incubate at 37°C or 37°C ± 1°C. Put up
conrol test tube wihout Rabbit plasma Examine for clotting of the
plasma after 4 to 6h of incubation by tilting the tube, if the test is
negative, re-examine at 24h of incubation. Consider the coagulase test to
be positive if the volume of clot occupies more than half of original
volume of the liquid. Control plasma shall show no sign of clotting.
Calculation 1. Select petri dishes having 15 to 150 typical/atypical colonies for

243
 enumeration.
2. Spreading colony shall be considered as single colony.
3. If a 1 ml inoculum was spread over three plates, treat these plates
as one in all subsequent counting and confirmation procedures.
4. Use the following formula for calculation

N is Number of colonies per ml or g of product

∑ C is the sum of colonies counted on all the dishes retained
n1 is the no. of dishes retained in the first dilution
n2 is the no of dishes retained in the second dilution
d is the dilution factor corresponding to first dilution
5. In case petri dishes have typical/atypical colonies less than 15,
calculate the results using the following formula:
N= C/ (v × d)
N is Number of colonies per ml or g of product
C is the average of colonies on the petri dishes retained
v is the volume of inoculums used in each dish
d is the dilution corresponding to the dilution retained.
Expression of Results  Results shall be expressed as a number between 1.0 and 9.9
multiplied by 10x, where x is power of 10.
 In case of 0.1ml inoculum, if plates of initial dilution have no
colonies, the result is expressed as less than 10 cfu/ml or 100
cfu/g or mL (if primary suspension prepared)
 In case of 1ml inoculum, if plates of initial dilution has no
colonies, the result is expressed as less than 1 cfu/ml or 10 cfu/g
or mL (if primary suspension prepared)
Reference 1) ISO 6888-1:1999/AMD 1 (2003): Microbiology of food and animal
feeding stuffs — Horizontal method for the enumeration of
coagulase-positive staphylococci (Staphylococcus aureus and other
species) — Part 1: Technique using Baird-Parker agar medium —
Amendment 1: Inclusion of precision data.
2) IS 5887-8-1 (2002): Methods for Detection of Bacteria Responsible

244
 fo Food Poisoning, Part 8: Horizontal Method for Enumeration of
Coagulase-Positive Staphylococci (Staphylococcus aureus and other
Species), Section 1: Tecnique Using Baird-Parker Agar Medium.
Approved by Scientific Panel on Methods of Sampling and Analysis

245
 Method for Detection of Coagulase Positive Staphylococcibased on
ISO 6888-2 and IS 5887- Part 8 (Sec 2)

Method No. FSSAI 15.021:2023 Revision No. & Date 0.0

Introduction Staphylococci are aerobic Gram-positive bacteria, with diameters of 0.5
– 1.5 µm and characterized by individual cocci, in grape-like clusters.
They are non-motile, non-spore forming facultative anaerobesSome of
species of Staphylococcus genera have ability to produce coagulase
enzyme i.e Staphylococcus aureus, Staphylococcus hyicus,
Staphylococcus intermedius etc. Coagulase positive
Staphylococciproduce Enterotoxins, which is responsible for food-
poisoning if contaminated food is consumed.S. aureus is the species
with the more pathogenic of the genus Staphylococcus.
Scope This method is applicable to those food product categories and their
sub-categories as mentioned in the Appendix B tables of Food Safety
and Standards (Food Products Standards and Food Additives)
Regulations, 2011 and amendments (Gazette notifications) issued from
time to time.
Caution Carry out the test under the control of skilled microbiologist and great
care shall be taken in the disposal of all the incubated material. Follow
safe and good laboratory practices to avoid cross contamination.
Principle Pre enrichment of Staphylococci is carried out in salt medium. High
salt content inhibit the growth of most of contaminating micro flora.
After enrichment selective isolation is done on Baird Parker agar
(BPA) and sheep blood agar. BPA contain Lithium chloride and
potassium tellurite which inhibit most of the contaminating micro flora
except Staphylococci. Staphylococci can reduce tellurite to telluride,
which results in grey to black coloration of the colonies. With the
addition of egg yolk, the medium becomes yellow, slightly opaque. A
clear halo develops around colonies from coagulase positive
Staphylococci. Grey-black colonies and a halo on this medium are
presumed to be indicative of coagulase positive Staphylococci which

246
 are confirmed by doing coagulase test. On blood agar, colonies appear
as golden yellow colour with haemolytic activity.
Equipment 1. Laminar airflow
2. Biosafety cabinet
3. Hot air oven
4. Autoclave
5. Incubator (Operating at 37 °C ± 1°C))
6. Water bath (at 44 °C to 47 °C)
7. pH meter with measuring accuracy ±0.1
8. Microscope and glass slides
9. Refrigerator (at 2 °C– 8 °C)
10. Petri dishes (Glass or plastic of 90-100mm diameter or 140mm)
11. Graduated pipettes (0.1 ml divisions) of capacity 1 ml (Class A)
12. Micropipette with tips
13. Tubes and glass bottles
14. Vortex
15. Mechanical stirrer
16. Spreader (glass or plastic)
17. Inoculation loops and straight wire
18. Spiral plater/rotator
19. Inoculation loops and straight wire
Culture Media and  Primary Diluent
Reagents  Baired Parker Agar (BPA)
 Sheep Blood Agar
 Nutrient Agar
 Cooked Meat Salt Medium
 Gram Stain kit
 Coagulase (Rabbit) Plasma
For media preparation and other details refer Chapter 3
Reference Cultures Staphylococcus aureus
Sample Preparation Refer Chapter 2
Procedure Enrichment
1) Inoculate specified amount of food sample/primary suspension

247
 prepared into blood agar and cooked meat salt medium (1:9) and on
Baird Parker medium.
2) Incubate the blood agar and salt medium at 37 °C for 18-24 hr
and Baird Parker medium at 37 °C for 30 h

Selective Isolation
Streak a loopful from primary suspension prepared from cooked salt
medium onto BPA and sheep blood agar and incubate BPA plate at 37
°C for at least 30 hr and sheep blood agar plate at 37 °C for 18-24 hr.
Observation
On sheep blood agar, typical colonies appear as yellow color
surrounded by clear zone of haemolysis.
On BPA, typical colonies appear as 1-2.5 mm diameter shiny
black/grey color and surrounded by clear zone which may be partially
opaque. Atypical colonies may appear as grey colonies free of clear
zone or shiny black colonies with or without narrow white edge and
free from clear zone.
Confirmation
Pick 5 suspected colonies from each plate and streak on nutrient agar,
prior to confirmation. Perform Gram staining and coagulase test for
confirmation.
Gram Staining: If there are typical/suspected colonies, pick up the
colonies and confirm by gram staining. Staphylococci are Gram
positive cocci which appear in clusters.
Coagulase test: The test may be performed using one of following
method.
(i) Slide method: Emulsify a portion of suspected colony in normal
saline or water on a clean slide. Mix it with a straight wire dipped in
rabbit plasma. Coagulase positive Staphylococci produce visible
clumping immediately.
Positive control with a known coagulase positive strain of S. aureus
and a control of rabbit plasma without inoculum should be included in
the test.

248
 In case of nergative result, perform the test using tube method.
(ii) Tube method: Emulsify a portion of suspected colony from 24 hr
growth on blood agar in 1 ml citrated rabbit plasma diluted 1 in 5 in
0.85% saline. Incubate at 37 °C preferably in a water bath or incubator.
Observe every hour to observe clotting of plasma. Positive control with
a known coagulase positive strain S. aureus and a control of rabbit
plasma without inoculum should be included in the test.
Expression of Results Results shall be expressed as Present/Absent per x ml/gm, where x is
quantity of food sample enriched in salt medium.
Reference 1) IS 5887-8-2 (2002): Methods for Detection of Bacteria
Responsible for Food Poisoning, Part 8: Horizontal Method for
Enumeration of Coagulase-Positive Staphylococci (Staphylococcus
Aureus and other species), Section 2 Technique using rabbit
plasma fibrinogen agar medium
2) ISO 6888-2 (1999): Microbiology of food and animal feeding
stuffs — Horizontal method for the enumeration of coagulase-
positive staphylococci (Staphylococcus aureus and other species)
— Part 2: Technique using rabbit plasma fibrinogen agar medium
Approved by Scientific Panel on Methods of Sampling and Analysis

249
 Method for Determination of Vibrio cholera and Vibrio
parahaemolyticus

Method No. FSSAI 15.022:2023 Revision No. & Date 0.0

Introduction The members of the genus Vibrio are primarily aquatic bacteria, found
both in freshwater and in seawater, in addition to being frequently
associated with marine animals. Several species cause diarrhea or
infections of the gastrointestinal tract but the most frequent enteric
pathogens are Vibrio cholerae and Vibrio parahaemolyticus.
Cholera, a disease caused by strains of V. cholerae of serotypes O1 and
O139, is classified by the International Commission on Microbiological
Specifications for Foods (ICMSF, 2002) into risk group IA: “diseases of
severe hazard for general population; life threatening or resulting in
substantial chronic sequelae or presenting effects of long duration”.
The disease caused by V. parahaemolyticus is classified by the
International Commission on Microbiological Specifications for Foods
(ICMSF, 2002 ) into risk group III: “diseases of moderate hazard usually
not life threatening, normally of short duration without substantial
sequelae, causing symptoms that are self-limiting but can cause severe
discomfort.
Scope This method is applicable to those food product categories and their sub-
categories as mentioned in the Appendix B tables of Food Safety and
Standards (Food Products Standards and Food Additives) Regulations,
2011 and amendments (Gazette notifications) issued from time to time.
Special Caution In order to safeguard the health of laboratory personnel, it is essential
that tests for detection of Vibrio spp., and particularly toxigenic Vibrio
cholerae, Vibrio parahaemolyticus be conducted only in laboratories
equipped for this purpose and under the supervision of an experienced
microbiologist, and that great care is exercised in the disposal of
contaminated material.
Principle The detection of potentially enteropathogenic Vibrio spp. (V. cholera,
Vibrio parahaemolyticus) requires four successive phases. Recovery of

250
 certain Vibrio spp. from foodstuffs may be improved by the use of
different incubation temperatures depending upon the target species or
state of the food matrix. For example, recovery of V. cholera and Vibrio
parahaemolyticus in fresh products is enhanced by enrichment at 41.5
°C whereas for deep frozen, dried or salted products, recovery is
enhanced by enrichment at 37 °C.

If detection of V. cholerae and Vibrio parahaemolyticusis required, all

specified incubation temperatures should be used. If detection of V.
cholera and Vibrio parahaemolyticus together is not required, the
specific procedure(s) maybe selected according to the species being
sought.
Equipment 1. Laminar airflow
2. Biosafety cabinet
3. Hot air oven
4. Autoclave
5. Incubator (Operating at 37°C ± 1°C)
6. Incubator (Operating at 41.5°C ± 1°C)
7. Water bath (at 44 °C to 47 °C)
8. pH meter with measuring accuracy ±0.1pH units at 25°C
9. Microscope
10. Refrigerator (at 2 °C– 8 °C)
11. Freezer, adjustable to <−15 °C
12. Micro-centrifuge tubes (capacity of 1.5 ml and 2.0 ml).
13. Micro-centrifuge, for reaction tubes (capacity of 1.5 ml and 2.0 ml)
and capable of running at 10 000g.
14. Heating block (at 95 °C ± 2.0 °C).
15. Graduated pipettes and pipette filter tips, for volumes between 1 μl
and 1000 μl.
16. Associated consumables for conventional or real-time PCR, e.g.
optical plates and caps, optical plate holder, suitable for use with the
selected PCR machine.
17. Conventional or real-time PCR machine, gel electrophoresis and UV

251
 visualization equipment as appropriate.
18. Petri dishes (Glass or plastic of 90-100mm diameter or 140mm)
19. Graduated pipettes (0.1 ml divisions) of capacity 1 ml (Class A)
20. Micropipette with tips
21. Tubes and glass bottles
22. Vortex
23. Mechanical stirrer
24. pH meter with measuring accuracy ±0.1
25. Spreader (glass or plastic)
26. Inoculation loops and straight wire
27. Spiral plater/rotator
28. Inoculation loops and straight wire

Culture Media and  Enrichment medium: alkaline saline peptone water (ASPW)
Reagents
Solid selective isolation media
 First medium: Thiosulphate Citrate Bile and Sucrose agar medium
(TCBS)
 Second medium: The selection of the second medium is left to the
choice of the test laboratory. Preparation of the medium should be
strictly according to the manufacturer’s instructions. (Saline nutrient
agar) Reagent for detection of oxidase

Biochemical tests
 L-lysine decarboxylase saline medium (LDC)
 Arginine dihydrolase saline medium (ADH)
 Reagent for detection of β-galactosidase
 Saline medium for detection of indole
 Saline peptone waters
 Sodium chloride solution
 Oxidase Test
 Saline Nutient agar

252
 Details of preparation given in Chapter 3
Reference Cultures Further details of culturing given in Chapter 3

Sample Preparation Sample preparation based on the product categories are given in Chapter
2
Procedure Test portion and initial suspension
Primary selective enrichment:
Take 25 g or 25 ml of sample and homogenize in 225 ml of
alkaline saline peptone water (ASPW). Incubate the flasks
in the following conditions:
Fresh products at 41.5 ±1°C for 6 ±1 hours;
Processed (deep frozen, dried or salted) products at 37 ±1°C for 6 ±1
hours.

Target Vibrio spp. in fresh product

Incubation Vibrio parahaemolyticus Vibrio cholerae
temperature
41.5 °C ± 1 °C √ √
37 °C ± 1 °C ------ ------
Target Vibrio spp. in deep frozen, dried or salted product (such as
bacalhau, stock fish, bonefish, katsuobushi, obambo)
Incubation Vibrio parahaemolyticus Vibrio cholerae
temperature
41.5 °C ± 1 °C ----- ------
37 °C ± 1 °C √ √
Primary incubation and target species/product state

Secondary selective enrichment

Transfer 1 ml of the Pre-enrichment Culture obtained from the surface
into a tube containing 10 ml of ASPW. It is recommended that the
sample is not agitated before taking the aliquot. Incubate the ASPW at
41.5 °C ± 1 °C for 18 h ± 1 h according to table below.

Target Vibrio spp. in all product states

253
 Incubation Vibrio Vibrio cholerae
temperature parahaemolyticus
41.5 °C ± 1 °C √ √

Isolation and identification

From the cultures obtained in the Secondary selective enrichment
ASPW, inoculate with a 1 μl sampling loop the surface of two selective
agar paltes.
First selective agar plate is TCBS agar plate, so as to permit the
development of well-isolated colonies.
Second selective agar plate must be chosen by laboratory which is
complementary to TCBS using a fresh sampling loop.
Invert the agar plates and incubate as:
— for TCBS agar plates, incubate at 37 °C ± 1 °C for 24 h ± 3 h;
— for the second isolation medium, incubate according to the
manufacturer’s instructions.
After incubation, examine the TCBS and second selective medium for
the presence of typical colonies of presumptive pathogenic Vibrio spp.
Mark their position on the bottom of the plates.
On TCBS agar V. parahaemolyticus, and V. cholerae exhibit different
typical colony morphologies:
— typical colonies of V. parahaemolyticus is smooth, green (negative
sucrose) and of 2 mm to 3 mm in diameter;
— typical colonies of V. cholerae are smooth, yellow (positive sucrose)
and of 1 mm to 2 mm in diameter.
For the second selective medium, examine for the presence of colonies,
which, according to their characteristics, may be considered as possible
isolates of V. parahaemolyticus, and/or V. cholerae.

Confirmation
Using this manual, V. parahaemolyticus, and V. cholerae can be
confirmed by molecular PCR and/or biochemical approaches.
Confirmation may be carried out at the end user laboratory or by a

254
specialist reference laboratory. Not all V. parahaemolyticus possess
pathogenicity traits. In order to confirm the pathogenic character of the
strains, it is preferable to detect the presence of thermostable direct
haemolysin (tdh) or TDH- related haemolysin (trh) genes. This should
be carried out using PCR tests. PCR based confirmation also may reduce
the subjective interpretation of biochemical identification tests and
accelerate the identification process.

If shown to be reliable, commercially available biochemical test kits

(must be approved by FSSAI under RAFT scheme) may be used to
identify Vibrio to the species level provided they are inoculated with a
suspension of the bacteria to be identified in a sufficiently saline
medium or dilution fluid, and as long as the database or identification
table for the product has been based on reactions obtained using similar
media to those described in this document. These kits shall be used in
accordance with the manufacturer’s instructions.
Commercially available molecular detection kits (must be approved by
FSSAI under RAFT scheme) may be used to identify Vibrio to the
species level. These kits shall be used in accordance with the
manufacturer’s instructions.

Selection of colonies for confirmation and preparation of pure

cultures
For confirmation, subculture from each selective medium at least one
well isolated colony considered to be typical or similar to each of the
potentially pathogenic Vibrio spp. sought. If the result of the first
isolated colony tested is negative, a further four well isolated colonies
should be tested. In samples where it is considered important to optimize
the detection of potentially pathogenic V. parahaemolyticus based upon
the presence of thermostable direct and thermostable direct related
haemolysins, it is recommended that at least five and, where possible, all
colonies exhibiting typical V. parahaemolyticus colony morphology are
sub-cultured for downstream testing. Inoculate the colonies selected
onto the surface of plates of saline nutrient agar (SNA) or suitable

255
medium of the laboratory’s choice to obtain isolated colonies. Incubate
at 37 °C ± 1 °C for 24 h ± 3 h.

Tests for presumptive identification

Oxidase test
Using an inoculating loop (platinum iridium straight wire) or glass rod,
take a portion of the pure culture from the saline nutrient agar and streak
onto the filter paper moistened with oxidase reagent or use a
commercially available test, following the manufacturer’s instructions.
Neither a nickel-chromium sampling loop nor a metallic wire shall be
used. Nickel or chrome wire sampling loops may give false-positive
results, and should be avoided.

Microscopic examination (optional)

a) For each pure culture obtained, perform gram staining.
Observe under a microscope and record the results.
b) Inoculate a tube of ASPW. Incubate at 37 °C ± 1 °C for 1 h to 6
h. Perform motility using concave glass slide, and examine for
the motility under the microscope.
c) Note Vibrio cultures showing a positive result for motility.

Selection of the cultures

For confirmation, retain the oxidase-positive colonies.
For confirmation, also retain Gram-negative colonies which give a
positive result in the motility test (if examined).

Biochemical confirmation
Halotolerance test: Prepare a suspension from each SNA cultures and
inoculate a series of tubes of Halotolerance Saline Peptone Water (0, 2,
6, 8 and 10% NaCl). Incubate the tubes at 37 ± 1°C/24 ± 3 h. Growth
(tolerance) at the corresponding NaCl concentration present in the tube
is indicated by turbidity.
L- Lysine decarboxylase and Arginine dehydrolasetests: From each
SNA cultures inoculate a tube of Saline Decaboxylase Broth with 0.5%

256
of L-Lysine and a tube of Saline Decaboxylase Broth with 0.5% of
Arginine. Cover both tubes with a layer (1 ml) of sterile mineral oil and
incubate at 37 ± 1°C/24 ± 3 h. A violet color after incubation indicates a
positive reaction. A yellow color indicates a negative reaction.
β-Galactosidase test: From each SNA cultures inoculate a tube
containing 0.25 ml of sterile 1% aqueous NaCl solution. Add one drop
of toluene, shake and incubate at 37 ± 1°C/5 min (water bath). Add 0.25
ml of the β-Galactosidase reagent (ONPG reagent), mix and incubate at
37 ± 1°C/24 ± 3 h (water bath). Examine the tubes periodically for the
development of a yellow color (often after 20 min) indicative of positive
reaction. If the yellow color is not observed after 24 h the test is
considered negative.
Indole test: From each SNA cultures inoculate a 5 ml tube of Saline
Tryptophan Broth and incubate at 37 ± 1°C/24 ± 3 h. Test for indole by
adding 1 ml of Indole Kovacs Reagent to each 5 ml culture. Appearance
of distinct red color in upper layer is a positive test. A yellow brown
color is a negative test.

Interpretation of biochemical tests

Test Vibrio Vibrio

cholerae a parahaemolyticus a
Oxidase + +
LDC(Lysine + +
decarboxylase)
ADH (Arginine - -
dehydrolase)
ONPG hydrolysis + -
(β-Galactosidase)
Production of indole + +
Growth in peptone
water with
0 % NaCl + -

257
 6 % NaCl - +
10 % NaCl - -
a
The sign + denotes 76 % to 89 % positive reactions. b
Provided for reference purposes.

 The reactions given in the table above are a guide to the

identification of the listed species. Additional phenotypic tests can
be required to fully distinguish these species from each other and
from non pathogenic Vibrio spp.
 It is preferable to conduct serology for V. cholerae (at least to
determine whether they are serogroups O1 or O139) or an
appropriate PCR – based test to determine toxigenic strains such as
those that carry the cholera toxin gene ctx. This can be carried out by
the end user laboratory or a specialist reference laboratory.
Expression of Results Depending on the interpretation of results, indicate that potentially
enteropathogenic Vibrio spp. Is detected or not detected in a test portion
of x grams or x ml of product, specifying the name of the relevant
species and any pathogenicity characteristics if they have been tested.
Reference ISO 21872-1- Microbiology of the food chain — Horizontal method for
the determination of Vibrio spp. — Part 1: Detection of potentially
enteropathogenic Vibrio parahaemolyticus, Vibrio cholerae and Vibrio
vulnificus
Approved by Scientific Panel on Methods of Sampling and Analysis

258
 Method for Enumeration of Yeast and Mould

Method No. FSSAI 15.023:2023 Revision No. & Date 0.0

Introduction Yeast and moulds are classified under the Kingdom Fungi. Yeast and
mould are heterotrophic in nature and of great importance to the food
industry. Yeast and mould can cause various degree of spoilage in food
chain. Acid/alkaline requirements for yeast and mold growth in wide
range food products are quite broad, ranging from pH 2 to above pH 9.
Scope This method is applicable to those food product categories and their
sub-categories as mentioned in the Appendix B tables of Food Safety
and Standards (Food Products Standards and Food Additives)
Regulations, 2011 and amendments (Gazette notifications) issued from
time to time.
Caution Carry out the test under the control of skilled microbiologist and great
care shall be taken in the disposal of all the incubated material. Follow
safe and good laboratory practices to avoid cross contamination.
Principle Chloramphenicol Yeast Glucose Agar is a selective medium
recommended for isolation and enumeration of Yeast & Moulds. The
medium contains yeast extract, which provides nitrogenous nutrient
and vitamin B complex. Dextrose is the energy source.
Chloramphenicol a thermos table antibiotic, suppresses the bacterial
flora. Aerobic incubation of plates is done at 25 °C ± 1 °C and count is
taken on 3rd, 4th or 5th day.
Equipment 1. Laminar airflow
2. Hot air oven
3. Autoclave
4. Incubator (Operating at 25 °C ± 1°C)
5. Water bath (at 44 °C to 47 °C)
6. pH meter with measuring accuracy ±0.1pH units at 25°C.
7. Microscope
8. Refrigerator (at 2 °C– 8 °C)

259
 9. Petri dishes (Glass or plastic of 90-100mm diameter Graduated
pipettes (0.1 ml divisions) of capacity 1 ml (Class A)
10. Micropipette with tips
11. Tubes and glass bottles
12. Vortex
13. Mechanical stirrer
14. pH meter with measuring accuracy ±0.1
15. Spreader (glass or plastic)
16. Inoculation loops and straight wire
17. Spiral plater/rotator
18. Inoculation loops and straight wire
19. Colony counter

Culture Media and  Primary Diluent

Reagents  Chloramphenicol Yeast Glucose Agar (CGYEA)
 Sodium Propionate
For media preparation and other details refer Chapter 3
Reference Cultures Candida albicans, Saccharomyces, Aspergillus niger

Sample Preparation Refer Chapter 2

Procedure Preparation of the samples and serial dilutions: Follow the

procedures as described in Chapter 2. Homogenize 10 g of sample with
90 ml of saline peptone water (SPW) or buffered peptone water (BPW)
to make (10-1 dilution). From this first dilution prepare serial decimal
dilutions

Prepare subsequent serial dilutions as required by pipetting 1 ml of

previous dilution into a subsequent tube containing 9ml of the diluent.
Shake each dilution. Vortex mixing can be performed to prepare
homogenate.

Inoculation

260
Label all petri dishes with the sample code, dilution, date and any other
information.
A) Spread plate
Take plate of Chloramphenicol Yeast Glucose Agar prepared
previously. Using sterile pipette/auto pipette, spread 0.1ml of test
sample (liquid or primary suspension if prepared) over the surface of
medium plate. Similarly prepare plates from subsequent dilution as
required.
 Carefully spread the inoculums as quickly as possible over the
surface of agar medium, using the spreader, trying not to touch
the sides of agar plate.
 Allow all the plates to dry with their lids on for 15 min at
laboratory temperature.

B) Pour Plate
 Transfer 1ml of test sample if the product is liquid, or 1 ml of
primary suspension if prepared using a sterile pipette to a Petri
plate in duplicate. Similarly prepare plates from subsequent
dilution as required. If only yeast colonies are required, add
0.25% sodium propionate solution to the plate at the time of
pouring to inhibit the growth of moulds. Pour approximately
15 to 20 ml molten Chloramphenicol Yeast Glucose Agar
(CGYEA) agar (cooled at 44 0C to 47 0C in water bath).
Time elapse between inoculation and addition of agar into plates
should not exceed 15 min. The total time between the end of the
preparation of the initial suspension and pouring of plates (dishes) shall
not exceed 45 min.
Carefully mix the inoculum with the medium by horizontal
movements and allow to solidify.

Incubation
Incubate all the petri dishes at 25 °C ± 1 °C for 3 to 5 and 7 days in
upright position.

261
 Observation
Count the colonies on each plate after 3, 4 and 5 days of incubation.
Yeast colonies grow as smooth, moist, elevated or surface colonies.
Mould colonies grow as fuzzy, thread like profuse growth of hyphae
and may appear in different color.
Calculation 1. Select the Petri dishes having 10 to 150 colonies for enumeration.
2. Use the following formula for calculation

N -Number of colonies per ml or g of product

∑ C - Sum of colonies counted on all the dishes retained.
n1 - Number of dishes considered for the first dilution
n2 - Number of dishes considered forthe second dilution
d -Dilution factor corresponding to first dilution
3. In case Petri dishes have total/typical colonies less than 10,
calculate the results using the following formula:
N= C/(v × d)
N -Number of colonies per ml or g of product
C -Average of colonies on the Petri dishes retained.
v -Volume of inoculum used in each dish.
d -Dilution from which first counts were obtained.
Expression of Results  Results shall be expressed as number between 1.0 and 9.9
multiplied by 10x, where x is power of 10.
 If plates from all dilutions have no colonies, the result is expressed
as less than 1 cfu/ml or 10 cfu/g or ml (if primary suspension
prepared)
Reference 1) ISO 6611 (2004) [IDF 94:2004] Milk and milk products —
Enumeration of colony-forming units of yeasts and/or moulds —
Colony-count technique at 25 degrees C
2) IS 5403: 1999 (RA 2013) Method for Yeast and Mould Count of
Foodstuffs and animal feeds
3) ISO 21527-1 (2008): Microbiology of food and animal feeding

262
 stuffs — Horizontal method for the enumeration of yeasts and
moulds — Part 1: Colony count technique in products with water
activity greater than 0.95
4) ISO 21527-2 (2008): Microbiology of food and animal feeding
stuffs — Horizontal method for the enumeration of yeasts and
moulds — Part 2: Colony count technique in products with water
activity less than or equal to 0.95
Approved by Scientific Panel on Methods of Sampling and Analysis

263
 Part C

ANALYSIS OF WATER

264
 Method for Enumeration of Aerobic Microbial count/Standard Plate
Count

Method No. FSSAI 15.024:2023 Revision No. & Date 0.0

Scope This method is applicable to Packaged Drinking water
Caution Carry out the test under the control of skilled microbiologist and great
care shall be taken in the disposal of all the incubated material. Follow
safe and good microbiology laboratory practices to avoid cross
contamination.

Principle Plate count agar (PCA) is a general purpose growth medium commonly
used to assess "total" or “viable” bacterial growth of a water sample. The
number of microorganisms per milliliter of sample is calculated from the
number of colonies obtained on PCA plate from selected dilution. Poured
plates are prepared using a specified culture medium and a specified
quantity of the sample. The plates are aerobically incubated at two
different temperatures i.e. 37 °C for 24 hr. and 20 – 22 °C for 72 hr.
Apparatus/Instruments a) Laminar Air flow / Biosafety Cabinet
b) Universal Incubator (37 °C), and BOD Incubator 20± 2 °C)
c) Thermostatically controlled water bath (capable of being maintained
at 44 °C to 47 °C)
d) Autoclave
e) Sterilizing oven
f) Petri dishes (Glass or plastic of 90-100mm diameter or 140mm)
g) Total delivery graduated pipettes, of nominal capacity 1 mL,
graduated in 0.1 mL divisions (Class A), or Micropipette with sterile
tips
h) Mechanical stirrer
i) Tubes, flasks or bottles, of appropriate capacity
j) Colony-counting device (optional)
k) Sterile Glass or plastic spreaders
Culture Media and 1) Plate count Agar (PCA)
Reagents 2) Buffered Peptone water (BPW)
3) Overlay Medium

265
 (Overlay medium : 12 to 18 g Agar + 1000 ml water)
Preparation of Reagents Not Applicable
Sample Preparation Not Applicable
Method of analysis 1) Disinfect the surface of the bottle/pouch/cups containing sample with
70% ethanol. Thoroughly mix the sample by vigorous shaking to
achieve uniform distribution.
2) Aseptically inoculate 1 mL of the water sample using sterile pipette
into sterile petri plates in duplicate in two sets. The petri plates should
be labeled with the sample number, date and any other desired
information.
3) Pour into each plate 15–18 mL of the molten sterilized PCA media
(cooled to 44 °C–47 °C). Avoid pouring of molten medium directly
onto the inoculum. Mix the media and inoculum by swirling gently
clockwise and anti-clockwise so as to obtain homogenous distribution
of inoculum in the medium.
4) Allow to cool and solidify. In case, where in sample microorganism
having spreading colonies is expected, add 4 mL of overlay medium
onto the surface of solidified plates.
5) After complete solidification, invert the prepared plates and incubate
one set at 37 °C for 24 hr. and second set at 20 – 22 °C for 72 hr.
6) After specified incubation period count all colonies including pinpoint
colonies. Spreading colonies shall be considered as single colony. If
less than one quarter of dish is overgrown by spreading, count the
colonies on the unaffected part of the dish and calculate corresponding
number in the entire dish. If more than one quarter is overgrown by
spreading colonies discard the plate.
Calculation with units of Case 1: Plates having microbial count between 10 and 300cfu
expression ColoniesPl ate1  ColoniesPl ate2
N
2
Case 2: Plates having microbial count less than 10cfu but at least 4.
Calculate the results as given in Case 1.
Case 3: If microbial load is from 3 to 1 then reporting of results shall be:
“Microorganisms are present, but, less than 4 per mL”

266
 Case 4: When the test sample/plates contains no colonies then reporting
of results shall be:
“Less than 1 cfu/ mL”.
Inference NA
(Qualitative Analysis)
Reference 1) IS 5402:2012 - Microbiology of Food and Animal Feeding Stuffs —
Horizontal Method for the Enumeration of Microorganisms —
Colony-Count Technique at 30°C (Second Revision)
2) ISO 7218:2007 Amd. 1: 2013 - Microbiology of food and animal
feeding stuffs — General requirements and guidance for
microbiological examinations — Amendment 1
3) IS 14543:2016 - Packaged Drinking Water (Other Than Packaged
Natural Mineral Water) - Specification (Second Revision)
Approved by Scientific Panel on Methods of Sampling and Analysis

267
 Method for Detection of Escherichia coli and Coliform

Method No. FSSAI 15.025:2023 Revision No. & Date 0.0

Scope This method is applicable to Packaged Drinking water
Caution Carry out the test under the control of skilled microbiologist and great care
shall be taken in the disposal of all the incubated material. Follow safe and
good microbiology laboratory practices to avoid cross contamination.
Principle A test portion of water sample is passed through a membrane filter, which is
then placed for incubation on Chromogenic coliform agar plate. All
presumptive coliform bacteria grow as pink to red colonies which are than
confirmed by negative oxidase test. Due to β-D-galactosidase and β-D-
glucuronidase activity E. coli bacteria appear as Dark blue to violet in color.
This method is not applicable to β-D-glucuronidase negative E. coli strains
like E.coli O157.
Apparatus/Instruments a) Protective cabinet with vertical laminar airflow
b) Membrane Filtration Assembly
c) Incubators (36±2°C)
d) Thermostatically controlled water bath (capable of being maintained at
44 °C to 47 °C)
e) Autoclave
f) Sterilizing oven
g) Petri dishes (Glass or plastic of 90 – 100mm diameter or 140mm)
h) Tubes, flasks or bottles, of appropriate capacity
i) Colony-counting device (optional)
Culture Media and a) Chromogenic coliform Agar (CCA)
Reagents b) Tryptone Soya agar (Soyabean Casein digest agar)
Preparation of a) Oxidase Reagent (1% α-nepthol in 95% ethanol and equal amount of
Reagents para-amino diamethylaniline hydrochloride in water) or Oxidase Disc
commercially available
Sample Preparation Not Applicable
Method of analysis a) Disinfect the surface of the bottle/pouch/cups containing sample with
70% ethanol. Thoroughly mix the sample by vigorous shaking to achieve

268
 uniform distribution

b) Filter the sample (required volume) through a sterile membrane filter

(0.45µm pore size) and place the filter in CCA plate and Incubate
overnight at 36±2°C for 18 – 24hr.

a) Count all pink to red colonies as presumptive coliform.

b) Confirm all presumptive colonies by negative oxidase test.
c) Count all dark blue to violet colonies as E. coli.
d) Oxidase test: Add2-3 drops of Oxidase reagent on to a filter paper in a
Petridish. Take a colony to be confirmed by inoculation loop on
pretreated filter paper. A positive reaction is indicated by the appearance
of blue color within 30 s. commercially available oxidase disc can be
used as an alternative
e) Further subculture the presumptive colonies on non-selective agar
(Tryptone Soy Agar) and incubate at 36±2°C for 21±2 hr. to carry out
oxidase test.
f) Consider all colonies giving negative oxidase reaction as Coliform.
Calculation with units Presence/Absence of E. coli and Coliform given after confirmation of
of expression presumptive colonies in the sample examined.
Test for E.coli and Coliform= Present/absent per X mL of sample
Reference 1) ISO 9308-1:2014 Amd. 1:2016 - Water quality — Enumeration of
Escherichia coli and coliform bacteria — Part 1: Membrane filtration
method for waters with low bacterial background flora — Amendment 1
2) IS 15185:2016 - Water Quality - Detection and Enumeration of
Echerichia Coli and Coliform Bacteria - Membrane Filtration Method
For Water With Low Bacterial Background Flora (First Revision)
3) IS 14543:2016 - Packaged Drinking Water (Other Than Packaged
Natural Mineral Water) - Specification (Second Revision)
4) IS 15188:2022 - Water quality — General requirements and guidance for
microbiological examinations by culture
Approved by Scientific Panel on Methods of Sampling and Analysis

269
 Method for Detection of Coliform (Alternative method)

Method No. FSSAI 15.026:2023 Revision No. & Date 0.0

Scope This method is applicable to Packaged Drinking water
Caution Carry out the test under the control of skilled microbiologist and great care
shall be taken in the disposal of all the incubated material. Follow safe and
good microbiology laboratory practices to avoid cross contamination.
Principle A test portion of water sample is passed through a membrane filter, which is
then placed for incubation on Violet red Bile lactose Agar plate. The medium
is selective due to the presence of the inhibitors - bile salts and crystal violet.
Crystal violet inhibits gram-positive microorganisms especially
Staphylococci. Organisms which rapidly ferment lactose produce red
colonies surrounded by red purple halo. Lactose non-fermenters and late
lactose fermenters produce pale colonies.
Apparatus/Instruments a) Protective cabinet with vertical laminar airflow
b) Membrane Filtration Assembly
c) Incubators (36±1°C)
d) Thermostatically controlled water bath (capable of being maintained at
44 °C to 47 °C)
e) Autoclave
f) Sterilizing oven
g) Petri dishes (Glass or plastic of 90 – 100mm diameter or 140mm)
h) Tubes, flasks or bottles, of appropriate capacity
i) Colony-counting device (optional)
Culture Media and a) Violet Red Bile Lactose Agar (VRBL)
Reagents
Preparation of Not Applicable
Reagents
Sample Preparation Not Applicable

Method of analysis a) Disinfect the surface of the bottle/pouch/cups containing sample with
70% ethanol. Thoroughly mix the sample by vigorous shaking to achieve
uniform distribution

270
 b) Filter the sample (required volume i.e X) through a sterile membrane
filter (0.45µm pore size) and place the filter in VRBL agar plate and
Incubate overnight at 36±1°C for 24±2hr.
c) Coliform bacteria grow as purplish red colonies surrounded by a reddish
zone of precipitated bile.
d) Count all purplish red colonies as coliform.
Calculation with units Presence/Absence of Coliform given in per unit of the sample examined.
of expression Test for Coliform= Present/absent per X mL of sample
Reference 1) IS 5401 (Part 1): 2012 - Microbiology of food and animal feeding stuffs -
Horizontal method for the detection and enumeration of coliforms: Part 1
colony count technique (Second Revision)
2) IS 14543:2016 - Packaged Drinking Water (Other Than Packaged
Natural Mineral Water) - Specification (Second Revision)
3) IS 15188: 2022 - Water quality — General requirements and guidance
for microbiological examinations by culture
Approved by Scientific Panel on Methods of Sampling and Analysis

271
 Method for Determination of Enterococci (Faecal Streptococci)

Method No. FSSAI 15.027:2023 Revision No. & Date 0.0

Scope This method is applicable to Packaged Drinking water
Caution a) Sodium Azide is highly toxic and mutagenic; precautions shall be taken
to avoid contact with it, especially through the inhalation of fine dust
during the preparation of media.
b) Azide containing media should not be mixed with strong inorganic acids,
as toxic hydrogen azide (HN3) may be produced.
c) Solution containing azide can also form explosive compounds when in
contact with metal pipework e.g. from sinks.
Principle Slantez and Bartley medium contains sodium azide (to suppress the growth
of gram negative bacteria) and 2,3,5-triphenyltetrazolium chloride that is
reduced to red formazon by Enterococci. Confirmation is done by transfer of
membrane with all the colonies on Bile Aesculin Azide agar where asculin is
hydrolyzed within 2 hr. to form 6,7-dihydroxycoumarin, and combines with
iron (III) to give tan colored to black compound which diffuses into the
medium.
Apparatus/Instruments a) Protective cabinet with vertical laminar airflow
b) Membrane Filtration Assembly
c) Incubators (36±1°C, 44 ± 0.5°C)
d) Thermostatically controlled water bath (capable of being maintained at
44 °C to 47 °C)
e) Autoclave
f) Sterilizing oven
g) Petri dishes (Glass or plastic of 90 – 100mm diameter or 140mm)
h) Tubes, flasks or bottles, of appropriate capacity
i) Colony-counting device (optional)
Culture Media and a) Slantez and Bartley medium
Reagents b) Bile-aesculin-azide agar
Preparation of Not Applicable
Reagents

272
Sample Preparation Not Applicable

Method of analysis a) Disinfect the surface of the bottle/pouch/cups containing sample with
70% ethanol. Thoroughly mix the sample by vigorous shaking to achieve
uniform distribution
b) Filter the sample (requisite volume) through a sterile membrane filter
(0.45µm pore size) and place the filter on Slantez and Bartley medium
plates.
c) Incubate the plates at 36±2°C for 44±4hr. After incubation observe the
plates showing red, maroon or pink colonies as presumptive Enterococci.
d) If there are presumptive colonies, confirmation can be done by
transferring the membrane with sterile forceps without inverting it on
plate of bile-aseculin-azide agar which has been preheated to 44°C.
e) Incubate at 44 ± 0.5°C for 2hr and observe the plates immediately.
f) Consider all colonies showing tan to black color as Enterococci.
Calculation with units Presence/Absence of Enterococci is given after confirmation on Bile -
of expression aesculin-azide agar and given as.
Enterococci: Present/Absent per X mL of sample.
Reference 1) IS 15186:2002 - Water Quality - Detection and Enumeration of Intestinal
Enterococci - Membrane Filtration Method
2) IS 14543:2016 - Packaged Drinking Water (Other Than Packaged
Natural Mineral Water) - Specification (Second Revision)
3) IS 15188: 2022 - Water quality — General requirements and guidance
for microbiological examinations by culture -
Approved by Scientific Panel on Methods of Sampling and Analysis

273
 Method for Detection of Enterococci (Faecal Streptococci) (Alternative
method)

Method No. FSSAI 15.028:2023 Revision No. & Date 0.0

Scope This method is applicable to Packaged Drinking water
Caution a) Sodium Azide is highly toxic and mutagenic; precautions shall be taken
to avoid contact with it, especially through the inhalation of fine dust
during the preparation of media.

b) Azide containing media should not be mixed with strong inorganic acids,
as toxic hydrogen azide (HN3) may be produced.

c) Solution containing azide can also form explosive compounds when in

contact with metal pipework e.g. from sinks.

Principle Ethyl violet azide dextrose Agar contain sodium azide and ethyl violet that
inhibit gram-positive bacilli and gram-positive cocci other than Enterococci.
Confirmation is done by Gram stain reaction and colony chracterstics on
MacConkey Agar at 44°C.

Apparatus/Instruments a) Protective cabinet with vertical laminar airflow

b) Membrane Filtration Assembly
c) Incubators (36±1°C, 44 ± 0.5°C)
d) Thermostatically controlled water bath (capable of being maintained at
44 °C to 47 °C)
e) Autoclave
f) Sterilizing oven
g) Petri dishes (Glass or plastic of 90 – 100mm diameter or 140mm)
h) Tubes, flasks or bottles, of appropriate capacity
i) Colony-counting device (optional)
Culture Media and i. Ethyl Violet Azide Dextrose Agar
Reagents ii. MacConkey Agar
iii. Nutrient Agar
Preparation of Not Applicable
Reagents

274
Sample Preparation Not Applicable

Method of analysis a) Disinfect the surface of the bottle/pouch/cups containing sample with
70% ethanol. Thoroughly mix the sample by vigorous shaking to
achieve uniform distribution

b) Filter the sample (requiste volume) through a sterile membrane filter

(0.45µm pore size) and place the filter on Ethyl Violet Azide Dextrose
Agar plates.

c) Incubate the plates at 37±1°C for 48hr. After incubation observe the
plates showing dark red colonies or colonies having red or pink centres
as presumptive Enteroccci.

d) If there are presumptive colonies, confirmation can be done by Gram

stain reaction and colony chracterstics on MacConkey Agar at 44°C.

e) On MacConkey agar Enterococci appear as small pink colonies. Streak

suspected or typical colonies on nutrient agar prior to Gram staining.

f) Enterococci usually appear as Gram positive cocci in pairs or short

chains.

Calculation with units Presence/Absence of Enterococci is given after confirmation as.

of expression Enterococci: Present/Absent per X mL of sample.
Reference 1) IS 5887 (Part 2): 1976 - Methods for detection of bacteria responsible for
food poisoning: Part 2 Isolation, identification and enumeration of
staphylococcus aureus and faecal streptococci (First Revision)
2) IS 14543:2016 - Packaged Drinking Water (Other Than Packaged
Natural Mineral Water) - Specification (Second Revision)
3) IS 15188: 2022 - Water quality — General requirements and guidance
for microbiological examinations by culture
Approved by Scientific Panel on Methods of Sampling and Analysis

275
 Method for Detection of Salmonella

Method No. FSSAI 15.029:2023 Revision No. & Date 0.0

Scope This method is applicable Packaged Drinking water
Caution In order to safeguard the health of laboratory personnel, it is essential that
tests for detecting Salmonella spp. are only undertaken in properly
equipped laboratories, under the control of a skilled microbiologist and
that great care is taken in the disposal of all incubated materials. It is also
the responsibility of the user to establish appropriate safety and health
practices in the laboratory
Principle Detection of Salmonella is based on pre-enrichment, selective enrichment
followed by isolation on selective media. Presumptive Salmonella
colonies are confirmed by biochemical and serological tests. Pre-
enrichment broth is necessary to enable injured cells to grow. Further
selective enrichment is done to increase the proportion of Salmonella in
relation to background flora. Selective media are used for further isolation
and preliminary confirmation.
Apparatus/Instruments a) Protective cabinet with vertical laminar airflow
b) Incubators (36±2°C, 41.5±1.0°C)
c) Thermostatically controlled water bath (capable of being maintained
at 44 °C to 47 °C)
d) Membrane filtration unit (Filter size 0.45 µ)
e) Autoclave
f) Sterilizing oven
g) Petri dishes (Glass or plastic of 90 – 100mm diameter or 140mm)
h) Total delivery graduated pipettes, of nominal capacity 1 mL,
graduated in 0.1 mL divisions (Class A), or Micropipette with sterile
tips
i) Mechanical stirrer
j) Tubes, flasks or bottles, of appropriate capacity
Culture Media and a) Buffered peptone water
Reagents b) Rappaport-Vassiliadis (RVS) broth

276
 c) Brilliant green/phenol red lactose agar (BGA) or any additional
selective Medium
d) Xylose lysine deoxycholate agar (XLD)
e) Nutrient agar
f) MkTTn Broth

Preparation of Reagents As per manufacturer instructions

Sample Preparation Not Applicable

Method of analysis a) Pre-enrichment: Aseptically clean the surface of bottle/pouch/cups

containing sample with 70% ethanol and filter 250 mL (or as
specified) of water sample through a membrane filter of 0.45µm pore
size using sterile membrane filtration assembly. Place the filter disk in
50 mL buffered peptone water and incubate at 36±2°C for 16-20 hrs.
b) Selective enrichment: Transfer 0.1mL of the pre-enrichment culture to
10mL (RVS broth) and incubate at 41.5±1.0°C for 24±3h. Transfer 1
mL of the pre-enrichment culture to 10mL MkTTn Broth and
incubate at 36±2.0°C for 24±3h. Confirmation on Selective agar
media: After incubation streak a loopful from RVS broth on selective
medium i.e. BGA and XLD. Incubate the plates at 36±2°C for 24±3
hr.
c) In order to detect slow growing Salmonella reinoculate BGA, XLD
and BSA (optional) after continued incubation of RVS broth for
further 24 hr.
d) Observation: Colonies on BGA are red or slightly pink-white and
opaque with red surrounding. Colonies on XLD agar are colorless but
appear red usually with a black centre. Salmonella H2S negative
strains appear on XLD agar as pink with a darker pink center. Lactose
positive Salmonella strains grow on XLD agar as yellow with or
without blackening
e) Confirmation: If there are typical/suspected colonies, plate at least 1
selected colony from each positive agar medium and further four
colonies if the first colony tests negative on nutrient agar plates and

277
 incubate at 36±2°C for 18-24 hr. and thereafter proceed for further
biochemical & serological confirmation.
f) Biochemical confirmation:
i. Lactose/Glucose fermentation & Hydrogen Sulphide
formation: Streak a colony on Iron/two-sugar agar (Kligler Iron
agar slant) and stab the butt. Incubate at 36±2°C for 24 hr. Typical
Salmonella show red slant with gas formation and yellow butts
with blackening of agar.
ii. Urea degradation: Incubate a colony on slant of urea agar and
incubate at 36±2°C for 24hr. Typical Salmonella culture show a
negative reaction i.e. no rose pink color followed by deep cerise.
iii. Lysine decarboxylase medium: Inoculate a colony just below
the surface of the liquid Lysine decarboxylase medium. Overlay
the medium with sterile liquid paraffin or oil. Incubate at 36±2°C
for 24hr. Typical Salmonella show a purple color.
Table1 Biochemical characterization of Salmonella
Sr. Biochemical test Reaction Observation
No.
1. Lactose - Red slant with gas
2. Glucose + formation and yellow
butts with blackening of
3. Hydrogen sulfide +
the agar.
No rose-pink color
4. Urea -
followed by deep cerise.
Development of purple
5. Lysine decarboxylase +
color.
Note: (+) means positive reaction, (-) means negative reaction
g) Serological confirmation: Carry out slide agglutination test to check
the presence of Salmonella O-, Vi- and H-antigens with the
appropriate antisera from pure colonies on Nutrient agar as per
manufacturer’s instructions after elimination of auto-agglutinable
strain.

278
Calculation with units of Presence/Absence of Coliform given in per unit of the sample examined.
expression
Test for Salmonella= Present/absent per X mL of sample

1) IS 15187:2016 - Water Quality - Detection of Salmonella Species

Reference
(First Revision)
2) IS 14543:2016 - Packaged Drinking Water (Other Than Packaged
Natural Mineral Water) - Specification (Second Revision)
3) IS 15188: 2022 - Water quality — General requirements and
guidance for microbiological examinations by culture

Approved by Scientific Panel on Methods of Sampling and Analysis

279
 Method for Detection of Shigella

Method No. FSSAI 15.030:2023 Revision No. & Date 0.0

Scope This method is applicable to Packaged Drinking water
Caution In order to safeguard the health of laboratory personnel, it is essential that
tests for detecting Shigella spp. are only undertaken in properly equipped
laboratories, under the control of a skilled microbiologist, and that great
care is taken in the disposal of all incubated materials. It is also the
responsibility of the user to establish appropriate safety and health
practices.

Principle Detection of Shigella is based on pre enrichment, selective enrichment

followed by isolation on selective media. Presumptive Shigella colonies
are identified by biochemical and serological test. Pre enrichment broth
enables injured cells to grow. A selective enrichment is necessary to
increase the proportion of Shigella sp. in relation to background flora.
Apparatus/Instruments a) Protective cabinet with vertical laminar airflow
b) Incubators (36±2°C)
c) Thermostatically controlled water bath (capable of being maintained
at 44 °C to 47 °C)
d) Membrane filtration unit (Filter size 0.45 µ)
e) Autoclave
f) Sterilizing oven
g) Petri dishes (Glass or plastic of 90 – 100mm diameter or 140mm)
h) Total delivery graduated pipettes, of nominal capacity 1 mL,
graduated in 0.1 mL divisions (Class A), or Micropipette with sterile
tips
i) Mechanical stirrer
j) Tubes, flasks or bottles, of appropriate capacity
Culture Media and a) Nutrient broth
Reagents b) Kauffmann-Muller’s Tetrathionate broth
c) Selenite F broth
d) Deoxycholate citrate agar

280
 e) MacConkey agar
f) Nutrient agar No. 2
Preparation of Reagents Commercially available reagents/Disc

Sample Preparation Not Applicable

Method of analysis a) Pre-enrichment: Aseptically clean the surface of bottle/pouch/cups

containing sample with 70% ethanol and filter 250mL (or as specified)
of water sample through a membrane filter of 0.45µm pore size using
sterile membrane filtration assembly and place the filter in 50mL of
Nutrient broth. Incubate at 37°C for 18-24 hr.
b) Selective enrichment: Transfer 1mL from pre-enriched nutrient broth
to each of 100mL Selenite-F broth and tetrathionate broth. Incubate at
37°C for 24hr.
c) After incubation streak out a loopful from Selenite F broth and
tetrathionate broth deoxycholate citrate agar (DCA). Incubate the
plates at 37°C for 24 hr. If there is no typical/suspected growth on
DCA plate, reincubate further for 24 hr.
d) Observations: Shigella colonies on deoxycholate citrate agar (DCA)
appear opaque with a ground-glass appearance and with even margins.
e) Confirmation: If there are typical/suspected colonies, pick at least 5
colonies from each plate and streak on MacConkey agar Incubate the
plate(s) overnight at 37°C to isolate pure colonies. Pure colonies are
transferred on to Nutrient agar No. 2 prior to biochemical and
serological confirmation.
f) Biochemical Confirmation:
i. Gram staining: Typical colonies of Shigella are gram negative
rods.
ii. Motility: Nutrient agar tubes are inoculated by stabbing with a
straight wire to a depth of 5 mm and incubated at 37°C for 18-24 hr
to check the motility. Out growth in whole tube indicates motility.
If no motility is observed within 24 hr. the tubes are incubated at
room temperature for further 4-6 days to check the Motility.

281
 Shigella is non-motile.
iii. Catalase Test: Streak a colony on nutrient agar slant and incubate
at 37°C for 24 hr. Add 1 mL of hydrogen peroxide over the growth.
Shigella is Catalase positive and show release of oxygen, as bubbles
of hydrogen peroxide indicating the presence of catalase.
iv. Oxidase Test: Streak a colony on nutrient agar slant and incubate
at 37 °C for 24 hr. To nutrient agar slant containing the culture, add
a few drops of mixed test reagents (1% α-nepthol in 95% ethanol
and equal amount of para-amino diamethylaniline hydrochloride in
water). Shigella being an Oxidase negative does not develop blue
color within two minutes of addition of reagent.
v. Hugh-Leifson’s test: Stab a fresh culture from nutrient agar slant
into two tubes of Hugh Leifson medium. One tube is over layered
with a small amount of sterile paraffin liquid to create anaerobic
conditions, whereas, other is incubated as such. Both are incubated
at 37°C and examined daily up to 4 days. In case of Shigella Acid
formation in both the tubes indicates fermentative reaction.
vi. Test for H2S Production: Inoculate Triple sugar iron agar (TSI) by
stabbing the butt and streaking the slope. Incubate at 37°C and
examine daily up to 7 days. In case of H2S production blackening of
butt is observed.
vii. Test for Urease: Inoculate culture in Nutrient broth and incubate
for 37°C for 24hr. Add inoculated culture from Nutrient broth to
urea agar slant and incubate at 37°C for 18-24 hr. A positive urease
is shown by the medium becoming pink or red on incubation. If no
color formed, continue incubation for 4 days and record for color
development.
viii. Phenyl pyruvic acid production: Suspend overnight grown
culture in 0.5 mL of normal saline and transfer to a test tube (dia
1.5 cm). Add 0.5 mL of 0.2% di-phenylalanine and mix. Keep it in
horizontal position for at least 3 hr. at room temperature. Add few
drops of half saturated ferric chloride solution. A positive reaction
is indicated by the formation of immediate deep color which fades
on keeping.

282
 ix. Test for Citrate utilization: Inoculate the culture on Simmons
citrate agar slant using a straight wire. Incubate at 37°C and
examine daily up to 4 days. No change in color of media indicates
negative reaction.
x. Test for Indole: Inoculate Peptone water medium with a loopful of
24hr grown culture in Nutrient broth and incubate at 37°C for 48 hr.
Add 0.5mL of KOVAC’S reagent and shake the tube gently.
Observe for the appearance of red color which indicates the
presence of Indole.
xi. Test for Fermentation of carbohydrates: Inoculate each of
Andrade peptone water medium tubes for carbohydrates i.e.
Glucose, Lactose, Sucrose, Salicin, Dulcitiol, Mannitol (@ 1%
concentration) with freshly grown culture from Nutrient broth/agar
and incubate at 37°C for 18-48 hr. Record the presence of acid from
pink color and that of gas in Durham tube.
xii. Test for Dihydrolase & decarboxylase activity: Inoculate each of
the tubes of Dihydrolase and Decarboxylase medium through liquid
paraffin with freshly grown culture from Nutrient agar. Incubate at
37ºC and examine upto 4 days. Medium first become yellow due to
acid production from the glucose. Later if dehydrolation or
decarboxylation of respective amino acid occur, the medium
become violet in color.
xiii. Test for utilization of Malonate: Inoculcate the medium with
freshly grown culture from Nutrient broth/agar for Malonate test
and incubate at 37°C for 24hr. Positive Malonate function is
indicated by deep blue color and negative reaction by unchanged
greenish or yellow color of medium.
xiv. Gelatin liquefaction test: Inoculate the culture into gelatin
liquefaction test medium and as a stab culture. Incubate at 22°C for
4 days in an upright position. Before taking reading keep the tube
at refrigerated temperature to check liquefaction of the media.

283
Table 1 Biochemical characteristics of Shigella sp
S. Tests Positive Test Sh. Sh. Sh.
No. observations dysenter Flexneri, sonnei
iae Sh. boydii
1. Gram Gram negative, Gram Gram Gram
reaction Rods negative negative, negative,
, rods rods rods
2. Motility Growth present - - -
outside of
inner glass
tube
3. Catalase Oxygen + + +
released as
bubble
4. Oxidase Blue color - - -
within 2 min
5. Hugh- Fermentative F F F
Leifson’s (Acid &
test yellow color
formation in
both tube)
6. TSI for H2S Blackening of - - -
Butt
7. Urease Medium color - - -
change to pink
or red
8. Phenyl Deep green - - -
pyruvic acid color formed
9. Citrate Medium color - - -
change to Blue
10. Glucose Acid formation + + +
fermentation (Pink color),
gas +

284
 11. Lactose(1%) - - (++)
fermentation
12. Sucrose - - (++)
fermentation
13. Salicin - - -
fermentation
14. Mannitol - + +
fermentation
15. Lysine First yellow - - -
color(Acid
formation)
then purple
color due to
decarboxylatio
n of amino
acid
16. Ornithine - -/+ +
17. Malonate Medium color - - -
change to deep
Blue
18. Gelatin Gelatin is - - -
liquefied
Note: (+) means positive reaction, (-) means negative reaction, (++)
means late positive, (F) means fermentative reaction
xv. Serotyping: Use slide agglutination with the appropriate antisera
from discrete single colonies on Nutrient agar as per
manufacturer’s instructions after elimination of auto-agglutinable
strain. Shigella strain is confirmed by Polyvalent Shigella sera, on
the basis of serotyping.
Calculation with units of Presence/Absence of Shigella species given after Biochemical &
expression Serological confirmation of presumptive colonies in the sample examined.
Test for Shigella = Present/absent per X mL of sample

Reference 1) IS 5887 (Part-7):1999 (Reaffirmed 2018) - Methods for detection of

285
 bacteria responsible for food poisoning: Part 7 general guidance on
methods for isolation and identification of Shigella
2) IS 14543:2016 - Packaged Drinking Water (Other Than Packaged
Natural Mineral Water) - Specification (Second Revision)
3) IS 15188: 2022 - Water quality — General requirements and guidance
for microbiological examinations by culture
Approved by Scientific Panel on Methods of Sampling and Analysis

286
 Method for Detection of Pseudomonas aeruginosa

Method No. FSSAI 15.031:2023 Revision No. & Date 0.0

Scope This method is applicable to Packaged Drinking water
Caution Carry out the test under the control of skilled microbiologist and great
care shall be taken in the disposal of all the incubated material. Follow
safe and good microbiology laboratory practices to avoid cross
contamination.

Principle Detection of Pseudomonas aeruginosa is based on selective enrichment

followed by isolation on confirmation media. Asparagine proline broth is
used as selective medium for cultivation of P. aeruginosa. Milk Agar is
used for selective isolation of Pseudomonas aeruginosa. Strains of
Pseudomonas aeruginosa are identified by their pigment i.e. pyocyanin
production. Pseudomonas aeruginosa hydrolyzes casein and produces a
yellow to green diffusible pigment on Milk agar.
Apparatus/Instruments a) Protective cabinet with vertical laminar airflow
b) Incubators (36±2°C, 42±0.5°C)
c) Thermostatically controlled water bath (capable of being maintained
at 44 °C to 47 °C)
d) Membrane filtration unit (Filter size 0.45 µ)
e) Autoclave
f) Sterilizing oven
g) Petri dishes (Glass or plastic of 90 – 100mm diameter or 140mm)
h) Total delivery graduated pipettes, of nominal capacity 1 mL,
graduated in 0.1 mL divisions (Class A), or Micropipette with sterile
tips
i) Mechanical stirrer
j) Tubes, flasks or bottles, of appropriate capacity
Culture Media and a) Pseudomonas asparagine proline broth
Reagents b) Milk agar with cetrimide
Preparation of Reagents Commercially available reagents/Disc

Sample Preparation Not Applicable

287
Method of analysis a) Aseptically clean the surface of bottle/pouch/cups containing sample
with 70% ethanol and filter 250 mL (or as specified) of water sample
through a membrane filter of 0.45µm pore size using sterile
membrane filtration assembly and place the filter in 50 mL of
concentrated Asparagine proline broth. Incubate at 37±1°C for 48 hr.
b) After incubation examine the medium showing either growth or
fluorescence under U.V. light (360±20 nm).
c) Subculture a loopful on Milk agar plate and incubate for 24h at
42±0.5°C.
d) Observation: Observe the plates for culture growth, pigment
production and casein hydrolysis (clearing of medium around the
colonies).
Table 1 Characteristics of Pseudomonas aeruginosa on Milk
agar
S. Reaction Pseudomonas aeruginosa
No. characteristics
Typical Atypical
1. Casein hydrolysis + +
2. Growth at 42 °C + +
3. Fluorescence under UV light + -
4. Pyocyanine (Blue/green) + -
pigment production
Note: (+) means positive reaction, (-) means negative reaction
i. Culture showing either growth or fluorescence in Asparagine
proline broth, which further produce colonies on Milk agar plates
and show pigment production and casein hydrolysis are regarded
positive for presence of Pseudomonas aeruginosa.
ii. The colonies (atypical) showing casein hydrolysis, but no
florescence or pigment productions are further confirmed by
biochemical tests.
e) Confirmation of Atypical colonies: If there are Atypical/suspected
colonies, streak out at least 5 selected colonies further on Milk agar
medium and incubate at 37±1°C for 24 hr. Proceed for further

288
 biochemical confirmation.
i. Catalase Test: Streak a colony on Nutrient agar slant and allow to
grow at 37°C for 24 hr. Add 1 mL of hydrogen peroxide over the
growth in slanting position. Release of oxygen, as bubbles, from
hydrogen peroxide indicates presence of catalase.
ii. Oxidase Test: Streak a colony on Nutrient agar slant and allow to
grow at 37°C for 24 hr. Add a few drops of mixed test reagents
(1% alpha nepthol in 95% ethanol and equal amount of para-
amino diamethylaniline hydrochloride in water) to Nutrient agar
slant of culture. A positive reaction is indicated by the appearance
of blue color within two minutes of addition of reagent.
iii. Hugh-Leifson’s test: Stab a fresh culture from Nutrient agar slant
into two tubes of Hugh Leifson medium, one of which is then
layered over with a small amount of sterile liquid paraffin.
Incubate at 37°C and examine daily up to 4 days. Acid formation,
in both the tubes indicates fermentative reaction.
iv. Nitrate reduction: AddReagent A (Sulfanilic acid) and Reagent
B (N,N-dimethyl-1-naphthylamine) to 18hr old culture inoculated
in Nitrate broth. Formation of red color indicates nitrate reduction.
If no change in color is observed, add Zinc granules to tube, and
observe color change. If the broth turns to red, test is negative but
if no red color is developed after addition of zinc granules, then
test is positive.
v. Gelatin liquefaction test: Inoculate the strain into Gelatin
liquefaction test medium as a stab culture. Incubate at 22°C for 4
days in an upright position. Before taking reading keep the tube at
refrigerated temperature to check liquefaction of the media.
Table 2 Biochemical characteristics Pseudomonas aeruginosa
S. Tests Observation
No.
1. Catalase test +
2. Oxidase test +
3. Growth in Hugh and Liefson medium Oxidative reaction

289
 4. Nitrate reduction to ammonia +
5. Gelatine Liquefication +
Calculation with units of Presence/Absence of Pseudomonas sp. is given after Biochemical &
expression Serological confirmation of presumptive colonies in the sample
examined.
Test for Pseudomonas aeruginosa = Present/ absent per X mL of sample
Reference 1) IS 13428(Annex D):2005 - Packaged Natural Mineral Water -
Specification (Second Revision)
2) IS 14543:2016 - Packaged Drinking Water (Other Than Packaged
Natural Mineral Water) - Specification (Second Revision)
3) IS 15188: 2022 - Water quality — General requirements and guidance
for microbiological examinations by culture
Approved by Scientific Panel on Methods of Sampling and Analysis

290
 Method for detection of Yeast & Moulds

Method No. FSSAI 15.032:2023 Revision No. & Date 0.0

Scope This method is applicable to Packaged Drinking water
Caution Carry out the test under the control of skilled microbiologist and great
care shall be taken in the disposal of all the incubated material. Follow
safe and good microbiology laboratory practices to avoid cross
contamination.

Principle Chloramphenicol Yeast Glucose Agar is a selective medium

recommended for isolation and enumeration of Yeast & Moulds. The
medium contains yeast extract, which provides nitrogenous nutrient and
vitamin B complex. Dextrose is the energy source. Chloramphenicol a
thermostable antibiotic, suppresses the bacterial flora. Aerobic incubation
of plates is done at 25±1oC and count is taken on 3rd, 4th or 5th day.
Apparatus/Instruments a) Protective cabinet with vertical laminar airflow
b) Incubators (25±1oC)
c) Thermostatically controlled water bath (capable of being maintained
at 44 °C to 47 °C)
d) Autoclave
e) Sterilizing oven
f) Petri dishes (Glass or plastic of 90-100mm diameter or 140mm)
g) Total delivery graduated pipettes, of nominal capacity 1 mL,
graduated in 0.1 mL divisions (Class A), or Micropipette with sterile
tips
h) Mechanical stirrer
i) Tubes, flasks or bottles, of appropriate capacity
j) Colony-counting device (optional)
k) Sterile Glass or plastic spreaders
l) Spiral plater
Culture Media and Chloroamphenicol Glucose Yeast Extract Agar (CGYEA)
Reagents

291
Preparation of Reagents Not Applicable

Sample Preparation Not Applicable

Method of analysis a) Aseptically clean the surface of bottle/pouch/cups containing sample

with 70% ethanol and filter 250 mL (or as specified) of water sample
through a membrane filter of 0.45µm pore size using sterile
membrane filtration assembly.
b) Place the filter on CGYEA media and incubate at 25±1°C
c) Observation: Observe the plates for colonies of Yeast & Molds on
3rd, 4th and 5th days of incubation.
d) It is advisable to examine the plates at the end of three days for yeast
colonies, which are smooth, moist, elevated or surface colonies. Mold
colonies are easily recognized by the profuse growth of hyphae.
Calculation with units of Presence/ Absence of yeast & molds given after examining the plates of
expression sample.
Test for Yeast and molds = Present/absent per X mL of sample
Reference 1) IS 5403:1999 (Reaffirmed 2018) - Method for yeast and mould count
of foodstuffs and animal feeds (First Revision)
2) IS 14543:2016 - Packaged Drinking Water (Other Than Packaged
Natural Mineral Water) - Specification (Second Revision)
3) IS 15188: 2022 - Water quality — General requirements and guidance
for microbiological examinations by culture
Approved by Scientific Panel on Methods of Sampling and Analysis

292
 Method for Detection of Sulphite-Reducing anaerobes (Clostridia)

Method No. FSSAI 15.033:2023 Revision No. & Date 0.0

Scope This method is applicable to Packaged Drinking water
Caution Carry out the test under the control of skilled microbiologist and great
care shall be taken in the disposal of all the incubated material. Follow
safe and good microbiology laboratory practices to avoid cross
contamination.

Principle Detection of sulphite reducing anaerobes by inoculating 50 mL of sample

into equal volume of double strength of Differential Reinforced
Clostridial Medium (DRCM), followed by anaerobic incubation at
37±1°C for 44±4 hr. As a result of reduction of sulphite and the
precipitation of iron (II) sulphide in medium developing, Black color in
medium, sample is considered as positive.
Apparatus/Instruments a) Protective cabinet with vertical laminar airflow
b) Incubators (37±1°C, 75±5°C)
c) Thermostatically controlled water bath (capable of being maintained
at 44 °C to 47 °C)
d) Autoclave
e) Sterilizing oven
f) Petri dishes (Glass or plastic of 90-100mm diameter or 140mm)
g) Total delivery graduated pipettes, of nominal capacity 1 mL,
graduated in 0.1 mL divisions (Class A), or Micropipette with sterile
tips
h) Mechanical stirrer
i) Tubes, flasks or bottles, of appropriate capacity
Culture Media and Differential Reinforced Clostridial Medium (DRCM)
Reagents
Preparation of Reagents Not Applicable

Sample Preparation Not Applicable

293
Method of analysis a) Aseptically clean the surface of the bottle (1lt. /5lt. /20lt.) or water
pouch/cups containing sample with 70% ethanol. Aseptically
withdraw 100 mL sample to sterile bottle and heat at 75±5°C for 15
min.
b) Add 50 mL of sample after heat shock to 100 mL bottle containing 50
mL of the double strength DRCM. Cap the bottles tightly and incubate
under anaerobic conditions at 37±1°C for 44±4hr. Iron wire, heated to
redness can be placed in the medium before inoculation to enhance
anaerobic conditions.
Calculation with units of Test for Sulphite-Reducing anaerobes (Clostridia): = Present/ absent per
expression X mL of sample
Reference 1) IS 13428(Annex C):2005 - Packaged Natural Mineral Water -
Specification (Second Revision)

2) IS 14543:2016 - Packaged Drinking Water (Other Than Packaged

Natural Mineral Water) - Specification (Second Revision)
Approved by Scientific Panel on Methods of Sampling and Analysis

294
 Method for Detection of Vibrio parahaemolyticus

Method No. FSSAI 15.034:2023 Revision No. & Date 0.0

Scope This method is applicable to Packaged Drinking water
Caution In order to safeguard the health of laboratory personnel, it is essential that
tests for detection of Vibrio spp., and particularly toxigenic Vibrio cholerae,
Vibrio parahaemolyticus be conducted only in laboratories equipped for
this purpose and under the supervision of an experienced microbiologist,
and that great care is exercised in the disposal of contaminated material.

Principle Glucose salt teepol broth is used to enrich Vibrio parahaemolyticus.

Glucose is utilized by the organism while teepol inhibits the migration of
halophilic organisms and the growth of the gram-positive organisms. After
overnight incubation at 35±2°C, inoculation on TCBS agar plates gives
round green or bluish colonies.
Apparatus/Instruments a) Protective cabinet with vertical laminar airflow
b) Incubators (36±2°C)
c) Thermostatically controlled water bath (capable of being maintained
at 44 °C to 47 °C)
d) Sterile membrane filter (0.45µm pore size)
e) Autoclave
f) Sterilizing oven
g) Petri dishes (Glass or plastic of 90-100mm diameter or 140mm)
h) Total delivery graduated pipettes, of nominal capacity 1 mL,
graduated in 0.1 mL divisions (Class A), or Micropipette with sterile
tips
i) Mechanical stirrer
j) Tubes, flasks or bottles, of appropriate capacity
Culture Media and Glucose-salt-teepol broth
Reagents Thiosulphate-Citrate-Bile salts-Sucrose (TCBS)
Preparation of Reagents Commercially available reagents/Disc
Sample Preparation Not Applicable
Method of analysis a) Disinfect the surface of the bottle/pouch/cups containing sample with

295
 70% ethanol. Thoroughly mix the sample by vigorous shaking to
achieve uniform distribution
b) Filter the sample (required volume) through a sterile membrane filter
(0.45µm pore size) and place the filter in 50 mL of Glucose-salt-Teepol
broth. Incubate overnight at 37°C.
c) After incubation streak a loopful from pre-enrichment culture on TCBS
prepared plates and incubate for 18hr at 37°C.
d) Suspicious colonies of V. parahaemolyticus on TCBS medium are 2-3
mm and round with green or blue centers.
e) Identification: Suspicious growth of V. parahaemolyticus is confirmed
through biochemical confirmation.
f) Biochemical tests: If there are typical/suspected colonies, plate out at
least 5 selected colonies from each positive agar medium on nutrient
agar plates and incubate at 36±2°C for 18-24 hr. Proceed for further
biochemical & serological confirmation
i. Gram Staining: Gram stain the isolated colony. Typical Vibrio
parahaemolyticus are Gram negative rods.
ii. Test for Oxidase: Streak a colony on Nutrient agar slant (with 2-3%
added NaCl) and allow to grow for 24 hr. To Nutrient agar slant of
culture, add a few drops of mixed test reagents (1% alpha nepthol in
95% ethanol and equal amount of para-amino diamethylaniline
hydrochloride in water). A positive reaction is indicated by the
appearance of blue color within two minutes of addition of reagent.
iii. Hugh-Leifson’s test: Stab a fresh culture from Nutrient agar slant into
two tubes of Hugh Leifson medium (with 2-3% added NaCl), one of
which is then over layered with a small amount of sterile liquid
paraffin. Incubate at 37°C and examine daily up to 4 days. Acid
formation, shown by yellow color in the tube without paraffin indicates
oxidative utilization of glucose. Acid in both tubes indicates
fermentative reaction.
iv. Test for Fermentation of carbohydrates: Inoculate each of Andrade
Peptone water medium tubes for carbohydrates i.e. Sucrose, Mannitol
(@ 1% concentration) and incubate at 37°C for 18-48 hr. observe color
change for carbohydrate fermentation

296
 v. TSI for H2S production: Inoculate Triple Sugar Iron Agar (TSI)
medium (with 2-3% added NaCl) by stabbing into the butt and
streaking the slope. Incubate at 37°C and examine daily up to 7 days.
In case of H2S production blackening of butt is observed.
vi. Test for Dihydrolase & Decarboxylase activity: Inoculate each of
the tubes of Dihydrolase & Decarboxylase medium (with 2-3% added
NaCl) through liquid paraffin from freshly grown culture on Nutrient
agar medium. Incubate at 37°C and examine up to 4 days. Medium
first turns yellow due to acid production from glucose and later if
dehydration or decarboxylation of respective amino acids occur, the
medium changes to violet in color.
vii. Test for Voges-Proskauer reaction: Inoculate the medium with
added NaCl (2-3%) and incubate for 48hr at 37°C. To 1 mL of growth,
add 0.6mL of 5% alpha nephthol. Shake and add 0.2 mL of KOH
solution 40%. Shake and slope the tube for upto 4 hr. for color change.
Pink color indicate positive reaction.
viii. Test for the growth in Tryptone broth: Inoculate the culture in
Tryptone broth with different concentration (0%, 1%, 8% & 10%) of
Sodium chloride.
ix. Growth in Tryptone broth with added NaCl incubated at 42°C for 24 hr
shows positive growth. Growth in 1% Tryptone broth, with added 8%
NaCl and incubated at 37°C is positive. Growth in 1% Tryptone broth,
with added 10% NaCl and incubated at 37°C is negative. Growth in
1% Tryptone broth, without NaCl and incubated at 37°C is negative
x. Kanagawa test: Grow the culture in Trypticase-Soy-Sodium Chloride
broth by incubating for 18hr at 37°C. Further streak on blood agar and
incubate at 37°C for not more than 24hr. A positive result in case of V.
parahaemolyticus consists of a zone of transparent clearing of the red
blood cells around the colony. It is important that the reading is not
taken beyond 24hr of incubation as any haemolysis seen beyond this
time is not to be recorded as Kanagawa positive.
Table 1Biochemical characteristics of Vibrio parahaemolyticus
S. Tests Reaction

297
 No.
1. Gram Reaction Gram Negative , Rods
2. Oxidase test +
3. Hugh-Leifson’s test F
4. H2S production -
5. Glucose fermentation + (without gas)
6. Mannitol fermentation +
7. Sucrose fermentation -
8. Voges –Proskauer test -
9. Lysine decarboxylase +
10 Arginine dihydrolase -
11 Ornithine decarboxylase +
12 Growth in 1% tryptone brothwith +
added NaCl at 420C
13 Growth in 1% tryptone broth + 8% +
NaCl
14 Growth in 1% tryptone broth + 10% -
NaCl
15 Growth in 1% tryptone broth without -
naCl
Note: (+) means positive reaction, (-) means negative reaction, (F) means
fermentative reaction
Calculation with units of Presence/Absence of V. parahaemolyticus given after confirmation of
expression presumptive colonies in the sample examined.
Test for V. parahaemolyticus = Present/absent per X mL of sample.
Reference 1) IS 5887 (Part-5) -1976 (Reaffirmed 2018) - Methods for detection of
bacteria responsible for food poisoning, Part 5: Isolation, identification
and enumeration of Vibrio cholerae and Vibrio parahaemolyticus
2) IS 14543:2016 - Packaged Drinking Water (Other Than Packaged
Natural Mineral Water) - Specification (Second Revision)
3) IS 15188: 2022 - Water quality — General requirements and guidance
for microbiological examinations by culture
Approved by Scientific Panel on Methods of Sampling and Analysis

298
 Method for Detection of Vibrio cholerae

Method No. FSSAI 15.035:2023 Revision No. & Date 0.0

Scope This method is applicable to Packaged Drinking water
Caution In order to safeguard the health of laboratory personnel, it is essential that
tests for detection of Vibrio spp., and particularly toxigenic Vibrio
cholerae, Vibrio parahaemolyticus be conducted only in laboratories
equipped for this purpose and under the supervision of an experienced
microbiologist, and that great care is exercised in the disposal of
contaminated material.
Principle Bile Salt agar and Thiosulphate Citrate Bile salt sucrose agar (TCBS) is
used selectively for the isolation, identification and enumeration of Vibrio
cholerae. Vibrio species grow in the presence of relatively high levels of
bile salts which inhibit the growth of gram-positive microorganisms. TCBS
has a very high pH (8.5-9.5) which suppresses growth of intestinal flora
other than Vibrio sp. V. cholerae ferment sucrose, which results in a pH
shift and production of yellow colonies.
Apparatus/Instruments a) Protective cabinet with vertical laminar airflow
b) Incubators (36±2°C)
c) Thermostatically controlled water bath (capable of being maintained at
44 °C to 47 °C)
d) Sterile membrane filter (0.45µm pore size)
e) Autoclave
f) Sterilizing oven
g) Petri dishes (Glass or plastic of 90-100mm diameter or 140mm)
h) Total delivery graduated pipettes, of nominal capacity 1 mL, graduated
in 0.1 mL divisions (Class A), or Micropipette with sterile tips
i) Mechanical stirrer
j) Tubes, flasks or bottles, of appropriate capacity
Culture Media and a) Alkaline peptone water
Reagents b) Thiosulphate-Citrate-Bile salts-Sucrose agar (TCBS)
c) Bile salt agar (BSA)

299
 d) Nutrient agar No. 2

Preparation of Reagents Commercially available reagents/Disc

Sample Preparation Not Applicable
Method of analysis a) Disinfect the surface of the bottle/pouch/cups containing sample with
70% ethanol. Thoroughly mix the sample by vigorous shaking to
achieve uniform distribution.
b) Filter the sample (requisite volume) through a sterile membrane filter
(0.45µm pore size) and place the filter in 50 mL of alkaline peptone
water. Incubate at 37°C overnight.
c) After incubation streak a loopful on TCBS and BSA medium plates and
incubation at 37°C overnight.
d) Observation: Suspicious colonies of V. cholerae on TCBS appear as
opaque yellow colored with entire round margins.
V. cholerae colonies show a distinctive appearance on BSA medium.
e) Biochemical Confirmation: If there are typical/suspected colonies,
plate out at least 5 selected colonies from each positive agar medium on
nutrient agar No. 2 plates and incubate at 36±2°C for 18-24 hr. Proceed
for further biochemical & serological confirmation
i. Gram Staining: Gram stain the isolated colony. Typical Vibrio
cholerae are Gram negative curved shaped rods when observed
under microscope.
ii. Test for Motility: Nutrient agar for motility is used to check the
motility of V. cholera. Inoculate by stabbing with a straight wire
into glass tube to a depth of 5 mm and incubate at 37°C for 18-24
hr. Motile strain shall be found to show growth on the surface of
the medium, outside the inner glass tube; travelled through entire
medium. If no motility observed keep at room temperature for 4-6
days to see if any evidence of motility is present.
iii. Test for Catalase: Streak a colony on Nutrient agar slant and allow
to grow for 24 hr. at 37°C. Add 1 mL of hydrogen peroxide over
the growth in slanting position. Release of oxygen, shown as
bubbles, from hydrogen peroxide indicates presence of catalase.

300
 iv. Test for Oxidase: Streak a colony on Nutrient agar slant and allow
to grow at 37°C for 24 hr. To the slant culture, add a few drops of
mixed test reagents (1% α-napthol in 95% ethanol and equal
amount of para-amino diamethylaniline hydrochloride in water). A
positive reaction is indicated by the appearance of blue color within
two (2) minutes of addition of reagent.
v. Hugh-Leifson’s test: Stab a fresh culture from Nutrient agar slant
into two tubes of Hugh Leifson medium, one of which is then
layered over with a small amount of sterile paraffin liquid. Incubate
at 37°C and examine daily up to 4 days. Acid formation, shown by
yellow color in the tube without paraffin indicates oxidative
utilization of glucose. Acid in both tubes indicates fermentative
reaction.
vi. Test for Fermentation of carbohydrates: Inoculate each of
Andrade Peptone water medium tubes for carbohydrates i.e.
Glucose, inositol, Mannitol (@ 1% concentration) and incubate at
37°C for 18-48 hr. observe color change for carbohydrate
fermentation. Ferment glucose without gas production and in case
of mannitol ferment with acid production
vii. TSI for H2S production: Inoculate Triple Sugar Iron agar (TSI)
medium by stabbing into the butt and streaking the slope. Incubate
at 37°C and examine daily upto 7 days. In case of H2S production
blackning of butt is observed.
viii. Test for the growth in 1% Tryptone Broth: Inoculate the culture
in Tryptone broth (without sodium chloride) and incubate at 37°C
for 18hr. V. cholerae grows in 1% tryptone broth.
ix. Test for Dihydrolase & Decarboxylase activity: Inoculate each of
the tubes of Dihydrolase & Decarboxylase medium through liquid
paraffin from freshly grown culture from Nutrient agar. Incubate at
37ºC and examine up to 4 days. Medium first become yellow due to
acid production from the glucose. Later if dehydration or
decarboxylation of respective amino acid occur, the medium
become violet in color.

301
 Table 1 Biochemical characteristics of Vibrio cholera
S. Tests Reaction
No.
1. Gram Reaction Gram Negative , Rods
2. Motility test +
3. Catalase test +
4. Oxidase test +
5. Hugh-Leifson’s test F
6. H2S production -
7. Glucose fermentation + (without gas)
8. Mannitol fermentation +
9. Inositol fermentation -
10. Growth in 1% tryptone broth +
11. Lysine decarboxylase +
12 Arginine dihydrolase -
13 Ornithine decarboxylase +
Note: (+) means positive reaction, (-) means negative reaction, (F) means
fermentative reaction
f) Serological Confirmation: Suspicious growth of V. cholera is tested
by slide agglutination using polyvalent cholera typing serum (high titre
serum of combined Ogawa & Inaba Serotypes). The growth is
emulsified in a drop of normal saline and smooth suspensions mixed
with a drop of Cholera serum. Positive reaction is shown by the
appearance of clumps within 30 seconds.
Calculation with units of Presence/Absence of V. cholerae species given after confirmation of
expression presumptive colonies in the sample examined.
Test for V. cholerae = Present/absent per X mL of sample filtered
Reference 1) IS 5887 (Part-5): 1976 (Reaffirmed 2018) - Methods for detection of
bacteria responsible for food poisoning, Part 5: Isolation, identification
and enumeration of Vibrio cholerae and Vibrio parahaemolyticus
2) IS 14543: 2016 - Packaged Drinking Water (Other Than Packaged
Natural Mineral Water) - Specification (Second Revision)
3) IS 15188: 2022 - Water quality — General requirements and guidance

302
 for microbiological examinations by culture
Approved by Scientific Panel on Methods of Sampling and Analysis

303
 Method for Detection of Staphylococcus aureus

Method No. FSSAI 15.036:2023 Revision No. & Date 0.0

Scope This method is applicable to Packaged Drinking water
Caution Carry out the test under the control of skilled microbiologist and great care
shall be taken in the disposal of all the incubated material. Follow safe and
good microbiology laboratory practices to avoid cross contamination.

Principle Baird Parker agar is used for the isolation and differentiation of coagulase-
positive Staphylococci. Staphylococci can reduce tellurite to telluride,
which results in grey to black coloration of the colonies. With the addition
of egg yolk, the medium becomes yellow, slightly opaque. A clear halo
develops around colonies from coagulase positive Staphylococcusaureus.
Grey-black colonies and a halo on this medium are presumed to be
indicative of coagulase positive staphylococci.
Apparatus/Instruments a) Protective cabinet with vertical laminar airflow
b) Incubators (36 ±2°C)
c) Thermostatically controlled water bath (capable of being maintained at
44 °C to 47 °C)
d) Sterile membrane filter (0.45µm pore size)
e) Autoclave
f) Sterilizing oven
g) Petri dishes (Glass or plastic of 90-100mm diameter or 140mm)
h) Total delivery graduated pipettes, of nominal capacity 1 mL, graduated
in 0.1 mL divisions (Class A), or Micropipette with sterile tips
i) Mechanical stirrer
j) Tubes, flasks or bottles, of appropriate capacity
Culture Media and a) Cooked Salt Meat medium
Reagents b) Baired-Parker agar
c) Blood agar
Preparation of Reagents Commercially available reagents

304
Sample Preparation Not Applicable

Method of analysis a) Disinfect the surface of the bottle/pouch/cups containing sample with
70% ethanol. Thoroughly mix the sample by vigorous shaking to
achieve uniform distribution
b) Filter the sample (requisite volume) through a sterile membrane filter
(0.45µm pore size) and place the filter in 50 mL of Cooked Salt Meat
medium. Incubate overnight at 37°C.
c) After incubation streak a loopful from Cooked Salt Meat medium on
Baired-Parker agar for at least 30 hr. at 37°C and overnight on Blood
agar at 37°C.
d) S. aureus show shiny black colonies with or without grey-white margins
on Baired-Parker agar
e) Usually golden yellow colonies on Blood agar.
f) Identification:
i. Gram Staining: If there are typical/suspected colonies, pick up the
colonies and confirm by gram staining. S. aureus are Gram positive
cocci which appear in clusters.
ii. Coagulase test: The test may be performed using one of following
method.
1. Slide method: Emulsify a portion of suspected colony in normal
saline water on a clean slide. Mix it with a straight wire dipped in
rabbit plasma. Coagulase positive staphylococci produce visible
clumping immediately. Positive control with a known coagulase
positive strain of S. aureus and a control of rabbit plasma without
inoculum should be included in the test.
2. Tube method: Emulsify a portion of suspected colony from 24 hr
growth on blood agar in 1 mL citrated rabbit plasma,diluted 1 in 5,
0.85% saline. Incubate at 37°C, preferably in a water bath. Observe
every hour to observe clotting of plasma. Positive control with a
known coagulase positive strain S. aureus and a control of rabbit
plasma without inoculum should be included in the test. Tube method
shall be preferred

305
Calculation with units of Presence/Absence of S. aureus given after confirmation of colonies in the
expression sample examined.
Test for S. aureus= Present/absent per X mL sample
Reference 1) IS 5887 (Part-II): 1976 (Reaffirmed 2018) - Methods for Detection of
bacteria responsible for Food Poisoning- Part II Isolation, Identification
and Enumeration of Staphylococcusaureus and faecal Streptococci
(First Revision)
2) IS 14543: 2016 - Packaged Drinking Water (other than packaged
natural mineral water)- Specification (first revision)
3) IS 15188: 2022 - Water quality — General requirements and guidance
for microbiological examinations by culture
Approved by Scientific Panel on Methods of Sampling and Analysis

306
 Method for Detection of Viruses

Method No. FSSAI 15.037:2023 Revision No. & Date 0.0

Scope This method is applicable to Packaged Drinking water
Caution Carry out the test under the control of skilled microbiologist and great care
shall be taken in the disposal of all the incubated material. Follow safe and
good microbiology laboratory practices to avoid cross contamination.
Principle MS2 Phages are indicator of viral contamination in drinking water.
Detection procedure is based on RNA extraction followed by c-DNA
synthesis, which is identified after PCR amplification.
Apparatus/Instruments 1.1 CONCENTRATION OF DRINKING WATER
Apparatus
Pressure Pump
Membrane Filter Assembly with 144mm Diameter with Tripod Stand
Pressure Vessel (50 litre capacity) with Pressure Gauge
Inter-connecting Pressure Tubes
RNA EXTRACTION
Apparatus
Cooling Centrifuge
Deep Freezer (-20°C)
Vortex Mixer
Pipette Man
COMPLEMENTARY DNA (cDNA) SYNTHESIS
Apparatus
PCR Machine
Deep Freezer (-20°C)
PCR AMPLIFICATION
PCR Machine
Deep Freezer (-20°C)
Micropippette
AGROSE GEL ELECTROPHORESIS

307
 Micropippette
Electrophoresis Apparatus
Gel Documentation System
Culture Media and CONCENTRATION OF DRINKING WATER
Reagents a) Autoclaved double distilled water
b) Aluminum Chloride
c) HCl/NaOH Urea (Extra Pure)
d) Disodium Hydrogen Phosphate (Na2HPO4.2H2O) - 0.2M filter
sterilized.
e) Sodium Dihydrogen Phosphate (NaH2PO4.2H2o) - 0.2M filter
sterilized.
f) Citric Acid - 0.1M filter sterilized.
g) L-Arginine - 0.5M filter Sterilized.
h) Urea-Arginine Phosphate Buffer (U-APB)
i) Magnesium Chloride (MgCl2) - 1M.
j) McII Vaines Buffer (pH 5.0)
RNA EXTRACTION
a) Cetyl Trimethyl Ammonium Bromide (CTAB) Buffer CTAB:
b) Phenol, Chloroform and Isoamylalcohol in the ratio of 25:24:1 (PCI)
c) Ethanol
d) TE Buffer (pH 8.0)
COMPLEMENTARY DNA (c DNA) SYNTHESIS
cDNA synthesis kit
PCR AMPLIFICATION
a) Primers for EV and HAV - EV sense primer 5’ – TCC TCC GGC CCC
TGA ATG CG – 3’; antisense primer 5’- ATT GTC ACC ATA
AGC AGC CA - 3’; HAV sense primer 5’ –GTTTTGCTCCTCTTT
ATCATGCTATG- 3’; antisense primer 5’- GGA AATGTC TCAGGT
ACTTTCTTTG-3’
b) PCR Master Mix
c) Mineral Oil
AGROSE GEL ELECTROPHORESIS
a) Running Buffer

308
 b) Tracking Dye – 6X bromophenol blue.
c) Ethidium Bromide – 0.5 µg/mL
Preparation of Reagents a) Urea-Arginine Phosphate Buffer (U-APB)- Mix 4.5 gm of urea with
2mL of 0.2M NaH2PO4 and 2mL of 0.5 M L-Arginine and make up the
volume to 50mL with sterile distilled water. The pH of the eluent shall
be 9.0.
b) McII Vaines Buffer (pH 5.0)- Mix 9.7 mL of 0.1 M citric acid with
10.3mL of 0.2M Na2HPO4.2H2O under sterile conditions.
c) Cetyl Trimethyl Ammonium Bromide (CTAB) Buffer CTAB: 1 percent;
Sodium Dodecyl Sulphate (SDS): 1 percent EDTA: 20 mM; Sodium
Chloride: 1 M
d) TE Buffer (pH 8.0) - Tris base:1M; EDTA: 0.5 M; Sodium Acetate: 3M.
d) Running Buffer – 50X TAE buffer -Tris base/ Tris buffer: 121.00 gm;
Glacial acetic acid: 28.55 mL; 0.5 M EDTA: 50.00 mL; Distilled
water: 300.45 mL
Make the final volume up to 1000mL with deionized distilled water,
sterilize and store at 4°C. The final concentration for the preparation of
agarose gel and to run gel shall be 1X.
Sample Preparation CONCENTRATION OF DRINKING WATER: Filter 100 litre of
drinking water sample through membrane filter assembly using either
positively charged membrane of 144mm diameter or 0.22micron diameter
pore size nitrocellulose membrane. For positively charged membrane the
test water pH need not be adjusted. But for the 0.22 µ nitrocellulose
membrane adjust the pH to 3.5 after adding the aluminium chloride as a
coagulant to a final concentration of 0.0005M. At lower pH pass the water
through the membrane. The flow rate shall be 40lt/h approximately. After
the completion of the filtration, elute the adsorbed particles using 100 mL
of urea-arginine phosphate buffer using 1 mL of magnesium chloride (1M).
Dissolve the resultant precipitate centrifuged out of the sample in 800 - 1.0
mL of McII vaines buffer. The processed sample can be stored at
refrigerator until required.
Method of analysis a) RNA EXTRACTION: Treat 300µL of concentrated water sample with
equal volume of CTAB and 1/10thvolume of PCI. Vortex and centrifuge

309
 at 5000 x gm for 30 min at 4°C. Add 1/10thvolume of 3M sodium
acetate and double the volume of cold ethanol to the aqueous layer.
Keep the mixture at either -20°C for overnight or in liquid nitrogen for
2 – 5 min. Centrifuge at 10000 X gm for 30 min at 4°C. Discard the
supernatant and air dry the pellet and dissolve it in 20 µL TE buffer.
b) COMPLEMENTARY DNA (cDNA) SYNTHESIS: Suspend the
extracted RNA in 20 µL of cDNA reaction mixture, which consists of
4µL of 5X reverse transcriptase reaction buffer [250mM TRIS – HCl
(pH 8.5), 40mM KCl, 150mM MgCl2, 5mM dithiothreithol (DTT)],

0.5 L of 10mM deoxynucleotide phosphate (dNTP), 2µl of hexa

nucleotide mixture, 1µL of 25U of Maloney Murine Leukaema Virus
(M-MuLV) reverse transcriptase, 0.5µL of 20U of human placental
RNase inhibitor. Heat the reaction mixture to 95°C for 5 min and
rapidly chill on ice this is followed by the additional of 1 µL (25 U/µL)
of M-MuLV reverse transcriptase. Incubate the reaction mixture as
given by the manufacturer of the kit quickly chill the reaction tube on
ice.
c) PCR Amplification:
i. PCR Amplification for Hepatitis A Virus (HAV): In 5µL of
cDNA, add 95µL of a PCR Master Mix (10Mm tris-HCL (pH8.3),
50mMKCl, 2.5mM MgCl2, 0.01 percent gelatin (1X PCR buffer),
200µM of each dNTP, 1.5U of Thermus Aquaticus polymerase).
Add 25 pico moles of sense and antisense oligonucleotide primers
of HAV and overlay with mineral oil. Appropriate positive and
negative controls shall be included with each run. Set the following
reaction at thermo cycler:
Denaturation at 94°C for 2 min
Denaturation for 1.0 min at 94°C
Annealing for 1.0 min at 57°C, 35 cycles
Extension for 1.3 min at 72°C
Final extension at 72°C for 7 min
ii. PCR Amplification for Enterovirus: In 5µL of cDNA, add 95µL
of a PCR Master Mix (10 mM TRIS-HCL (pH 8.3), 50mM KCl,

310
 2.5mM MgCl2. 0.01 percent gelatin (1XPCR buffer), 200µM of
each dNTP, 1.5 U of Thermus aquaticus polymerase). Add 25
picomoles of sense and antisense oligonucleotide primers of EV and
overlay with mineral oil. Appropriate positive and negative controls
shall be included with each run. Set the following reaction at thermo
cycler:
Denaturation at 94°C for 2min
Denaturation for 1.0 min at 94°C
Annealing for 1.0 min at 42°C, 35 cycles
Extension for 2.0 min at 72°C
Final extension at 72°C for 7 min.
AGAROSE GEL ELECTROPHORESIS: Run the PCR amplified
product of EV and HAV on 1.5 percent agarose gel using 1X TAE buffer.
Load 10µL of amplified product after mixing it with 1µL 10X loading Dye.
Run the molecule weight marker along with the samples. Run the
electrophoresis at 100V for 30 min. Stain the gel with ethidium bromide
(0.5µL/mL) for 20 min. Wash it with distilled water and view under UV
transilluminator and photograph the gel to analyse the band pattern. EV
gives the band as 155 base pair and the HAV give band as 225 base pair.
Calculation with units of Entero Virus = Present/Absent
expression HAV virus = Present/Absent
Reference IS 10500:2012: Drinking Water — Specification (Second Revision)
Approved by Scientific Panel on Methods of Sampling and Analysis

311
 Method for Detection of MS2 Phage in Water by Enrichment Spot
Assay Technique

Method No. FSSAI 15.038:2023 Revision No. & Date 0.0

Scope This method is applicable to Packaged Drinking water

Caution Carry out the test under the control of skilled microbiologist and great care
shall be taken in the disposal of all the incubated material. Follow safe and
good laboratory practices to avoid cross contamination.

Principle This is a qualitative, presence- absence method. It employs an initial

enrichment step to amplify coliphage numbers in samples, prior to assay
with a spot technique. When a suspension of an infective phage (e.g. MS2
phage) is spotted over the lawn of host bacterial cells (e.g. Escherichia
coli), the phage attaches the bacterial cell, replicate inside it, which is
indicated by the formation of a zone of clearing or plaque within the lawn
of bacteria. In the absence of lytic phage, the bacteria form a confluent lawn
of growth.

Apparatus/Instruments a) Biosafety cabinet

b) Universal Incubator (36.5 ± 2°C)
c) Water bath (set temp. 45± 2 °C)
d) Centrifuge (0.5-1.0 mL sample capacity, 5-10,000x g performance
capability)
e) Autoclave
f) Sterile membrane filter (0.22µm pore size)
g) Erlenmeyer flasks, 125 mL, 250 mL and 2 L
h) Glass bottles, capped, 100 ml, 1L capacity
i) Graduated cylinders, 100 mL and 500 mL
j) Inoculating loop
k) Pipettes (1mL, 5mL and 10 mL)
l) Petri dishes (100 x 15 mm, 150 x 15 mm)
Culture Media and a) Tryptone enrichment broth
Reagents b) Tryptone agar slants

312
 c) Tryptone broth
d) Tryptone spot agar
e) Ampicillin solution
f) Streptomycin solution
g) Beef extract
h) Calcium chloride solution
Preparation of Reagents a) Tryptone enrichment broth — Add 10.0 g Tryptone, 1.0 g yeast
extract, 1.0 g glucose, 8.0 g NaCl, and 0.022 g CaCl2 per each 100
mL of dH2O. Sterilize by autoclaving at 121°C for 20 min.

After autoclaving, cool and add 0.1 mL of filtered ampicillin solution and
0.1 mL of Streptomycin solution per 100 mL of broth. Store at 4°C.

b) Tryptone agar slants — with gentle mixing, add 1.0 g Tryptone,

0.1 g yeast extract, 0.1 g glucose, 0.8 g NaCl, 0.022 g CaCl2, and
1.2 g of Bacto-agar to a total volume of 100 mL of dH2O in a 250
mL flask. Further dissolve and sterilize by autoclaving at 121°C for
20 min.

After autoclaving, allow the agar to equilibrate in water bath set at 44.5 °C
and then add 0.1 mL of filtered ampicillin solution and 0.1 mL of filtered
streptomycin solution. Dispense 8 mL aliquots into 16 × 150 mm test tubes
with. Prepare slants by allowing the agar to solidify with the tubes at about
a 20° angle. Slants may be stored at 4°C for up to three months.

c) Tryptone broth — Add 1.0 g Tryptone, 0.1 g yeast extract, 0.1 g

glucose, 0.8 g NaCl, and 0.022 g CaCl2 per each 100 mL of dH2O.
Sterilize by autoclaving at 121°C for 20 min.

After autoclaving, cool and add 0.1 mL of filtered ampicillin solution and
0.1 mL of Streptomycin cin solution per 100 mL of broth. Store at 4°C.

d) Tryptone spot agar dishes — Prepare one day prior to sample

analysis using the ingredients and concentrations listed for Tryptone
agar slants, except use 0.75 g of Bacto-agar for each 100 ml. After
autoclaving, place in water bath set at 44.5 °C and allow

313
 equilibrating.

Add 0.1 mL of filtered ampicillin solution and 0.1 mL of filtered

streptomycin solution to the 100 mL volume of warm agar. With gentle
mixing, add 2 mL of a 4 h culture of host E. coli. Pour the well mixed
suspension into five sterile 100 × 15 mm petri dishes (approximately 20 mL
per dish), swirl gently, and allow the agar to harden. Store the dishes
inverted at 4°C overnight and warm to room temperature for 1 h before use.

e) Ampicillin solution — Dissolve 1.5 g of ampicillin in 100 mL of

dH2O and filter with the 0.22 µm filter and store at 4°C.

f) Streptomycin solution — Dissolve 1.5 g of streptomycin sulfate in

100 mL of dH2O and filter with the 0.22µm filter and store at 4°C.

g) Beef extract — prepare buffered 1.5% beef extract by dissolving

1.5 g of beef extract powder and 0.375 g of glycine (final glycine
concentration =0.05 M) in 90 mL of dH2O. Adjust the pH to 7.0 -
7.5. Autoclave at 121°C for 15 min and use at room temperature. If
beef extract solutions are prepared for use at a later, store at 4°C.

h) Calcium chloride solution — Add 0.22 g of CaCl2 to 50 mL of

dH2O and sterilize by autoclaving at 121°C for 15 min. Use at room
temperature.

Reference Cultures a) Host Bacteria: E. coli Famp (ATCC #700891)

Inoculate 5 mL of Tryptone broth with host E. coli from a slant using a

sterile inoculating loop and incubate for 16 h at 36.5 ± 2°C. Transfer 1.5
mL of the 16 h culture to 30 mL of Tryptone broth in a 125 mL flask and
incubate for 4 h at 36.5 ± 2°C with gentle shaking. The amount of inoculum
and broth used can be proportionally altered according to need.

b) Coliphage MS2 (ATCC # 15597- B1)

Rehydrate the ATCC coli-phage MS2 and store at 4°C.

Prepare a 30 mL culture of the appropriate host as described above.
Incubate culture for 2 h at 36.5 ± 2°C with shaking. Add 1 mL of

314
 rehydrated phage stock and incubate for an additional 4 hours at
36.5 ± 2 °C. Filter the culture through a beef extract-treated 0.22µm
filter. Prepare 10-7, 10-8 and 10-9 dilutions of the filtrate using
tryptone dilution tubes. (These three dilutions should be sufficient
in most cases). Add 3 mL of melted tryptone top agarheld in the
44.5 ± 1°C water bath to fifteen 16 × 150 mm test tubes. These test
tubes should be kept in the heated water bath to avoid premature
solidifying of the agar. Add 0.1 mL of the host culture to each of
the 15 test tubes. Add 1 mL of the 10-9 dilution into each offive test
tubes. Add 1 mL of the 10-8 dilution into five additional tubes and 1
mL of the 10-7 dilution into the remaining five tubes. Be sure the
tubes are labelled with the appropriate dilution. For each tube, mix
and immediately pour the contents over the bottom agar of a
petridish labelled with the dilution assayed. Rotate the dish to
spread the suspension evenly over the surface of the bottom agar
and place it onto a level surface to allow the agar to solidify. Invert
and incubate the inoculated dishes at 36.5 ± 2°C overnight and
examine for plaques the following day. Count the number of
plaques on each of the 15 dishes. Five dishes from one of the
assayed dilutions should yield plaque counts of 20 to 100 plaques.
Average the plaque counts on these five dishes and multiply the
result by the reciprocal of the dilution to obtain the titer of the
undiluted stock. For use as a positive control in the coliphage assay,
dilute the filtrate to 30 to 80 PFU/mL in tryptone broth. Store the
original filtrate and the diluted positive control preparation at 4°C.
Before using the positive control preparation for the first time,
assay 10 mL by adding 1 mL volumes of the preparation to ten test
tubes containing agar and host culture, and pouring their contents
into ten petri dishes. Count the plaques on all dishes and divide by
10. If the result is not 30 to 80, adjust the dilution of the positive
controls am-ple and assay again.
Sample Preparation Not Applicable

315
Procedure A. Enrichment:

1. To 1000 ml of water sample add 5ml of 4h active E. coli culture,

12.5ml CaCl2, 50ml Tryptone enrichment broth, 1ml streptomycin
and 1ml ampicillin solution.

2. Forpositive control, first spike 100ml of sterile distilled water with

500 µl MS2 phage positive control. To the spiked sample add 500
µl of 4h E. coli culture, 1.25ml CaCl2, 5ml Tryptone enrichment
broth, 100µl streptomycin and 100µl ampicillin solution.

3. For the negative control, to 100ml of sterile distilled water add 500
µl of 4h E. coli culture, 1.25ml CaCl2, 5ml Tryptone enrichment
broth, 100µl streptomycin and 100µl ampicillin solution.

Incubate the water samples including positive and negative control

overnight at 36.5 ± 2°C.

B. Spot assay:

1. Remove enrichment cultures from incubator and mix well.

2. Draw 2ml of enriched culture from each sample (including positive
and negative control)
3. Take 10µl and carefully deposit the volume to a preselected area on
a Tryptone spot agar dish.
4. Allow the sample to absorb into agar for up to 60 min.
5. Incubate the spot agar plates overnight at 36.5 ± 2°C.
6. Examine dish for lysis (clearing) at the spot where the enrichment
inoculum was applied.
7. Sometime interfering bacterial growth may be observed at spot area
and can prevent accurate assessment of lysis zone formation. Such
interference may be addressed by including filtration via beef
extract treated 0.45um sterile filtration or by centrifugation at 5000-
10000x g for 10 mins.
Expression of results Presence/Absence of MS2 Phage given after confirmation of zone of lysis
in the sample examined.

316
 Test for MS2 Phage= Present/absent per 1000 mL water sample

Reference USEPA Manual of Methods for Virology, Chapter 16, June 2001

Approved by Scientific Panel on Methods of Sampling and Analysis

317
 Isolation and Identification of Giardia and Cryptosporidium in Water-
Method A

Method No. FSSAI 15.039:2023 Revision No. & Date 0.0

Scope This method is applicable to Packaged Drinking water (volume 10L to 1000
L)
Caution a) This method is not appropriate in case of turbid water.
b) Cartridge filtration can be used for small volumes of water (typically 10
L) but, based on the cost of these filters, this may be an expensive
approach in some circumstances.
Principle a) Concentration from water: The isolation of Cryptosporidium and
Giardia from water requires the use of a procedure which allows the
volume of the sample to be reduced whilst retaining any oocysts and
cysts. The concentration procedure used however, is dependent upon the
water type which is to be analysed, the volume of sample and the
amount of particulate material in the sample.
Table 1 — Membrane filters/filtration systems used for the
concentration of parasites from water samples Membrane filter/filtration
system
Membrane filter/filtration Application
system
Pall EnvirochekTM STD * Concentration of 10-litre to 200-litre
or equivalent (or more) samples of water
Pall EnvirochekTM HV or Concentration of 10-litre to 1000-litre
equivalent samples of water
IDEXX Filta-Max® or Concentration of 10-litre to 1000-litre
equivalent samples of water
* It has been shown by some laboratories that this technique may be
used successfully for larger volumes of water although the
manufacturers’ instructions may only include volumes up to 200
litres.

b) Purification and further concentration: After concentration of

318
 particulate material from filter eluates, oocysts and cysts are isolated
using immunomagnetic separation (IMS). Oocysts and cysts are
attached to para-magnetic beads coated with specific antibody, the beads
are separated from the unwanted particulate material using a magnet and
then the oocysts and cysts are dissociated from the beads using acid and
neutralized using alkali before immunostaining.
c) Detection of Cryptosporidium and Giardia: After IMS, organisms are
labelled with monoclonal antibody (mAb) conjugated to a fluorochrome,
usually fluoroscein isothiocyanate (FITC). In addition, any nuclear
material is labelled with a nucleic acid stain to aid identification. Each
sample is then examined for the presence of labelled Cryptosporidium
oocysts and Giardia cysts using epifluorescence and differential
interference contrast (DIC) microscopy.
Apparatus/Instruments a) Scientific apparatus, required for concentration using Pall
EnvirochekTM STD or HV
i) Sampling capsule, EnvirochekTM STD or HV
ii) Peristaltic pump, capable of a flow rate of 2 l/min.
iii) Silicon tubing, for use with the peristaltic pump.
iv) Seeding container, 10 l, if seeding filters is required.
v) Wrist-action shaker, with arms for the agitation of the
EnvirochekTM STD or HV sample capsules.
vi) Centrifuge, capable of a minimum of 1100 g.
vii) Centrifuge tubes, conical, plastic, screwtop, 250 ml capacity.
viii) Centrifuge tubes, conical, plastic, screwtop, 50 ml capacity.
NOTE A flow meter and flow restrictor are required for taking water
samples with the filter.
b) Specific apparatus, required for concentration using IDEXX Filta-Max®
or equivalent.
i) Sampling housing, Idexx Filta-Max® or equivalent.
ii) Sampling module, Idexx Filta-Max® or equivalent.
iii) Filter membranes, Idexx Filta-Max® or equivalent.
iv) Laboratory pump, capable of supplying 500 kPa (5 bar) pressure.
v) Peristaltic pump, capable of flow rate of 4 l/min.

319
 vi) Silicon tubing, for use with peristaltic pump.
vii) Seeding container, 10 L, if seeding filters is required.
viii) Wash station, automatic or manual, and wash station clamp set,
Idexx Filta-Max® or equivalent.
ix) Vacuum set, includes plastic hand pump, waste bottle, tubing and
magnetic stirring bar. Idexx Filta-Max® or equivalent.
x) Tubing set, includes elution tube, and middle section,
concentrator tube and base, with line tap and steel rod Idexx Filta-
Max® or equivalent.
xi) Membrane, for tubing set.
xii) Plastic bag, for washing membrane.
xiii) Centrifuge, capable of 1100 g.
xiv) Centrifuge tubes, conical, plastic, 50 ml capacity.
xv) Forceps.
NOTE A flow meter and flow restrictor are required for taking water
samples with the filter
c) General apparatus
i) Incubator, at (36 ± 2) °C.
ii) Refrigerator, at (5 ± 3) °C
iii) Magnetic stirrer, and magnetic stirring bars.
iv) Vortex mixer.
v) Wash bottles, polypropylene, 250 ml.
vi) Calibrated micropipettes, adjustable: 1 µl to 10 µl with 1 µl to 10
µl tips; 20 µl to 200 µl with 10 µl to 200 µl tips; 200 µl to 1000
µl with 100 µl to 1000 µl tips.
vii) pH meter.
viii) Magnetic particle concentrators, with suitable tubes.
ix) Well microscope slides, with special hydrophobic coating and
coverslips.
x) Epifluorescence microscope, with a UV filter (350 nm excitation,
450 nm emission), FITC filter (480 nm excitation, 520 nm
emission) filters, TM differential interference contrast (DIC)
optics and an eye piece graticule. Total magnification 1000x.

320
 xi) Microscope stage micrometer, 1 mm, ruled in 100 units.
xii) Eyepiece graticule, ruled in 100 units.
xiii) Humidity chamber, e.g. consisting of a tightly sealed plastic
container containing damp paper towels on which the slides are
placed.
xiv) 10 L containers, graduated in 1 L.
xv) Neubauer haemocytometer slide.
Culture Media and a) Reagents required for eluting Pall EnvirochekTM STD capsule filters
Reagents i) Deionized water, 0.2 µm filtered at the point of use.
ii) Laureth 12 detergent.
iii) Tris buffer, pH 7.4
iv) EDTA solution, 0.5 mol/l, pH 8.0.
v) Antifoam A.
vi) Elution buffer
b) Reagents required for eluting Pall EnvirochekTM HV capsule filters
i) Deionized water, 0.2 µm filtered at point of use.
ii) Pre-treatment buffer
iii) Laureth 12 detergent
iv) Tris buffer, pH 7.4
v) EDTA solution, 0.5 mol/l, pH 8.0
vi) Antifoam A.
vii) Elution buffer
c) Reagents required for eluting IDEXX Filta-Max® filters or
equivalent
i) Phosphate buffered saline (PBS)
ii) Polyoxyethylene(20)sorbitan monolaurate (Tween 20). Store at
room temperature (20 ± 5) °C. Expiry date one year.
iii) Elution buffer
d) Concentration and detection reagents
i) Methanol, analytical grade.
ii) Magnetic beads, for the detection of Cryptosporidium and
Giardia.
iii) Fluorescently labelled monoclonal antibodies (mAbs) against

321
 Cryptosporidium and Giardia. Store at (5 ± 3) °C. Expiry date as
stated by the manufacturer. When stains are prepared from
concentrated material using a diluent supplied by the
manufacturer, the prepared solution is stored at (5 ± 3) °C for no
longer than 6 months.
iv) Immunofluorescence mounting medium
v) 4′,6′-Diamidino-2-phenylindole dihydrochloride dihydrate
(DAPI) freeze dried reagent.
vi) DAPI stock solution
vii) DAPI working solution
viii) Phosphate buffered saline (PBS)
ix) Non-fluorescing immersion oil. Store at room temperature (20 ±
5) °C.
x) Stock suspensions of Cryptosporidium parvum oocysts and
Giardia lamblia cysts: Store at (5 ± 3) °C, never allow the
suspension to freeze and check quality regularly. Ideally,
suspensions of oocysts and cysts should be no more than 3
months old. Stock suspensions should be checked
microscopically to confirm that they are monodispersed and
discarded if clumps or aggregates are detected. In addition, if
mAb and DAPI staining become weak and oocysts become
deformed, they should also be discarded
xi) Parasite storage medium
Preparation of a) Tris Buffer: To prepare, dissolve 121.1g Tris in 700 ml of deionized
Reagents water and adjust the pH to 7.4 with 1,0 mol/l HCl or NaOH. Make up to
1000 ml with deionized water. Filter sterilization is not necessary. Store
at room temperature (20 ± 5) °C. Expiry date 3 months.
b) EDTA solution: To prepare, dissolve 186.1g EDTA in 800 ml of hot
deionized water. Cool to room temperature (20 ± 5) °C and adjust pH to
8.0 with 6.0 mol/l NaOH for initial adjustment and 1,0 mol/l HCl or
NaOH for final adjustment. Make up to 1000 ml with deionized water.
Store at room temperature (20 ± 5) °C. Expiry date 3 months.
c) Elution buffers for Gelman EnvirochekTM STD and HV capsule

322
Laureth12 1g
Tris buffer 10 ml
EDTA solution 2 ml
Antifoam A 150 µl
Deionized water 1000 ml
To prepare, weigh Laureth-12 in a glass beaker and add 100 ml of
filtered deionized water. Heat the beaker to melt the Laureth-12
(approximately 1 min) and transfer the solution to a 1 000 ml
graduated cylinder. Rinse the beaker several times to ensure the
transfer of the detergent to the cylinder. Add 10 ml of Tris solution,
pH 7.4, 2 ml of EDTA solution, pH 8.0 and 150 µl of Antifoam A.
Dilute to 1 000 ml with filtered deionized water. Store the solution at
room temperature (20 ± 5) °C, expiry 2 months.
d) Pre-treatment buffer
Sodium polyphosphate (NaPO3)n 5g
Deionized water 1000 ml
To prepare, dissolve the sodium polyphosphate in the water. Store at
room temperature (20 ± 5) °C. Expiry date 1 week.
e) Phosphate buffered saline (PBS) for IDEXX Filta-Max® filters
or equivalent
Sodium chloride 8,0 g
Di-sodium hydrogen phosphate (Na2HPO4) 1.15 g
Potassium di-hydrogen phosphate (KH2PO4) 0.2 g
Potassium chloride 0.2 g
Deionized water 1000 ml
Dissolve the ingredients in the water and adjust the pH to 7.3 ± 0.2
with 1,0 mol/l HCl or NaOH. Store at room temperature (20 ± 5) °C.
Expiry date 3 months.
f) Elution buffer for IDEXX Filta-Max® filters or equivalent
Polyoxyethylene sorbitan monolaurate (Tween 20) 1 ml
PBS 10 L
To prepare, add approximately 8 L of PBS to a 10 L container (with
tap). Stir this liquid using a magnetic stirrer and stir bar. Dispense 1

323
 ml of Tween 20 to a 50 ml centrifuge tube and dissolve in
approximately 10 ml of warm deionized water.
Carefully pour the contents of the centrifuge tube into the 10 L
container.
Rinse the tube twice with 10 ml of deionized water, adding the
contents of the tube to the 10 L container each time.
Finally, fill the 10 L container to the 10 L mark using PBS. Store at
room temperature (20 ± 5) °C, expiry date 1 month.
g) Immunofluorescence mounting medium
1,4-Diazabicyclo[2.2.2]octane (DABCO) 2.0 g
Glycerol 60 ml
PBS 40 ml
To prepare, dissolve the DABCO in the glycerol and the PBS. Adjust
the pH to 7.1 ± 0.2 with 0.1 mol/l HCl or NaOH.
Store vials in use at room temperature (20 ± 5) °C. Store additional
vials at (5 ± 3) °C.
h) DAPI stock solution
DAPI 1 mg
Methanol 0.5 ml
To prepare, add 0.5 ml of methanol to a vial containing 1 mg of
DAPI.
Store at (5 ± 3) °C. Expiry date 1 month.
i) DAPI working solution
Prepare by diluting 10 µl of DAPI stock solution in the 50ml of PBS.
Store at (5 ± 3) °C. Expiry date one day.
j) Parasite storage medium — stock solution
Sodium azide (NaN3) 100 mg
Deionized water 5 ml
To prepare, dissolve the sodium azide in the water. Store at (5 ± 3)
°C. Expiry date 1 month.
k) Parasite storage medium — working solution
To prepare, add 1 ml of the parasite storage medium stock solution to
100 ml of deionized water. Store at (5 ± 3) °C. Expiry date 1 month.

324
Sample Preparation a) Sampling and Transport

i) Take small volume grab samples (10 L) and transport them to the
laboratory in the dark at ambient temperature. Once at the
laboratory, samples should be stored at (5 ± 3) °C unless they are
to be analysed immediately. Samples should be analysed
preferably within 24 h of collection and no longer than 4 d.
ii) If the samples are filtered in the field (in case of large volume),
connect the device in-line with the water supply, making sure that
the flow through the filter is in the direction indicated on the
housing by the manufacturer. A flow meter should be included
with the filter and this should be read before and after sampling.
Transport the filters in the dark at ambient temperature. Once at
the laboratory, samples should be stored at (5 ± 3) °C unless they
are to be analysed immediately. Samples should be analysed
preferably within 24 h of collection and no longer than 4 d. If
filters are stored at (5 ± 3) °C, they shall be allowed to warm to
room temperature before elution starts.
iii) A pre-treatment step using sodium polyphosphate before the
elution buffer was introduced to improve the removal of
particulate material bound to the filter.

NOTE 1 The EnvirochekTM STD or equivalent filter consists of a pleated

polyether sulfone membrane sealed in a polycarbonate shell. The filter is
supported on a loose polypropylene support. It is supplied packaged with
two end caps which can be used to seal the filter. The filter can be connected
to a water supply by connecting to a ribbed inlet and the direction of flow
through the filter is clearly marked. The flow through the filter should not
exceed 2.3 l/min and the differential pressure across the filter should not
exceed 210 kPa (2.1 bar).

NOTE 2 The EnvirochekTM HV capsule or equivalent is comprised of 1 µm

pore size polyester track-etched membrane permanently enclosed in a

325
polycarbonate housing. The polyester membrane is directly laminated to a
polypropylene support which offers a significant strength improvement over
the standard EnvirochekTM STD. The capsule housing burst strength
exceeds 1 000 kPa (10 bar) and the differential pressure across the filter
membrane is rated to 410 kPa (4.1 bar). Each EnvirochekTM HV capsule is
100 % integrity tested after assembly to ensure product performance. The
effective filtration area of the EnvirochekTM HV is 1 300 cm2. The filter is
supplied with two end caps which can be used to seal the filter for transport
to the laboratory. The filter can be supplied with a tamper evident label
containing a unique identification number. The flow through the
EnvirochekTM HV or equivalent should not exceed 3.4 l/min.

NOTE 3 The Filta-Max® or equivalent filter consists of a foam filter

module comprising 60 open cell reticulated foam discs with an external
diameter of 55 mm and an internal diameter of 15 mm. The discs are
sandwiched between two retaining plates and compressed by tightening a
retaining bolt to give a nominal porosity of 1 µm. The filter module fits into
a filter housing which has a screw top and seal. The filter housing has
stainless steel barbed inlet and outlet ports. The sample enters through the lid
of the housing and exits through the base. Water flows into the housing,
through the compressed foam rings into the centre of the module and through
the outlet port. Removal of the retaining bolt during the elution stage allows
the filter to expand during washing. Filter housings are supplied with two
tools for the removal of the top and two rubber bungs to seal water in the
sample. After sampling, Filta-Max® should be kept wet during storage and
transportation. If stored or transported in the filter housing, the inlet and
outlet should be securely plugged with the rubber stoppers provided. During
transportation or storage, the filter module may be removed from the housing
and aseptically placed in an airtight container along with several milliliters of
additional deionized water.

b) Sample Concentration

326
i. Pall EnvirochekTM STD or equivalent Filtration: Support the
filter vertically with the white bleed valve uppermost. Remove the
two end caps and allow any water in the sample to drain out through
the filter. Replace the bottom end cap, fill the cartridge with elution
buffer through the inlet fitting until it covers the filter by
approximately 1 cm. Replace the upper end cap and secure the
cartridge horizontally into the wrist shaker with the white bleed valve
in the 12 o’clock position. Shake at 600 cycles per minute (cpm) ± 25
cpm for 5 min ± 30 s. Remove the upper end cap and pour the
washings into a 250 ml conical centrifuge tube. Add a further aliquot
of elution buffer into the capsule, replace the upper end cap and
shake for a further 5 min ± 30 s. Ensure that the white bleed valve is
in the 3 o’clock or 9 o’clock position. After 5 min of shaking, remove
the upper end cap and decant the washings into the 250 ml centrifuge
tube and centrifuge at 1 100 × g for 15 min without braking during
the deceleration phase. Record the pellet volume (volume of solids)
immediately after centrifugation. A second centrifugation step may
be required in a 50 ml centrifuge tube in order to measure the
volume. Alternatively, 50 ml centrifuge tubes may be used to
concentrate the particulate material eluted from the filter. Using a
pipette and a vacuum source of less than 20 kPa (0.2 bar), carefully
aspirate off the supernatant leaving 2 ml to 5 ml above the pellet. If
no pellet is visible, extra care shall taken to avoid aspirating oocysts
and cysts during this step. Add deionized water to the centrifuge tube
to bring the total volume to 9 ml. Vortex the tube for 10 s to 15 s to
resuspend the pellet and either store the sample at (5 ± 3) °C for
future or proceed directly for IMS. If the pellet volume exceeds that
recommended by the manufacturer of the IMS test kit, centrifuge the
sample a second time in a tube that permits the pellet volume to be
measured accurately. Subdivide the sample into aliquots for IMS
such that each aliquot represents the maximum pellet volume
recommended by the manufacturer and make up each aliquot to 9 ml
with deionized water.

327
ii. Pall EnvirochekTM HV or equivalent Filtration: Support the filter
vertically with the white bleed valve uppermost. Remove the two end
caps and allow any water in the sample to drain out through the filter.
Replace the bottom end cap, fill the cartridge with pre-treatment
buffer through the inlet fitting until it covers the filter by
approximately 1 cm. Replace the upper end cap and secure the
cartridge horizontally into the wrist shaker with the white bleed valve
in the vertical position. Shake at 600 cycles per minute (cpm) ± 25
cpm for 5 min ± 30 s. Secure the filter vertically with the white bleed
valve uppermost, remove the end caps and allow the pre-treatment
buffer to drain out through the filter. Replace the bottom end cap and
fill the cartridge as above with deionized water. Replace the upper
end cap and rinse the membrane by gently rotating the filter for 30 s.
Secure the filter vertically, remove the end caps and allow the
deionized water to drain out through the filter. Replace the bottom
end cap, fill the cartridge with elution buffer through the inlet fitting
until it covers the filter by approximately 1 cm. Replace the upper
end cap and secure the cartridge into the wrist shaker with the white
bleed valve in the 12 o’clock position. Shake at 600 cpm ± 25 cpm
for 5 min ± 30 s. Remove the upper end cap and pour the washings
into a 250 ml conical centrifuge tube. Add a further aliquot of elution
buffer into the capsule, replace the upper end cap and shake for a
further 5 min ± 30 s. Ensure that the white bleed valve is in the 4
o’clock position. After 5 min of shaking, turn the filter such that the
white valve is in the 8 o’clock position and shake for a further 5 min.
Remove the upper end cap and decant the washings into the 250 ml
centrifuge tube and centrifuge at 1100 g for 15 min without braking
during the deceleration phase. Record the pellet volume (volume of
solids) immediately after centrifugation. A second centrifugation step
may be required in a 50 ml centrifuge tube in order to measure the
volume. Alternatively, 50 ml centrifuge tubes may be used to
concentrate the particulate material eluted from the filter. Using a
pipette and a vacuum source of less than 20 kPa (0.2 bar), carefully

328
 aspirate off the supernatant leaving 2 ml to 5 ml above the pellet. If
no pellet is visible, extra care shall be taken to avoid aspirating
oocysts and cysts during this step. Add deionized water to the
centrifuge tube to bring the total volume to 9 ml. Vortex the tube for
10 s to 15 s to re-suspend the pellet and either store the sample at (5 ±
3) °C for IMS or proceed directly to IMS. If the pellet volume
exceeds that recommended by the manufacturer of the IMS test kit,
centrifuge the sample a second time in a tube that permits the pellet
volume to be measured accurately. Subdivide the sample into
aliquots for IMS such that each aliquot represents the maximum
pellet volume recommended by the manufacturer and make up each
aliquot to 9 ml with deionized water.

NOTE Warming all the elution solutions to 37 °C improves the removal of

particulate material. Elution is also helped by increasing the shaking speed to
900 cpm.

iii. Idexx Filta-Max® or equivalent Filtration: The elution apparatus

consists of an upper and lower wash tube, a wash station and a
vacuum set designed to reduce the volume of the eluate to 50 ml
through a membrane. The elution procedure is as follows: Place a
membrane filter (rough surface uppermost) in the base of the lower
wash tube and put the tube into the base. Make sure that the
membrane is held securely and that the tap on the base is closed.
Unscrew the housing top using the tools provided, remove the filter
module from its housing and screw it into the plunger head of the
wash station. Pour any residual water in the filter housing into the
lower wash tube. Place the upper part of the wash tube into the jaws
of the wash station and lower the filter module down through the
tube. Using the key provided, remove the retaining screw from the
filter module. The filter should begin to expand. Screw the stainless
steel tube into the base of the upper wash tube. Pour approximately
600 ml of wash buffer into the lower wash tube and run a small
volume of buffer through the membrane by opening the tap on the

329
base of the lower wash tube. Attach the lower wash tube to the upper
wash tube. Pump the plunger up and down four or five times to help
the filter module expand. If the filter does not expand, leave it
soaking in elution buffer for 5 min, occasionally pumping the plunger
to help filter expansion. Pump the plunger up and down as far as it
will travel 20 times only. Disconnect the lower wash tube, pressing
the plunger 5 times to remove any residual elution buffer from the
foam rings. Rinse the stainless steel tube with elution buffer and plug
the end with the small rubber bung provided. Place the elution tube
on a magnetic stirrer. Locate the magnetic stirring bar into the top of
the elution tube and set the stirrer such that the liquid in the tube is
mixed. Connect the vacuum pump and open the tap on the base of the
wash tube. If the sample has little turbidity and the catch bottle is
placed below the wash tube, liquid will flow by gravity through the
membrane. For turbid liquids, apply a vacuum of no greater than 40
kPa (30 cm of mercury) to filter the washings through the membrane.
Should the membrane become blocked, decant washings into a clean
bottle, remove the membrane to a plastic bag and place a fresh
membrane into the lower wash tube with the smooth surface
uppermost. Pour the liquid back into the wash tube, rinsing the bottle,
and continue the filtration process. Each membrane shall be washed
in a separate bag. When the washings fall to approximately half way
up the stirring bar (approximately 30 ml), close the tap and
disconnect the vacuum pump and water trap. Pour the liquid in the
wash tube into a 50 ml centrifuge tube. Add a further 600 ml of
elution buffer to the lower wash tube and attach it to the wash station.
Repeat the washing procedure using only 10 strokes of the plunger.
Remove the lower wash tube, rinsing the stainless steel tube, place it
on the stirrer and attach the stirring bar. Concentrate the filter
washing down to approximately one inch above the stirring bar as
described above. Add the contents of the first elution to the wash tube
and continue reducing the volume of eluate until it is again half way
up the magnetic bar. Remove the stirring bar and pour the filter

330
 washings (approximately 30 ml) into the centrifuge tube. Unscrew
the wash tube from the base and carefully remove the membrane
filter with fine forceps. Add the filter to the bag provided together
with 5 ml of elution buffer. Seal the bag and wash the filter by
rubbing between fingers and thumb for (60 ± 10) s. Pipette off the
washings and add to the 50 ml centrifuge tube. Repeat the wash
procedure and add the second washings to the centrifuge tube. Make
up the volume in the tube to 50 ml with elution buffer. Centrifuge the
50 ml tube at 1100x g for 15 min without braking during the
deceleration phase. Record the pellet volume (volume of solids)
immediately after centrifugation. Using a Pasteur pipette and a
vacuum source of less than 20 kPa (0.2 bar), carefully aspirate off the
supernatant leaving 2 ml to 5 ml above the pellet. If no pellet is
visible, extra care shall be taken to avoid aspirating oocysts and cysts
during this step. Add deionized water to the centrifuge tube to bring
the total volume to 9 ml. Vortex the tube for 10 s to 15 s to re-
suspend the pellet and either store the sample at (5 ± 3) °C for IMS or
proceed directly to IMS. If the pellet volume exceeds that
recommended by the manufacturer of the IMS test kit, centrifuge the
sample a second time in a tube that permits the pellet volume to be
measured accurately. Subdivide the sample into aliquots for IMS
such that each aliquot represents the maximum pellet volume
recommended by the manufacturer and make up each aliquot to 9 ml
with deionized water.

NOTE: Clean the wash tubes with hot water and detergent followed by
thorough rinsing in warm water and filtered deionized water.

NOTE: Where a number of samples from different sources are examined

routinely, it is advantageous to have a separate wash tube set and plunger
dedicated to each site to minimize cross contamination.

Method of analysis a) Immunomagnetic separation (IMS):

i) This technique involves the attachment of oocysts and cysts to
magnetic beads coated with antibodies to either Cryptosporidium

331
 or Giardia.
ii) The oocyst or cyst-bead complex is separated from interfering
particles in the water concentrate by using a magnet.
iii) After separation, the oocysts and cysts are dissociated from the
beads by acid treatment.
iv) Oocysts and cysts are transferred in suspension to a microscope
slide and the magnetisable beads are discarded.
Note: Commercial test kits are the only validated methods available for
IMS. The test kits shall be used according to the manufacturer's
instructions.
b) Sample staining:
i) Label an appropriate well slide with the sample number and the
sample volume analysed (the whole of the sample should be
analysed).
ii) After addition of NaOH to the wells of the slide, distribute
aliquots of the suspension containing the separated oocysts and
cysts onto the wells.
NOTE: The volume of the NaOH and the sample added to each well will
depend on the size of the wells.
iii) Prepare two separate well slides with positive and negative
controls. The positive control shall consist of a suspension of
Cryptosporidium and Giardia containing a known number of
parasites. The negative control shall consist of filtered deionized
water or PBS. Further positive and negative controls shall be
included with each batch of samples stained.
iv) Place the well slides containing the samples in an incubator at
(36 ± 2) °C or no higher than 42 °C and evaporate to dryness.
v) Apply a drop of methanol to each well containing the dried
sample and allow to air dry at (20 ± 5) °C. Overlay the sample
well with FITC fluorescently labelled monoclonal antibodies
(mAb).
vi) Place the slides in a humidity chamber, if required, and incubate
at (36 ± 2) °C for the time specified by the manufacturer of the

332
 conjugated antibodies.
NOTE: The exact volumes and times depend on the type of antibodies and
well slides used.
vii) After incubation, remove the slides from the humidity chamber
and gently aspirate excess labelled mAb from the side of each
well. When performing this step, ensure that the pipette tip does
not touch the well surface.
viii) Apply 1 drop of 4′,6′-diamidino-2-phenylindole (DAPI)
solution to each well. Allow to stand at room temperature (20 ±
5) °C for 2 min.
NOTE: This timing applies only to slides that have been methanol fixed and
subsequently dried.
ix) Remove excess DAPI solution by aspiration (as described
above). Apply a drop of filtered deionized water to each well
and then aspirate the excess deionized water (as described
above).
NOTE: An additional washing step using 0.01 M PBS, pH 7.2 is sometimes
used before washing with deionized water.
x) Allow slides to dry at room temperature (20 ± 5) °C or in an
incubator at (36 ± 2) °C. Store the slides in the dark at (5 ± 3) °C
until ready for examination.
xi) The slides should be examined as soon after processing as
possible and shall be examined the next day.
NOTE: Slides have been kept for up to three months in the dark and retained
their fluorescence. No detailed investigations have been carried out,
however, concerning the loss of fluorescence or DAPI staining upon storage.
Keep the slide dry and mount it before examination.
xii) Before the examination, apply approximately 20 µl of slide
mountant to the edge of the well on the sample slide, taking care
not to touch the slide with the pipette tip. Place a coverslip onto
the slide, taking care not to create bubbles in the slide mountant.
Seal the edges of the coverslip with clear nail polish.
xiii) Alternatively, the mounting medium may be pipetted onto the

333
 centre of the coverslip and the slide carefully inverted and
placed on the coverslip. The slide can then be carefully turned
over with the coverslip uppermost. Take care to avoid trapping
air bubbles between the slide and the coverslip.
c) Microscopy
i) General:
 Use an epifluorescence microscope fitted with DIC for analysis
of all sample preparations. Use objectives and eyepieces to a total
magnification of 200x or 400x and 1000x. Refer to the
manufacturer’s instruction manual for details of microscope
configuration.
 Calibrate the eyepiece graticule at regular intervals.
 Use a magnification of 800x to 1000x for the confirmation of
oocysts and cysts.
 Within this procedure, oocyst and cyst detection relies upon the
manual examination of sample preparations using
epifluorescence/DIC microscopy. Although this technique is
widely employed, it is time consuming, can cause operator
fatigue and, as a result, is open to human error. Consequently, a
reliable automated procedure is of considerable benefit.
Presently, several instruments that can automatically scan sample
preparations are available (e.g. laser scanning cytometry) or are
in development. When properly validated, such equipment may
be employed.
ii) Examination of fluorescent sample preparations using
epifluorescence microscopy
 Using the epifluorescence microscope and a 200x or 400x
magnification, examine the stained control slides to ensure that,
on the positive control slide, oocysts and cysts have been
correctly labelled by the mAb and that the negative control slide
is free from oocysts and cysts. Repeat this examination at 1000x
magnification to confirm the staining, the size and appearance of
the oocysts and cysts. Examine the contents using the UV

334
 excitation filter to ensure that the nuclear material has been
correctly labelled by DAPI.
 If the positive control slide is negative, repeat the stain before any
samples are processed. If the negative control slide is positive,
undertake an investigation to determine the source of the
contamination. Prepare fresh reagents and stain the control slides
again before any samples are processed.
 Providing that these checks are satisfactory, examine the samples
by scanning each well systematically using epifluorescent
microscopy (FITC). Use a side-to-side or top-to-bottom scanning
pattern.
 When a horizontal row has been completed, identify a feature
situated at the bottom centre of the field of view (i.e. sample
debris or the edge of the well slide coating). Move the
microscope stage so that this feature appears near the top of the
field of view. If the scanning has been carried out using a top-to-
bottom pattern (vertical rows), then identify a feature situated at
the right hand side, centre of the field of view. Move the
microscope stage so that this feature appears near the left hand
side of the field of view.
⎯ With side-to-side scanning, move the stage horizontally so
that the boundary of the well is completely in view, then scan
horizontally back across the well.
⎯ With top-to-bottom scanning, move the stage vertically so that
the boundary of the well is completely in view, then scan up
or down the well as necessary.
 Repeat until the whole well has been scanned. Scan using a
magnification of 200 × or 400 × and note the number of objects
which are presumptive Cryptosporidium or Giardia. Where there
are only one or two objects, examine each object at 800 × to 1
000 × using water or oil immersion objectives to confirm that
they are oocysts or cysts. Where there are more presumptive
oocysts or cysts, examine the whole slide at 800 × to 1 000 × and

335
 confirm each object. This process is easier than switching from a
dry low power objective to a high power objective to examine
each suspect body.
 All objects with typical characteristics of Cryptosporidium or
Giardia should be further examined and measured using DAPI
and DIC
 When labelled with FITC-mAb and examined using
epifluorescence microscopy (FITC, filter block), organisms
should exhibit the following characteristics.
Cryptosporidium Cryptosporidium oocysts Giardia
oocysts Giardia cysts cysts
Apple green fluorescence Apple green fluorescence (often with
(often with bright edges) bright edges)
Spherical or slightly Spherical or slightly ovoid in shape
ovoid in shape Some cysts will exhibit creases and
Some oocysts will exhibit folds
creases, splits and suture
lines
Diameter of 4 µm to 6 Dimensions of (8 µm to 12 µm) × (7
µm µm to 10 µm)
 Count badly distorted and damaged objects with care, particularly
when no typical oocysts or cysts are observed on a slide.
NOTE: The majority of Cryptosporidium oocysts appear spherical or slightly
ovoid with brighter even staining around the entire circumference. Some
oocysts can deviate from this description. Those which have been in the
environment for some time can be weakly stained or appear fuzzy. They may
still have contents and sporozoite nuclei can be identified. Often oocysts are
split as if a segment has been removed. Under these circumstances, the
oocyst may have ruptured during drying on the slide and sporozoites and
sporozoite nuclei may be evident adjacent to the oocyst. In addition, oocysts,
especially those without contents, may appear to be distorted or partially
folded.

NOTE: The majority of Giardia cysts appear ovoid (8 µm to 12 µm × 7 µm

to 10 µm), however, on occasion, cysts may appear spherical with
dimensions of approximately 10 µm × 10 µm. Cysts which have been in the
environment for some time may stain weakly and be badly distorted,

336
especially those without contents.
NOTE: A number of species of both Cryptosporidium and Giardia have been
classified (Annex G). The given size ranges target primarily C. parvum and
G. intestinalis. However other species may be in that size range which may
or may not be pathogenic to humans. Alternatively, other species or single
bodies of the target species may not be identified as Cryptosporidium or
Giardia due to their size being larger or smaller than the given size range and
pathogenicity to humans cannot be ruled out.
 When an apple green fluorescent event is observed which is
characteristic of a Cryptosporidium oocyst or Giardia cyst,
examine the object with the UV filter block for DAPI staining
and subsequently with DIC. Other objects (e.g. algae) may mimic
the size, structure and staining of Cryptosporidium and Giardia. It
is therefore important to further confirm presumptive cells
(bodies) by DAPI and DIC.
iii) Identification of Cryptosporidium oocysts and Giardia cysts:
DAPI
 Each presumptive oocyst or cyst should be examined to confirm
the presence of DAPI staining nuclei using a 100x oil or water
immersion objective. Switch over to the UV filter block on the
microscope for DAPI visualization.
NOTE The nuclei of DAPI stained oocysts and cysts appear sky blue upon
examination with the epifluorescence microscopy (DAPI UV filter block).
 If the object exhibits one of the following characteristics,
consider it to be a Cryptosporidium oocyst or Giardia cyst:
⎯ two to four distinct, sky blue nuclei within a single body;
⎯ nuclear material that may be slightly diffuse giving it a fuzzy or
ragged appearance;
⎯ diffuse blue internal staining where distinct nuclear material
cannot be identified.
 Include the two sub-groups in the total count unless they show
atypical morphological characteristics such as greater than four
nuclei or where one or two large intensely stained nuclei are

337
 visible within the object.

iv) Identification of Cryptosporidium oocysts and Giardia cysts:

DIC
 Having examined the object using the FITC and DAPI filter
blocks, close the light stop for the UV light and switch on the
transmitted light source ensuring that the substage condenser is in
place. Ensure that the substage condenser turret plate has the
correct ICT condenser prism in place.
IMPORTANT — It is important that the light from the mercury
vapour lamp is blocked as UV light can damage the DIC filter.
 Slide the DIC filter and prism into position and optimize the
image by adjusting the light intensity and/or turning the
adjustment screw on the prism.
 Using DIC, measure the size and look for external or internal
morphological characteristics typical of a Cryptosporidium
oocyst or Giardia cyst. Confirmation of size and internal contents
should only be done at a magnification of 1000x (using a 100x oil
or water immersion objective).
 Cryptosporidium oocysts should exhibit one of the following
characteristics.
⎯ Spherical or slightly ovoid with a convex central area, the
surface of which is irregular in appearance. In addition, a
thickened oocyst wall may be observed. This is indicative of an
oocyst which contains sporozoites. It may be possible to see
sporozoites inside the oocyst as well as a distinct refractile point
which is the residual body.
⎯ Spherical or slightly ovoid objects with a thickened oocyst
wall. In addition, a refractile residual body may be observed. This
can be indicative of an oocyst after excystation.
⎯ Spherical or slightly ovoid object that is flat and indistinct. In
addition, a thickened oocyst wall is observed. This can also be
indicative of an oocyst after excystation.

338
  Giardia cysts should exhibit one of the following characteristics.
⎯ Ovoid with a thickened cyst wall and a convex central area.
This is indicative of a cyst with contents. In addition, the nuclei
demonstrated by DAPI staining may be observed together with
remnants of flagellar axonemes and the median body.
⎯ Ovoid with a thickened cyst wall, the central area appearing flat
and indistinct. This is indicative of an empty cyst.

NOTE: The identification of organisms using DIC requires much

experience. The characteristics given can only be used as guidelines for
identification purposes. Misidentification of objects that mimic oocysts and
cysts can occur even at this stage of identification.
NOTE: Cryptosporidium- and Giardia-like bodies can show external or
internal morphological characteristics atypical of oocysts or cysts (e.g.
spikes, stalks, appendages, pores, one or two large nuclei filling the cell, red
fluorescing chloroplasts, crystals, spores, etc.). The presence of such features
indicates that the object is not an oocyst or cyst. Be aware that oocyst
contents stained by Evans Blue (present in many mAb preparations) can also
fluoresce red.
 Under some circumstances, the examination of samples using
DIC may not be possible due to the presence of interfering debris.
In such circumstances, this should be reported and a decision on
the identity of the event should be based on the characteristics of
FITC-mAb and DAPI labelling
Calculation with units a) The whole of the sample pellet should be examined.
of expression b) Give the number of Cryptosporidium oocysts and/or Giardia cysts per
volume of sample examined. Then calculate the concentration of
Cryptosporidium oocysts and/or Giardia cysts for the standard
volume usually given (e.g. 10 L or 1000 L). Absence of the
organism, i.e. none detected, shall be expressed as “not detected” in
the sample volume examined.
NOTE: The numbers of oocysts or cysts in different aliquots of the pellet
can vary considerably. Calculation of the numbers of oocysts and/or cysts in

339
 the total volume from a number found in an aliquot of the pellet can
therefore result in an over- or underestimation of oocyst or cyst
concentration.

Inference -
(Qualitative Analysis)
Reference a) IS 10500:2012: Drinking Water — Specification (Second Revision)
b) ISO 15553:2006: Water quality — Isolation and identification of
Cryptosporidium oocysts and Giardia cysts from water
Approved by Scientific Panel on Methods of Sampling and Analysis

340
 Isolation and Identification of Giardia and Cryptosporidium in
Water- Method B

Method No. FSSAI 15.040:2023 Revision No. & Date 0.0

Scope This method is applicable to Packaged Drinking water
Caution Flocculation technique is not suitable for particle-free water. Water
containing a substantial amount of particulate material can be difficult to
process by membrane filtration.
Principle This method is based on chemical flocculation. Three different
concentration techniques are described here. These are calcium carbonate
flocculation, ferrous (II) sulfate flocculation and membrane filtration.
These techniques allow the analysis of small volumes of water which can
be collected and delivered to the laboratory in a relatively short period of
time. The principles of IMS and staining and detection described in part
A method can be followed once oocysts and cysts have been concentrated
from water samples.

Apparatus/Instruments a) General apparatus: As given in Part A method

b) Specific apparatus required for concentration using calcium
carbonate flocculation / iron (II) sulfate flocculation
i) Measuring cylinders, 10 ml, 100 ml, 1 000 ml.
ii) Aspiration tubes.
iii) Vacuum source, with vacuum gauges and vacuum catch
bottles/reservoirs.
iv) Centrifuge, capable of 7 200 g and 1 100 g.
v) Centrifuge bottles, plastic, screwtop, 1 000 ml capacity.
vi) Centrifuge tubes, conical, plastic, 50 ml capacity.
vii) Flocculation container, 10 l carboy.
c) Specific apparatus required for concentration using 142 mm
membrane filtration
i) 142 mm stainless steel filter housing.
ii) 142 mm filter membranes, cellulose acetate, no greater than
2.0 µm pore size.

341
 iii) Peristaltic pump, capable of a flow rate of 1 l/min.
iv) Silicon tubing, for use with the peristaltic pump.
v) Seeding container, 10 l, if seeding filters is required.
vi) Suitable polythene bag, for washing the filter, e.g. Stomacher
bag.
vii) Centrifuge, capable of 1100 g.
viii) Centrifuge tubes, conical, plastic, capacity 50 ml.
Culture Media and a) Reagents required for use with calcium carbonate flocculation
Reagents i) Deionized water, 0.2 µm filtered at the point of use.
ii) Calcium chloride dihydrate, 1 mol/l.
iii) Sodium hydrogen carbonate, 1 mol/l.
iv) Sodium hydroxide, 1 mol/l,
v) Sulfamic acid, 10 %.
vi) Polyoxyethylene(20)sorbitan monolaurate (Tween 20),
volume fraction 0.01 %.
b) Reagents for use with iron (II) sulfate flocculation
i) Deionized water, 0.2 µm filtered at point of use.
ii) Sodium hydroxide, 1 mol/l.
iii) Iron(II) sulfate, 1 mol/l.
iv) Oxalic acid, 10 % (mass/volume).
v) Polyoxyethylene(20)sorbitan mono-oleate (Tween 80), 0.1 %
volume fraction.
vi) PBS-Tween, 10 mmol/l PBS (pH 7.4) and 0.1 % Tween 80.
vii) Polyoxyethylene(20)sorbitan monolaurate (Tween 20), 0.01 %
volume fraction.
c) Reagents required for eluting 142 mm membrane filters
i) Deionized water, 0.2 µm filtered at point of use.
ii) Tween 80 in deionized water, 0.1 % volume fraction.
Preparation of Reagents a) Calcium chloride dihydrate, 1 mol/l: Add 1470 g of
CaCl2·2H2O to 10 L of water. Store at room temperature (20 ± 5)
°C. Expiry date 3 months.
b) Sodium hydrogen carbonate, 1 mol/l: Add 840 g of NaHCO3 to
10 L of water. Store at room temperature (20 ± 5) °C. Expiry date

342
 3 months.
c) Sodium hydroxide, 1 mol/l: 400 g of NaOH per 10 Lof water.
Store at room temperature (20 ± 5) °C. Expiry date 3 months.
d) Sulfamic acid, 10 %: Add 1000 g of NH2SO3H to10 L of water.
Store at room temperature (20 ± 5) °C. Expiry date 3 months.
e) Iron(II) sulfate, 1 mol/L: Add 2780 g FeSO4 · 7 H2O to 10 L of
water. Store at room temperature (20 ± 5) °C. Expiry date 3
months.
Sample Preparation a) Sampling and Transport: As given in Part A method

b) Sample Concentration

i) Procedure for concentration using calcium carbonate

flocculation
 To a 10 L water sample, add 100 ml of 1 mol/L CaCl2 and 100 ml
of 1 mol/L NaHCO3 and then shake the container to mix. Add
100 ml of 1 mol/L NaOH and then shake the container to mix.

 Allow the contents to stand at room temperature for a minimum of

4 h. Samples shall be left for no longer than 24 h. After the floc
has settled, aspirate the supernatant to just above the floc
precipitate using a suction vacuum pressure of no greater than
20 kPa (0.2 bar). Take care not to disturb the precipitate.

 Add sufficient (100 ml to 200 ml) 10 % sulfamic acid to dissolve

the floc completely. Swirl the contents of the container to mix
and pour into a 1 L centrifuge bottle labelled with the sample
number.

NOTE: Larger deposits may require the use of two centrifuge bottles.

 Add (200 ± 20) ml of 0.01 % volume fraction Tween 20 to the

container, shake vigorously to rinse and add to the 1 L
centrifuge bottle.

 Add a further (200 ± 20) ml of 0.01 % volume fraction Tween 20

343
 to the container, rotate the container slowly to pick up froth
from around the edges and add to the 1 L centrifuge bottle.

 Carefully adjust the contents of the 1 L centrifuge bottle to pH 6

to 6.5 by the addition of 1 mol/L NaOH. Ensure that the pH
does not exceed this level as this will result in re-formation of
the floc.

 Balance 1 L centrifuge bottles in pairs to within 1 g using filtered

deionized water. Centrifuge at 7200 g maximum for 12 min
without braking during the deceleration phase.

 Immediately after centrifugation, remove the bottles from the

centrifuge and carefully aspirate the supernatant liquid to just
above the pellet using a vacuum pressure of no greater then 20
kPa (0.2 bar).

 Shake the remaining liquid vigorously to re-suspend the deposited

material and transfer to a 50 ml centrifuge tube.

 Using a wash bottle add (20 ± 2) ml of 0.01 % volume fraction

Tween 20 to the 1 L centrifuge bottle to suspend any remaining
sample debris and transfer to the 50 ml centrifuge tube. Make up
the volume in each tube to approximately 50 ml with filtered
deionized water.

 Centrifuge the 50 ml tubes at 1100x g for 15 min without braking

during the deceleration phase. Record the pellet volume
(volume of solids) immediately after centrifugation.

NOTE: IMS test kits usually have a defined pellet volume to be used for
the test, e.g. between 0.5 ml and 2 ml.

 Using a Pasteur pipette and a vacuum source of less than 20 kPa

(0.2 bar), carefully aspirate off the supernatant leaving 2 ml to 5
ml above the pellet. If no pellet is visible, take extra care to
avoid aspirating oocysts and cysts during this step.

344
  Add deionized water to the centrifuge tube to bring the total
volume to 10 ml. Vortex the tube for 10 s to 15 s to re-suspend
the pellet and either store the sample at (5 ± 3) °C for IMS.

 If the pellet volume exceeds that recommended by the

manufacturers of the IMS test kit, centrifuge the sample a
second time in a tube which permits the pellet volume to be
measured accurately. Sub-divide the sample into aliquots for
IMS such that each aliquot represents no more than the
maximum pellet volume recommended by the manufacturer and
make each aliquot up to 10 ml with filtered deionized water

ii) Procedure for concentration using iron(II) sulfate flocculation

 Fill a flocculation container with 10 L of surface water. Adjust the
pH to 9.0 ± 0.2 with 1 mol/L NaOH. Add 20 ml of the ferrous
sulfate solution while stirring and mix for 5 min at 280 rev/min.

 After overnight sedimentation (18 h) at room temperature (20 ± 5)

°C, remove the supernatant carefully until 4 cm to 5 cm above
the pellet (sediment).

 Place a beaker under the flocculation tank and collect the

sediment (600 ml to 1200 ml according to the surface water
used).

 Rinse the flocculation tank with a sufficient volume (100 ml to

150 ml) of 10 % oxalic acid in order to allow a complete
dissolution of the ferric sulfate precipitate.

 Rinse the tank with 150 ml of the 0.1 % volume fraction Tween
80 solution and add this solution to the sediment.

 Centrifuge washings at 1500 g for 10 min in 250 ml or 500 ml

conical centrifuge tubes (without braking). Carefully aspirate
the supernatant until 1 cm above the pellet. Do not disturb the

345
 pellet as the supernatant is discarded.

 Measure the pellet volume and re-suspend the pellet with 5

volumes of 0.1 % Tween 80 (5 volumes of Tween 80 for 1
volume of pellet). Repeat the last washing-centrifugation step.

 Centrifuge at 1500 g for 10 min and resuspend the pellet with 1

volume of PBS-Tween.

NOTE: The volume of PBS-Tween added is equivalent to the volume of

the pellet, total volume 60 ml to 80 ml usually.

 Control the pH of the water concentrate and adjust it to pH 7.2 to

7.4 with PBS (10 mmol/l pH 7,4) if necessary.

 Transfer the suspension to 50 ml centrifugation tubes. Add (20 ±

2) ml of 0.01 % volume fraction Tween 20 to the 1 L centrifuge
bottle to suspend any remaining sample debris and transfer to
the 50 ml centrifuge tubes. Make up the volume in each tube to
approximately 50 ml with filtered deionized water.

 Centrifuge the 50 ml tubes at 1100 g for 15 min without braking

during the deceleration phase. Record the pellet volume
(volume of solids) immediately after centrifugation.

 Using a Pasteur pipette and a vacuum source of less than 20 kPa

(0.2 bar), carefully aspirate off the supernatant leaving 2 ml to 5
ml above the pellet. If no pellet is visible, extra care shall be
taken to avoid aspirating oocysts and cysts during this step.

 Add deionized water to the centrifuge tube to bring the total

volume to 10 ml. Vortex the tube for 10 s to 15 s to re-suspend
the pellet and either store the sample at (5 ± 3) °C for IMS.

 If the pellet volume exceeds that recommended by the

manufacturer of the IMS test kit, centrifuge the sample a second
time in a tube that permits the pellet volume to be measured
accurately. Subdivide the sample into aliquots for IMS such that

346
 each aliquot represents the maximum pellet volume
recommended by the manufacturer and make up each aliquot to
10 ml with deionized water.

iii) Procedure for concentration using membrane filtration

 Place the membrane filter into the housing and clamp on the upper
part. Pump the water sample through the filter at a rate of no
greater than 1.5 L/min. Rinse the container with 2 L of filtered
deionized water and pump the washings through the filter.

 Remove the filter from the filter housing and place into a suitable
clean polythene bag (e.g. a stomacher bag). Add 25 ml of 0.1 %
volume fraction Tween 80 and gently rub the surface of the
filter for 1 min through the bag to remove particulate material.

 Decant the washings into a 50 ml centrifuge tube. Repeat the

wash procedure with a further 25 ml of 0.1 % volume fraction
Tween 80 and add this to the centrifuge tube.

 Centrifuge the tube at 1100x g for 15 min.

 Using a Pasteur pipette and a vacuum source of less than 20 kPa

(0.2 bar), carefully aspirate off the supernatant leaving 2 ml to 5
ml above the pellet. If no pellet is visible, extra care shall be
taken to avoid aspirating oocysts and cysts during this step.

 Add deionized water to the centrifuge tube to bring the total

volume to 10 ml. Vortex the tube for 10 s to 15 s to re-suspend
the pellet and either store the sample at (5 ± 3) °C for IMS.

 If the pellet volume exceeds that recommended by the

manufacturer of the IMS test kit, centrifuge the sample a second
time in a tube that permits the pellet volume to be measured
accurately. Subdivide the sample into aliquots for IMS such

347
 each aliquot represents the maximum pellet volume
recommended by the manufacturer and make up each aliquot to
9 ml with deionized water.

Method of analysis As given in Method A

Calculation with units of As given in Method A

expression
Inference As given in Method A
(Qualitative Analysis)
Reference 1) IS 10500:2012 - Drinking Water — Specification (Second Revision)
2) ISO 15553:2006 - Water quality — Isolation and identification of
Cryptosporidium oocysts and Giardia cysts from water
Approved by Scientific Panel on Methods of Sampling and Analysis

348
 APPENDIX A

Table 1—MPN values per gram of sample and 95% confidence limits
(when three test portions of 1g, three of 0.1 g and three of 0.01g are used)

Number positive results for inoculums 95% Confidence limits

volume, ml or g MPN
/ml or /g
1.00 0.10 0.01 Lower Upper
0 0 0 0 0 1.1
0 1 0 0.30 0.04 2.3
1 0 0 0.36 0.05 2.7
1 0 1 0.72 0.17 3.0
1 1 0 0.74 0.18 3.1
1 2 0 1.1 0.35 3.7
2 0 0 0.92 0.21 4.0
2 0 1 1.4 0.42 4.8
2 1 0 1.5 0.43 5.0
2 1 1 2.0 0.69 6.0
2 2 0 2.1 0.71 6.2
3 0 0 2.3 0.55 9.7
3 0 1 3.8 0.93 16
3 1 0 4.3 0.95 19
3 1 1 7.5 1.9 30
3 1 2 12 3.6 37
3 2 0 9.3 2.2 40
3 2 1 15 4.4 51
3 2 2 21 7.2 64
3 3 0 24 5.6 100
3 3 1 46 9.6 220
3 3 2 110 25 480
3 3 3 ∞ 36 ∞

349
Table 2 – Most Probable Number (MPN) of organisms present per 100 ml of Sample
and Confidence Limits using 5 tubes of 10 ml, 5 tubes of 1 ml and 5 tubes of 0.1 ml

Number of Positive Tubes Most Limits within which MPN

Probable per 100 ml can lie
10 ml Tubes 1 ml Tubes 0.1 ml Tubes Number Lower Limit Upper Limit
(MPN) per
100 ml
(1) (2) (3) (4) (5) (6)
0 0 1 2 <0.5 7
0 0 2 4 <0.5 11
0 1 0 2 <0.5 7
0 1 1 4 <0.5 11
0 1 2 6 <0.5 15
0 2 0 4 <0.5 11
0 2 1 6 <0.5 15
0 3 0 6 <0.5 15
1 0 0 2 <0.5 7
1 0 1 4 <0.5 11
1 0 2 6 <0.5 15
1 0 3 8 1 19
1 1 0 4 <0.5 11
1 1 1 6 <0.5 15
1 1 2 8 1 19
1 2 0 6 <0.5 15
1 2 1 8 1 19
1 2 2 10 2 23
1 3 0 8 1 19
1 3 1 10 2 23
1 4 0 11 2 25
2 0 0 5 <0.5 13
2 0 1 7 1 17
2 0 2 9 2 21
2 0 3 12 3 28
2 1 0 7 1 17
2 1 1 9 2 21
2 1 2 12 3 28
2 2 0 9 2 21
2 2 1 12 3 28
2 2 2 14 4 34
2 3 0 12 3 28
2 3 1 14 4 34

350
 Number of Positive Tubes Most Limits within which MPN
Probable per 100 ml can lie
10 ml Tubes 1 ml Tubes 0.1 ml Tubes Number Lower Limit Upper Limit
(MPN) per
100 ml
(1) (2) (3) (4) (5) (6)
2 4 0 15 4 37
3 0 0 8 1 19
3 0 1 11 2 25
3 0 2 13 3 31
3 1 0 11 2 25
3 1 1 14 4 34
3 1 2 17 5 46
3 1 3 20 6 60
3 2 0 14 4 34
3 2 1 17 5 46
3 2 2 20 6 60
3 3 0 17 5 46
3 3 1 21 7 63
3 4 0 21 7 63
3 4 1 24 8 72
3 5 0 25 8 75
4 0 0 13 3 31
4 0 1 17 5 46
4 0 2 21 7 63
4 0 3 25 8 75
4 1 0 17 5 46
4 1 1 21 7 63
4 1 2 26 9 78
4 2 0 22 7 67
4 2 1 26 9 78
4 2 2 32 11 91
4 3 0 27 9 80
4 3 1 33 11 93
4 3 2 39 13 106
4 4 0 34 12 96
4 4 1 40 14 108
4 5 0 41 14 110
4 5 1 48 16 124
5 0 0 23 7 70
5 0 1 31 11 89
5 0 2 43 15 114

351
 Number of Positive Tubes Most Limits within which MPN
Probable per 100 ml can lie
10 ml Tubes 1 ml Tubes 0.1 ml Tubes Number Lower Limit Upper Limit
(MPN) per
100 ml
(1) (2) (3) (4) (5) (6)
5 0 3 58 19 144
5 0 4 76 24 180
5 1 0 33 11 93
5 1 1 46 16 120
5 1 2 63 21 154
5 1 3 84 26 197
5 2 0 49 17 126
5 2 1 70 23 168
5 2 2 94 28 219
5 2 3 120 33 281
5 2 4 148 38 366
5 2 5 177 44 515
5 3 0 79 25 187
5 3 1 109 31 253
5 3 2 141 37 343
5 3 3 175 44 503
5 3 4 212 53 669
5 3 5 253 77 788
5 4 0 130 35 302
5 4 1 172 43 486
5 4 2 221 57 698
5 4 3 278 90 479
5 4 4 345 117 999
5 4 5 426 145 1161
5 5 0 240 68 754
5 5 1 348 118 1005
5 5 2 542 180 1405

352
 Table 3: Most Probable Number (MPN) of Organisms present per 100 ml of Sample
and Confidence Limits using 5 tubes of 10 ml, 5 tubes of 1 ml and 5 tubes of 0.1 ml

Combination MPN 95 percent Combination MPN 95 percent

of Positives Index/ Confidence Limits of Positives Index/ Confidence Limits
100 ml Lower Upper 100 ml Lower Upper
0-0-0 <2 - - 420 22 9.0 56
0-0-1 2 1.0 10 421 26 12 65
0-1-0 2 1.0 10 430 27 12 67
0-2-0 2 1.0 13 431 33 15 77
440 34 16 80
500 23 9.0 86
1-0-0 2 1.0 11 501 30 10 110
1-0-1 4 1.0 15 502 40 20 140
1-1-0 4 1.0 15 510 30 10 120
1-1-1 6 2.0 18 511 50 20 150
1-2-0 6 2.0 18 512 60 30 180
2-0-0 4 1.0 17 520 50 20 170
2-0-1 7 2.0 20 521 740 30 210
2-1-0 7 2.0 21 522 90 40 250
2-1-1 9 3.0 24 530 80 30 250
2-2-0 9 3.0 25 531 110 40 300
2-3-0 12 5.0 29 532 140 60 360
3-0-0 8 3.0 24 533 170 80 410
3-0-1 11 4.0 29 540 130 50 390
3-1-0 11 4.0 29 541 170 70 480
3-1-1 14 6.0 35 542 220 100 580
3-2-0 14 6.0 35 543 280 120 690
3-2-1 17 7.0 40 544 350 160 820
550 240 100 940
4-0-0 13 5.0 38 551 300 100 1300
4-0-1 17 7.0 45 552 500 200 2000
4-1-0 17 7.0 46 553 900 300 2900
4-1-1 21 9.0 55 554 1600 600 5300
4-1-2 26 12 63 555 1600 - -

353
 Annexure 1 — Test microorganisms and performance criteria for culture media commonly used in food microbiology

Microorgan- WDCM Reference Method of

Mediaa Typee Function Incubation Control strain Criteria Characteristic reaction
ism numberc media control

Agar Listeria S Listeria Productiv- Listeria

00021b
according to monocytogenes ity monocytogenes 4b Quantita- Blue green colonies with opaque
Ottaviani and TSA PR ≥ 0,5
Listeria tive halo
Agosti 00109
monocytogenes 1/2a
Selectivity (44 ± 4) h/ 00012
(37 ± 1) °C Escherichia colid
or 00013 Total inhibi-
— Qualitative —
00009 tion (0)
Enterococcus faecalisd
or 00087
Specificity Blue green colonies without
Listeria innocua 00017 — Qualitative —
opaque halo
Baird- S Coagulase- Productiv- (24 ± 2) h
00034b Quantita- Black or grey colonies with clear
Parker positive ity to (48 ± 2) h/ Staphylococcus aureus TSA PR ≥ 0,5
00032 tive halo (egg yolk clearing reaction)
staphylococci (37 ± °1) °C
Selectivity (48 ± 2) h/ 00012 Total inhibi-
Escherichia colid — Qualitative —
(37 ± 1) °C 00013 tion (0)
Specificity Staphylococcus
(24 ± 2) h to 00159b
saprophyticus Black or grey colonies without egg
(48 ± 2) h/ — Qualitative —
Staphylococcus yolk clearing reaction
(37 ± 1) °C 00036
epidermidis
BGBLB L Coliforms Productiv- 00012b Turbidity
Escherichia coli
ity 00013 (2)f
— Qualitative and gas in Gas production and turbidity
(24 ± 2) h to Citrobacter freundii 00006 Durham
(48 ± 2) h/ tube
Selectivity (30 ± 1) °C Partial
00009 inhibition
Enterococcus faecalisd — Qualitative —
00087 without gas
production
CFC S Pseudomonas Productiv- Pseudomonas
00115b Quantita-
spp. ity fluorescens TSA PR ≥ 0,5 —
(44 ± 4) h/ tive
Pseudomonas fragi 00116
(25 ± 1) °C
Selectivity 00012 Total inhibi-
Escherichia colid — Qualitative —
00013 tion (0)

354
 Annexure 1 — (continued)

Microorgan- WDCM Reference Method of

Mediaa Typee Function Incubation Control strain Criteria Characteristic reaction
ism numberc media control

DG18 S Yeasts and Productiv- Saccharomyces

00058b
moulds ity cerevisiae
Wallemia sebi 00182b Quantita- Characteristic colony/propagules
SDA PR ≥ 0,5
tive according to each species
Aspergillus restrictus 00183
5 days/
(25 ± 1) °C Eurotium rubrum 00184
Selectivity 00012
Escherichia coli
or 00013g
— Qualitative No growth —
Bacillus subtilis subsp.
spizizenii 00003

DRBC S Yeasts and Productiv- Saccharomyces

00058b
moulds ity cerevisiae
Aspergillus brasiliensis 00053b Quantita- Characteristic colony/propagules
SDA PR ≥ 0,5
tive according to each species
Candida albicans 00054
5 days/
(25 ± 1) °C Mucor racemosus 00181
Selectivity 00012
Escherichia coli
or 00013g
— Qualitative No growth —
Bacillus subtilis subsp.
spizizenii 00003

EC L Escherichia coli Productiv- Turbidity

ity (2)f
00012b
(24 ± 2) h to Escherichia coli — Qualitative and gas in Gas production and turbidity
00013
(48 ± 2) h/ Durham
(44 ± 1) °C tube
Selectivity Pseudomonas
00025 — Qualitative No growth —
aeruginosa
IS (“TS”) S Sulfite-reducing Productiv- TSA or
bacteria ity other non-
(24 ± 3) h 00007b selective Quantita-
to (48 ± 2) h/ Clostridium perfringens PR ≥ 0,5 Black colonies
00080 medium tive
(37 ± 1) °C for anaer-
anaerobic obes
atmosphere
Specificity 00012
Escherichia colid — Qualitative — No blackening
00013
 Annexure 1 — (continued)

Microorgan- WDCM Reference Method of

Mediaa Typee Function Incubation Control strain Criteria Characteristic reaction
ism numberc media control

LST L Coliforms Productiv- 00012b Turbidity

Escherichia coli
ity 00013 (2)f
(24 ± 2) h to — Qualitative and gas in Gas production and turbidity
(48 ± 2) h/ Citrobacter freundii 00006 Durham
(30 ± 1) °C tube
Selectivity 00009
Enterococcus faecalisd — Qualitative No growth —
00087
Escherichia coli Productiv- Turbidity
ity (2)fi
00012b
(24 ± 2) h to Escherichia coli — Qualitative and gas in Gas production and turbidity
00013 Durham
(48 ± 2) h/
(37 ± 1) °C tube
Selectivity 00009
Enterococcus faecalisd — Qualitative No growth —
00087
mCCDA S Campylobacter Productiv- Campylobacter jejuni 00156b Blood agar Quantita- PR ≥ 0,5 Greyish, flat and moist, sometimes
ity (44 ± 4) h/ 00005 tive with metallic sheen
(41,5 ± 1) °C
microaerobic Campylobacter coli 00004
Selectivity atmosphere Escherichia colid 00012 Qualitative Total or
or 00013 — partial —
inhibition
(0-1)
— Qualitative Total inhibi- —
Staphylococcus aureus 00034 tion (0)
MRS S Lactic acid Productiv- Lactobacillus sakei 00015b Media
bacteria ity (72 ± 3) h/ batch MRS Quantita- Characteristic colonies according to
Lactococcus lactis 00016b PR ≥ 0,7
(30 ± 1) °C already tive each species
Pediococcus pentosaceus 00158 validated

Selectivity 00012
(72 ± 3) h/ Escherichia colid Total inhibi-
or 00013 — Qualitative —
(30 ± 1) °C tion (0)
Bacillus cereus 00001
MYP S Bacillus cereus Productiv- (24 ± 3) h to
Quantita- Pink colonies with precipitation
ity (44 ± 4) h/ Bacillus cereus 00001 TSA PR ≥ 0,5
tive halo
(30 ± 1) °C
Selectivity 00012 Total inhibi-
Escherichia colid — Qualitative —
(44 ± 4) h/ or 00013 tion (0)
Specificity (30 ± 1) °C Bacillus subtilis subsp. Yellow colonies without precipita-
00003 — Qualitative —
spizizenii tion halo
Annexure 1 — (continued)

Microorgan- WDCM Reference Method of

Mediaa Typee Function Incubation Control strain Criteria Characteristic reaction
ism numberc media control

RPFA S Coagulase- Productiv- (24 ± 2) h

00034b Quantita- Black or grey colonies with opac-
positive ity to (48 ± 2) h/ Staphylococcus aureus TSA PR ≥ 0,5
00032 tive ity halo
staphylococci (37 ± 1) °C
Selectivity (48 ± 2) h/ 00012 Total inhibi-
Escherichia colid — Qualitative —
(37 ± 1) °C or 00013 tion (0)
Specificity Staphylococcus
(24 ± 2) h to 00159b
saprophyticus Black or grey colonies without
(48 ± 2) h/ — Qualitative —
Staphylococcus opacity halo
(37 ± 1) °C 00036
epidermidis
PPA S Pseudomonas Productiv- Pseudomonas
00115b
spp. ity fluorescens Quantita-
TSA PR ≥ 0,5 —
(48 ± 2) h/ Pseudomonas tive
00025
(25 ± 1) °C aeruginosa
Selectivity 00012 Total inhibi-
Escherichia colid — Qualitative —
or 00013 tion (0)
TBX S β-d- Productiv- 00012d
Quantita-
Glucuronidase- ity Escherichia colih 00013d TSA PR ≥ 0,5 Blue colonies
tive
positive 00202b
Escherichia coli
Selectivity (21 ± 3) h/ 00009 Total inhibi-
Enterococcus faecalisd Qualitative —
(44 ± 1) °C 00087 tion (0)
Specificity Citrobacter freundii
00006b
Pseudomonas Qualitative — White to green-beige colonies
00025
aeruginosa
TSC (SC) S Clostridium Productiv- TSA or
perfringens ity other non-
00007b selective Quantita-
(20 ± 2) h/ Clostridium perfringens PR ≥ 0,5 Black colonies
(37 ± 1) °C 00080 medium tive
anaerobic for anaer-
atmosphere obes
Selectivity 00012 Total inhibi-
Escherichia colid — Qualitative —
or 00013 tion (0)
 Annexure 1 — (continued)

Microorgan- WDCM Reference Method of

Mediaa Typee Function Incubation Control strain Criteria Characteristic reaction
ism numberc media control

VRBG S Entero- Productiv- 00012b

Escherichia coli
bacteriaceae ity 00013
Quantita- Pink to red colonies with or with-
Salmonella TSA PR ≥ 0,5
(24 ± 2) h/ Typhimuriumd,i 00031 tive out precipitation halo
(37 ± 1) °C Salmonella Enteritidisd,i 00030

Selectivity 00009 Total inhibi-

Enterococcus faecalisd — Qualitative —
00087 tion (0)
VRBL S Coliforms Productiv- 00012b Quantita- Purplish-red colonies with or
Escherichia coli TSA PR ≥ 0,5
ity 00013 tive without precipitation halo
Selectivity (24 ± 2) h/ 00009 Total inhibi-
Enterococcus faecalisd — Qualitative —
(30 ± 1) °C 00087 tion (0)
Specificity Pseudomonas
00025 Qualitative — Colourless to beige colonies
aeruginosa
Mediaa Typee Micro- Function Incubation Control strains WDCM Reference Method of Criteria Characteristic reactions
organisms num- media control
bersc
PCA S Colony count Productiv- Bacillus subtilis subsp.
00003b
MPC ity spizizenii
A (72 ± 3) h/ Quantita-
00012b TSA PR ≥ 0,7 —
(30 ± 1) °C Escherichia coli tive
00013
Staphylococcus aureus 00034
Annexure 1 — (continued)

WDCM
Micro- Reference Method of Characteristic reactions of tar-
Mediaa Typee Function Incubation Control strains num- Criteria
organisms media control get microorganism
bersc
Bolton L Campylobacter Productiv- 00156
Campylobacter jejunid
ity or 00005
+ Escherichia colid 00012
or 00013
(5 ± 1) h/ + Proteus mirabilis 00023 > 10 colonies Greyish, flat and moist, sometimes
— Qualitative
(37 ± 1) °C then on mCCDA with metallic sheen
(44 ± 4) h/ Campylobacter coli 00004
(41,5 ± 1) °C + Escherichia colid 00012
microaerobic or 00013
atmosphere
+ Proteus mirabilis 00023
Selectivity 00012 Total inhibi-
Escherichia colid
or 00013 — Qualitative tion (0) on —
Proteus mirabilis 00023 TSA

EE L Entero- Productiv- 00012b

Escherichia coli
bacteriaceae ity 00013

+ Enterococcus faecalisd 00009

or 00087
> 10 colonies Pink to red colonies with or with-
Salmonella 00031 — Qualitative
on VRBG out precipitation halo
(24 ± 2) h/ Typhimuriumd,i or
(37 ± 1) °C Salmonella Enteritidisd,i 00030
00009
+ Enterococcus faecalisd
or 00087
Selectivity Total inhibi-
00009
Enterococcus faecalisd — Qualitative tion (0) on —
or 00087 TSA
Annexure 1 — (continued)

WDCM
Micro- Reference Method of Characteristic reactions of tar-
Mediaa Typee Function Incubation Control strains num- Criteria
organisms media control get microorganism
bersc
Fraser L Listeria Productiv- Listeria
00021b
monocytogenes ity monocytogenes 4b
00012
+ Escherichia colid
or 00013
> 10 colonies
+ Enterococcus faecalisd 00009 on Agar Lis-
or 00087 teria Blue green colonies with opaque
— Qualitative
according halo
Listeria
00109 to Ottaviani
monocytogenes 1/2a
(48 ± 2) h/ and Agosti
(37 ± 1) °C 00012
+ Escherichia colid
or 00013

+ Enterococcus faecalisd 00009

or 00087
Selectivity Total inhibi-
00012
Escherichia colid — Qualitative tion (0) on —
or 00013 TSA
00009 < 100 colo-
Enterococcus faecalisd — Qualitative —
or 00087 nies on TSA
Giolitti Cantoni L Coagulase- Productiv- (24 ± 2) h to Staphylococcus aureus 00034b
positive ity (48 ± 2) h/
staphylococci (37 ± 1) °C 00012 > 10 Characteristic colonies according to
+ Escherichia colid
or 00013 colonies on each medium for Baird Parker and
— Qualitative
Staphylococcus aureus 00032 Baird Parker RPFA)
or RPFA
00012
+ Escherichia colid
or 00013
Selectivity (48 ± 2) h/ Total inhibi-
00012
(37 ± 1) °C Escherichia colid — Qualitative tion (0) on —
or 00013 TSA
 Annexure 1 — (continued)

WDCM
Micro- Reference Method of Characteristic reactions of tar-
Mediaa Typee Function Incubation Control strains num- Criteria
organisms media control get microorganism
bersc
Half-Fraser L Listeria Productiv- Listeria
00021b
monocytogenes ity monocytogenes 4b
00012
+ Escherichia colid
or 00013
> 10 colonies
+ Enterococcus faecalisd 00009 on Agar Lis-
or 00087 teria Blue green colonies with opaque
— Qualitative
according halo
Listeria
00109 to Ottaviani
monocytogenes 1/2a
(24 ± 2) h/ and Agosti
(30 ± 1) °C 00012
+ Escherichia colid
or 00013

+ Enterococcus faecalisd 00009

or 00087
Selectivity Total inhibi-
00012
Escherichia colid — Qualitative tion (0) on —
or 00013 TSA
00009 < 100 colo-
Enterococcus faecalisd — Qualitative —
or 00087 nies on TSA
ITC L Yersinia Productiv- Yersinia enterocolitica 00038b
enterocolitica ity
00012
+ Escherichia colid
or 00013
+ Pseudomonas
aeruginosa 00025 > 10 colonies
Characteristic colonies according to
— Qualitative on CIN or
each medium
Yersinia enterocolitica 00160 SSDC
(44 ± 4) h/
(25 ± 1) °C 00012
+ Escherichia colid
or 00013
+ Pseudomonas
aeruginosa 00025

Selectivity Pseudomonas Total inhibi-

00025
aeruginosa — Qualitative tion (0) on —
Proteus mirabilis 00023 TSA
Annexure 1 — (continued)

WDCM
Micro- Reference Method of Characteristic reactions of tar-
Mediaa Typee Function Incubation Control strains num- Criteria
organisms media control get microorganism
bersc
MKTTn L Salmonella Productiv- Salmonella Enteritidisd,i
00030
ity Salmonella
Typhimuriumd,i 00031 > 10 colonies
on XLD
Characteristic colonies according to
00012 — Qualitative or other
+ Escherichia colid each medium
or 00013 medium of
choice
+ Pseudomonas aerugi- 00025
(24 ± 3) h/
nosa
(37 ± 1) °C
Selectivity Partial inhi-
00012 bition ≤ 100
Escherichia colid — Qualitative —
or 00013 colonies on
TSA
00009 < 10 colonies
Enterococcus faecalisd — Qualitative —
or 00087 on TSA
MSRVk SS Salmonella Productiv- Salmonella Enteritidisd,i 00030 Grey-white,
ity turbid zone
extending
out from
inoculated
drop(s).
After
Possible extra: characteristic colo-
Salmonella — Qualitative 24–48 h,
00031 nies after subculturing on XLDk
Typhimuriumd,i the turbid
zone(s) will
be (almost)
fully
2 × (24 ± 3) h/ migrated
(41,5 ± 1) °C over the
plate.

Selectivity Possible
growth at the
place of the
00012
Escherichia colid — Qualitative inocu- lated —
or 00013 drop(s)
without a
turbid zone.
00009
Enterococcus faecalisd — Qualitative No growth —
or 00087
Annexure 1 — (continued)

WDCM
Micro- Reference Method of Characteristic reactions of tar-
Mediaa Typee Function Incubation Control strains num- Criteria
organisms media control get microorganism
bersc
MMG L β-d- Productiv- 00012b Acid produc-
Glucuronidase- ity Escherichia coli — Qualitative Colour change to yellow
(24 ± 2) h/ 00013 tion
positive E. coli
(37 ± 1) °C
Selectivity 00009
Enterococcus faecalisd — Qualitative No growth —
or 00087
PSB L Yersinia Productiv- Yersinia enterocolitica 00038b
enterocolitica ity
00012
+ Escherichia colid
or 00013
+ Pseudomonas
aeruginosa 00025 > 10 colonies
Characteristic colonies according to
— Qualitative on CIN or
each medium
Yersinia enterocolitica 00160 SSDC
3 to 5 days/
(25 ± 1) °C 00012
+ Escherichia colid
or 00013
+ Pseudomonas
aeruginosa 00025

Selectivity Pseudomonas Total inhibi-

00025b
aeruginosa — Qualitative tion (0) on —
Proteus mirabilis 00023 TSA

RVS L Salmonella Productiv- Salmonella Enteritidisd,i

00030
ity Salmonella
Typhimuriumd,i 00031 > 10 colonies
on XLD
Characteristic colonies according to
00012 — Qualitative or other
+ Escherichia coli d each medium
or 00013 medium of
choice
+ Pseudomonas 00025
(24 ± 3) h/
aeruginosa
(41,5 ± 1) °C
Selectivity Partial inhi-
00012 bition ≤ 100
Escherichia colid — Qualitative —
or 00013 colonies on
TSA
00009 < 10 colonies
Enterococcus faecalisd — Qualitative —
or 00087 on TSA
Annexure 1 — (continued)

WDCM
Micro- Reference Method of Characteristic reactions of tar-
Mediaa Typee Function Incubation Control strains num- Criteria
organisms media control get microorganism
bersc
TSPB L Bacillus cereus Productiv- > 10 colonies
Characteristic colonies according to
ity Bacillus cereus 00001 — Qualitative on PEMBA
each medium
(48 ± 4) h/ or MYP
Selectivity (30 ± 1) °C Total inhibi-
00012
Escherichia colid — Qualitative tion (0) on —
or 00013 TSA
WDCM
Micro- Reference Method of
Mediaa Typee Function Incubation Control strains num- Criteria Characteristic reactions
organisms media control
bersc
BHI L Coagulase- Productiv- Turbidity
(24 ± 2) h/
positive ity Staphylococcus aureus 00034 — Qualitative —
staphylococci (37 ± 1) °C (1–2)f

Brucella L Campylobacter Productiv- 2 to 5 days/ 00156

Campylobacter jejunid Turbidity
ity (41,5 ± 1) °C 00005 — Qualitative —
microaerobic (1–2)f
atmosphere Campylobacter colid 00004
Diluents for L Dilution liquids Diluent 00012
Escherichia colid ±30 %
special pur- or 00013
poses e.g. colonies/
45 min – 1 h/ Quantita-
TSA T0 (±30 % —
BPW with 20 °C to 25 °C tive
Staphylococcus aureus 00034b of original
bromo-cresol- count)
purple
Quarter- L Dilution liquids Diluent
00012
strength Escherichia colid
Ringer’s or 00013
±30 %
Peptone colonies/
45 min – 1 h/ Quantita-
solution TSA T0 (±30 % —
20 °C to 25 °C tive
of original
Peptone-salt Staphylococcus aureus 00034b count)
Phosphate
buffer solution
Thioglycollate L Clostridium Productiv- (21 ± 3) h/ Turbidity
perfringens ity Clostridium perfringens 00007 — Qualitative —
(37 ± 1) °C (1–2)f
TSYEB L Listeria Productiv- Listeria
00021b
monocytogenes ity (21 ± 3) h/ monocytogenes 4b Turbidity
— Qualitative —
(25 ± 1) °C Listeria (1–2)f
00109
monocytogenes 1/2a
Annexure 1 — (continued)

WDCM
Micro- Reference Method of
Mediaa Typee Function Incubation Control strains num- Criteria Characteristic reactions
organisms media control
bersc
Agar Listeria S Listeria Productiv- Listeria
00021b
according to monocytogenes ity monocytogenes 4b Good Blue green colonies with opaque
Ottaviani and — Qualitative
Listeria growth (2) halo
Agostij 00109
monocytogenes 1/2a
Selectivity (44 ± 4) h/ 00012
(37 ± 1) °C Escherichia colid
or 00013 Total inhibi-
— Qualitative —
00009 tion (0)
Enterococcus faecalisd
or 00087
Specificity Blue green colonies without
Listeria innocua 00017 — Qualitative —
opaque halo
mCCDAj S Campylobacter Productiv- Campylobacter 00156b
ity jejuni 00005 Good Greyish, flat and moist colonies,
— Qualitative
growth (2) sometimes with metallic sheen
Campylobacter coli 00004
(44 ± 4) h/
Selectivity (41,5 ± 1) °C Total or
microaerobic 000012 partial inhi-
Escherichia colid — Qualitative bition No characteristic colonies
atmosphere or 00013
(0 − 1)
Total inhibi-
Staphylococcus aureus 00034 — Qualitative —
tion (0)
CT-SMAC S Escherichia coli Productiv- 00014
O157 ity Transparent colonies with a pale
Good
Escherichia coli O157:H7 (non- — Qualitative yellowish-brown appearance and a
toxigenic growth (2) diameter ~1 mm
(21 ± 3) h/ strain)
Selectivity (37 ± 1) °C 00032 Total inhibi-
Staphylococcus aureusd — Qualitative —
or 00034 tion (0)
00012 Partial inhi-
Escherichia colid — Qualitative Growth of some pink colonies
or 00013 bition (1)
Annexure 1 — (continued)

WDCM
Micro- Reference Method of
Mediaa Typee Function Incubation Control strains num- Criteria Characteristic reactions
organisms media control
bersc
CPC S Vibrio spp. Productiv- Good Yellow colonies surrounded by a
Vibrio vulnificus 00187b — Qualitative
other than ity growth (2) yellow coloration in the medium
mCP Vibrio
para- Vibrio cholerae Good Purple colonies surrounded by a
C (24 ± 3) h/ 00203b — Qualitative
haemolyticus/ non-O1/non-O139 growth (2) purple coloration in the medium
(37 ± 1) °C
V. cholerae Selectivity 00012
Total inhibi-
Escherichia colid or 00013 — Qualitative —
tion (0)
or 00090
MYPj S Bacillus cereus Productiv- (21 ± 3) h
Good Pink colonies with precipitation
ity to 48 h/ Bacillus cereus 00001 — Qualitative
growth (2) halo
(30 ± 1) °C
Selectivity 00012 Total inhibi-
Escherichia colid — Qualitative —
(44 ± 4) h/ or 00013 tion (0)
Specificity (30 ± 1) °C Bacillus subtilis subsp. Yellow colonies without precipita-
00003 — Qualitative —
spizizenii tion halo
PEMBA S Bacillus cereus Productiv- (21 ± 3) h to
Good Turquoise-blue colonies with
ity (44 ± 4) h/ Bacillus cereus 00001 — Qualitative
growth (2) precipitation halo
(37 ± 1) °C
Selectivity 00012 Total inhibi-
Escherichia colid — Qualitative —
(44 ± 4) h/ or 00013 tion (0)
Specificity (37 ± 1) °C Bacillus subtilis subsp. White colonies without precipita- tion
00003 — Qualitative —
spizizenii halo
SDS S Vibrio spp. Productiv- Good Purple/green colonies with an
Vibrio vulnificus 00187b — Qualitative
other than ity growth (2) opaque halo
Vibrio
para- Vibrio cholerae Good Yellow colonies with an opaque
(24 ± 3) h/ 00203b — Qualitative
haemolyticus/ non-O1/non-O139 growth (2) halo
(37 ± 1) °C
V. cholerae Selectivity 00012
Total inhibi-
Escherichia colid or 00013 — Qualitative —
tion (0)
or 00090
Annexure 1 — (continued)

WDCM
Micro- Reference Method of
Mediaa Typee Function Incubation Control strains num- Criteria Characteristic reactions
organisms media control
bersc
TBXj S β-d- Productiv- 00012d
Good
Glucuronidase- ity Escherichia colih 00013d — Qualitative Blue colonies
positive growth (2)
00202b
Escherichia coli
Selectivity (21 ± 3) h/ 00009 Total inhibi-
Enterococcus faecalisd — Qualitative —
(44 ± 1) °C or 00087 tion (0)
Specificity Citrobacter freundii
00006b
Pseudomonas — Qualitative — White to green-beige colonies
00025
aeruginosa
TCBS S Vibrio para- Productiv- Good Green colonies
Vibrio parahaemolyticus 00185b — Qualitative
haemolyticus / ity growth (2) (sucrose negative)
V. cholerae
Good Yellow colonies
(24 ± 3) h/ Vibrio furnissii 00186b — Qualitative
growth (2) (sucrose positive)
(37 ± 1) °C
Selectivity 00012
Total inhibi-
Escherichia colid or 00013 — Qualitative —
tion (0)
or 00090
VRBGj S Entero- Productiv- 00012b
Escherichia coli
bacteriaceae ity 00013
Salmonella Good Pink to red colonies with or with-
00031 — Qualitative
(24 ± 2) h/ Typhimuriumd,i growth (2) out precipitation halo
(37 ± 1) °C Salmonella
00030
Enteritidisd,i
Selectivity 00009 Total inhibi-
Enterococcus faecalisd — Qualitative —
or 00087 tion (0)
 Table E.1 — (continued)

WDCM
Micro- Reference Method of
Mediaa Typee Function Incubation Control strains num- Criteria Characteristic reactions
organisms media control
bersc
XLD S Salmonella Productiv- Salmonella Colonies with black centre and a
00031
ity Typhimuriumd,i Good lightly transparent zone of reddish
— Qualitative
growth (2) colour due to the colour change of
Salmonella Enteritidisd,i 00030 the medium
Selectivity (24 ± 3) h/ Growth or
(37 ± 1) °C 00012 partial
Escherichia colid — Qualitative Yellow colonies
or 00013 inhibition
(0 − 1)
00009 Total inhibi-
Enterococcus faecalisd — Qualitative —
or 00087 tion (0)
WDCM
Micro- Reference Method of
Mediaa Typee Function Incubation Control strains num- Criteria Characteristic reactions
organisms c media control
bers
Nutrient agarl S Entero- Productiv- (24 ± 2) h/ 00012b
Escherichia coli
bacteriaceae ity (37 ± 1) °C 00013
Salmonella Salmonella
(24 ± 2) h/ 00031 Good
Typhimuriumd,i — Qualitative —
(37 ± 1) °C growth (2)
Salmonella Enteritidisd,i 00030
Yersinia (24 ± 2) h/ 00038b
Yersinia enterocolitica
enterocolitica (30 ± 1) °C 00160
TSYEA S Listeria Productiv- Listeria
00021b
monocytogenes ity (21 ± 3) h/ monocytogenes 4b Good
— Qualitative —
(37 ± 1) °C Listeria growth (2)
00109
monocytogenes 1/2a
Annexure 1 — (continued)

WDCM
Micro- Reference Method of
Mediaa Typee Function Incubation Control strains num- Criteria Characteristic reactions
organisms media control
bersc
BPWm L Diluent for all Dilution 00012b ±30 %
Escherichia coli
enumerations 00013 colonies/
45 min – 1 h/ Quantita-
of microorgan- TSA T0 (±30 % —
20 °C to 25 °C Staphylococcus tive
isms 00034b of original
aureus count)
Diluent for Dilution Listeria ±30 %
00021b
Listeria monocytogenes 4b colonies/
(1 h ± 5°min) / Quantita-
monocytogenes TSA T0 (±30 % —
(20 ± 2) °C Listeria tive
enumeration 00109 of original
monocytogenes 1/2a count)
Pre-enrichment Productiv- Salmonella
(18 ± 2) h/ 00031 Turbidity
for Salmonella ity Typhimuriumd,i — Qualitative —
detection (37 ± 1) °C (1–2)f
Salmonella Enteritidisd,i 00030
Pre-enrichment Productiv- 00012b
Escherichia coli
for Entero- ity 00013
(18 ± 2) h/ Turbidity
bacteriaceae — Qualitative —
detection (37 ± 1) °C Salmonella
(1–2)f
Typhimuriumd,i 00031
Salmonella Enteritidisd,i 00030
Annexure 1 — (continued)

WDCM
Micro- Reference Method of
Mediaa Typee Function Incubation Control strains num- Criteria Characteristic reactions
organisms media control
bersc
Blood agar S Campylobacter Productiv- 00156 Media
Campylobacter jejunid
ity 00005 batch
(44 ± 4) h/ Quantita-
blood agar PR ≥ 0,7 —
(41,5 ± 1) °C tive
Campylobacter colid 00004 already
validated
TSAn S Colony count Productiv- Bacillus cereus 00001
ity
As specified in Bacillus subtilis subsp.
00003
the method in spizizenii Media
which TSA batch TSA Quantita- Characteristic colony according to
Escherichia coli 00012 PR ≥ 0,7
is used as already tive each species
reference Listeria validated
medium 00021
monocytogenes 4b
Staphylococcus aureus 00034

SDA S Colony count Productiv- As specified in Saccharomyces

00058b
ity the method in cerevisiae Media
which SDA batch SDA Quantita- Characteristic colony/propagules/
PR ≥ 0,7
is used as Aspergillus already tive germs according to each species
reference 00053b validated
brasiliensis
medium
Annexure 1 — (continued)

a Full names of media abbreviated terms.

b Strains to be used as a minimum.
c Make reference to the reference strain catalogue available on http://www.wfcc.info for information on culture collection strain numbers and contact details.
d Strain free of choice; one of the strains has to be used as a minimum.
e L: liquid medium, S: solid medium, SS: semi-solid medium.
f Growth/turbidity is categorized as: 0 — no growth/no turbidity; 1 — weak growth/slight turbidity; 2 — growth/good turbidity
g Escherichia coli WDCM 00013 is given by the specific standard.
h Escherichia coli WDCM 00013 is a strong β-d-glucuronidase producer and WDCM 00202 is a weak β-d-glucuronidase producer.
i Some national restrictions and directions may require the use of a different serovar. Make reference to national requirements relating to the choice of Salmonella serovars.
j In case of both quantitative and qualitative use for the medium, only results of the quantitative tests are required .
k More details for quality control of MSRV medium including final concentration of the inoculum and criteria are given in manual.
l If nutrient agar is used for two or three of these different applications: perform the Salmonella growth test as a minimum (if laboratory tests for this organism).
m If BPW is used for two or three of these different applications: perform the Salmonella enrichment test as a minimum (if laboratory tests for this organism).
n Choose the strain(s) according to the method for which TSA is used as a reference medium.
Table 2 — Abbreviated terms for media used in Annexure 1

Abbreviated media term Full name of the media

Baird –Parker Baird-Parker agar

BGBLB Brilliant green lactose bile broth

BHI Brain heart infusion broth

Bolton Bolton broth

BPW Buffered peptone water

Brucella Brucella broth

CFC Cephalothin fucidin cetrimide agar
CIN Cefsulodin, Irgasan novobiocin agar
CPC Cellobiose polymyxin B colistin agar
CT-SMAC Cefixime tellurite sorbitol MacConkey agar
DG18 Dichloran glycerol agar
DRBC Dichloran-rose bengal chloramphenicol agar
EC EC broth
EE Buffered brilliant green bile glucose broth
Fraser Fraser broth
Half-Fraser Half Fraser broth
Iron sulfite agar
IS (“TS”)
(“Tryptose sulfite agar”)
ITC Irgasan, ticarcillin chlorate broth

LST Lauryl sulfate broth, lauryl tryptose broth

mCCDA Modified charcoal cefoperazone deoxycholate agar

mCPC Modified cellobiose polymyxin B colistin agar
MKTTn Muller-Kauffmann tetrathionate novobiocin broth
Minerals-modified
MMG
glutamate medium
Plate count agar with skimmed milk/ milk plate count
MPCA
agar
MRS MRS medium (de Man, Rogosa and Sharpe)
Modified semi-solid
MSRV
Rappaport-Vassiliadis medium
MYP Mannitol egg yolk polymyxin agar
PCA Plate count agar
Polymyxin pyruvate egg yolk mannitol bromothy- mol
PEMBA
blue agar
PPA Penicillin and pimaricin agar
PSB Peptone, sorbitol and bile salts broth
RPFA Rabbit plasma fibrinogen agar
Table .2 (continued)

Abbreviated media term Full name of the media

Rappaport-Vassiliadis
RVS
soya peptone broth
SDA Sabouraud dextrose agar
Sodium dodecyl sulfate
SDS
polymyxin sucrose agar
Salmonella Shigella
SSDC
deoxycholate calcium agar
TBX Tryptone bile X-glucuronide agar
TCBS Thiosulfate citrate bile salts sucrose agar
Thioglycollate Fluid thioglycollate medium
TSA Tryptone soya agar
Sulfite cycloserine agar/
TSC/SC tryptose sulphite cycloserine agar
without egg yolk
TSPB Tryptone soya polymyxin broth
TSYEA Tryptone soya yeast extract agar
TSYEB Tryptone soya yeast extract broth
VRBG Violet red bile glucose agar
VRBL Violet red bile lactose agar
XLD Xylose lysine deoxycholate agar
 Annexure II — Test microorganisms and performance criteria for culture media commonly used in water microbiology

Micro- WDCM Reference Method of

Mediaa Typee Function Incubation Control strains Criteria Characteristic reactions
organisms numbersc media control

Colilert-18 L Escherichia Productiv- Escherichia coli 00013b TSA Quantita- PR ≥ 0,5 Yellow colour and fluorescence for
coli /coliform ity 00090 tive E. coli
bacteria
(20 ± 2)°h/ Klebsiella pneumoniae 00206 TSA Quantita- PR ≥ 0,5 Yellow colour equal or greater than
tive the comparator for coliform
(36 ± 2) °C
bacteria
Pseudomonas 00024 Total inhibi-
Selectivity — Qualitative Less yellow than the comparator
aeruginosad or 00025 tion (0)
GVPCf S Legionella Productiv- 2-5 days/ Legionella 00107b White-grey-blue-purple colonies
ity (36 ± 2) °C pneumophila 00180 Quantita- with an entire edge and exhibit- ing
BCYE PR ≥ 0,5
5-10 days/ tive a characteristic ground-glass
Legionella anisa 00106 appearance
(36 ± 2) °C
Selectivity Enterococcus 00009 Total inhibi-
— Qualitative —
faecalisd or 00087 tion (0)
3 days/ Pseudomonas 00026
Total or par-
(36 ± 2) °C aeruginosad or 00025
— Qualitative tial inhibition —
00012
Escherichia colid (0-1)
or 00013
Lactose S Escherichia Productiv- 00179b
TTC coli / coliform ity Escherichia coli 00012
bacteria 00013
Quantita- Yellow colour in the medium under
TSA PR ≥ 0,5
Enterobacter tive the membrane
aerogenes 00175
(21 ± 3) h/
(36 ± 2) °C Citrobacter freundii 00006
Selectivity 00009 Total inhibi-
Enterococcus faecalisd — Qualitative —
or 00087 tion (0)
Specificity Pseudomonas 00025 Red colonies, blue colour in the
— Qualitative —
aeruginosad or 00026 medium
mCP S Clostridium Productiv- TSA or
perfringens ity 00007b other non-
Clostridium Quantita- Yellow colonies; Phophatase test
00080 selective PR ≥ 0,5
perfringens tive positive
(21 ± 3) h/ 00174 medium for
(44 ± 1) °C anaerobes
anaerobic
Specificity Clostridium Blue colonies; Phosphatase test
atmosphere 00079 — Qualitative —
bifermentans negative
Selectivity 00012 Total inhibi-
Escherichia colid — Qualitative —
or 00013 tion (0)
Annexure II — (continued)

Micro- WDCM Reference Method of

Mediaa Typee Function Incubation Control strains Criteria Characteristic reactions
organisms numbersc media control

Pseudo- S Pseudomonas Productiv- 00024b

Pseudomonas Quantita- Blue-green colonies with fluores-
monas CN aeruginosa ity 00025 TSA PR ≥ 0,5
aeruginosa tive cence under UV light (360 ± 20 nm)
00026
(44 ± 4) h/
Selectivity 00012 —
(36 ± 2) °C Escherichia colid
or 00013 Total inhibi-
— Qualitative
00009 tion (0)
Enterococcus faecalisd
or 00087
Slanetz S Intestinal Productiv- 00009b
and enterococci ity Enterococcus faecalis 00087
Bartley 00176 Quantita-
TSA PR ≥ 0,5 Red-maroon-pink colonies
tive
Enterococcus faeciumd 00177
(44 ± 4) h/ 00178
(36 ± 2) °C
Selectivity 00012
Escherichia colid
or 00013 Total inhibi-
— Qualitative —
Staphylococcus 00032 tion (0)
aureusd or 00034
Sulfite Iron S Sulfite- Productiv- TSA or
reducing anaer- ity Blood agar
Tryptose obes (clostridia) or
Sulfite Clostridium 00007b Quantita-
(44 ± 4) h/ other non- PR ≥ 0,5 Black colonies
(TS) (37 ± 1) °C perfringens 00080 tive
selective
anaerobic medium for
atmosphere anaerobes
Specificity 00012
Escherichia colid — Qualitative — No blackening
or 00013
TSC S Clostridium Productiv- TSA or
perfringens ity Blood agar
00007b or
Clostridium Quantita-
(21 ± 3) h/ 00080 other non- PR ≥ 0,5 Black colonies
(44 ± 1) °C perfringens tive
00174 selective
anaerobic medium for
atmosphere anaerobes
Selectivity Bacillus subtilis subsp. Total inhibi-
00003 — Qualitative —
spizizenii tion (0)
Annexure II — (continued)

Micro- WDCM Reference Method of

Mediaa Typee Function Incubation Control strains Criteria Characteristic reactions
organisms numbersc media control

Colilert-18 L Escherichia Productiv- Escherichia coli 00013b previously Quantita- PR ≥ 0,7

coli /coliform ity 00090 validated tive Yellow colour and fluorescence for
bacteria batch E. coli
Colilert
(20 ± 2)h/ Klebsiella pneumoniae 00206 previously Quantita- PR ≥ 0,7
(36±2) °C Yellow colour equal or greater than
validated tive
the comparator for coliform
batch
bacteria
Colilert
Pseudomonas 00024 Total inhibi-
Selectivity — Qualitative Less yellow than the comparator
aeruginosad or 00025 tion (0)
GVPCf S Legionella Productiv- 2-5 days/ Legionella 00107b Media
ity (36 ± 2) °C pneumophila 00180 batch White-grey-blue-purple colonies
Quantita- with an entire edge and exhibit- ing
00106 GVPC PR ≥ 0,7
5-10 days/ tive a characteristic ground-glass
Legionella anisa already val- appearance
(36 ± 2) °C idated
Selectivity 00009 Total inhibi-
Enterococcus faecalisd — Qualitative —
or 00087 tion (0)
3 days/ Pseudomonas 00026
Total or par-
(36 ± 2) °C aeruginosad or 00025
— Qualitative tial inhibition —
Escherichia colid 00012 (0 - 1)
or 00013
Lactose S Escherichia Productiv- Escherichia coli 00179b
TTC coli / coliform ity 00012 Media
bacteria 00013 batch Lac-
Quantita- Yellow colour in the medium under
tose TTC PR ≥ 0,7
Enterobacter 00175 tive the membrane
already
aerogenes
(21 ± 3) h/ validated
(36 ± 2) °C Citrobacter freundii 00006
Selectivity Enterococcus 00009 Total inhibi-
— Qualitative —
faecalisd or 00087 tion (0)
Specificity Pseudomonas 00025 Red colonies, blue colour in the
— Qualitative —
aeruginosad or 00026 medium
Annexure II — (continued)

Micro- WDCM Reference Method of

Mediaa Typee Function Incubation Control strains Criteria Characteristic reactions
organisms numbersc media control

mCP S Clostridium Productiv- Media

perfringens ity batch
00007b
Clostridium Quantita- Yellow colonies; Phophatase test
00080 mCP PR ≥ 0,7
perfringens tive positive
(21 ± 3) h/ 00174
already
(44 ± 1) °C validated
anaerobic
Specificity atmosphere Clostridium Blue colonies; Phosphatase test
00079 — Qualitative —
bifermentans negative
Selectivity 00012 Total inhibi-
Escherichia colid — Qualitative —
or 00013 tion (0)
Pseudo- S Pseudomonas Productiv- Pseudomonas Media batch
monas CN aeruginosa ity aeruginosa 00024b Pseu-
Quantita- Blue-green colonies with fluores-
00025 domonas PR ≥ 0,7
tive cence under UV light (360 ± 20 nm)
00026 CN already
(44 ± 4) h/ validated
(36 ± 2) °C
Selectivity 00012
Escherichia colid
or 00013 Total inhibi-
— Qualitative —
00009 tion (0)
Enterococcus faecalisd
or 00087
Slanetz S Intestinal Productiv- 00009b Media batch
and enterococci ity Enterococcus faecalis 00087 Slanetz and
Bartley 00176 Bartley Quantita-
PR ≥ 0,7 Red-maroon-pink colonies
already tive
Enterococcus faeciumd 00177 validated
(44 ± 4) h/ 00178
(36 ± 2) °C
Selectivity 00012
Escherichia colid
or 00013 Total inhibi-
— Qualitative —
Staphylococcus 00032 tion (0)
aureusd or 00034
Annexure II — (continued)

Micro- WDCM Reference Method of

Mediaa Typee Function Incubation Control strains Criteria Characteristic reactions
organisms numbersc media control

Sulfite Iron S Sulfite- Productiv- Media

reducing anaer- ity batch
Tryptose obes (clostridia) Clostridium 00007b Sulfite iron Quantita-
Sulfite (44 ± 4) h/ PR ≥ 0,7 Black colonies
(37 ± 1) °C perfringens 00080 or TS tive
(TS) already
anaerobic
atmosphere validated
Specificity 00012
Escherichia colid — Qualitative — No blackening
or 00013
TSC S Clostridium Productiv- Media
00007b
perfringens ity Clostridium batch TSC Quantita-
(21 ± 3) h/ 00080 PR ≥ 0,7 Black colonies
(44 ± 1) °C perfringens already tive
00174 validated
anaerobic
Selectivity atmosphere Bacillus subtilis subsp. Total inhibi-
00003 — Qualitative —
spizizenii tion (0)

Micro- WDCM Reference Method of

Mediaa Typee Function Incubation Control strains Criteria Characteristic reactions
organisms numbersc media control

YEA S Total flora Productiv- 00012 Media

Escherichia colid
ity (44 ± 4) h/ or 00013 batch YEA Quantita-
PR ≥ 0,7 —
(36 ± 2) °C Bacillus subtilis subsp. 00003 already tive
spizizenii validated
 Annexure II —
(continued)

Micro- WDCM Reference Method of

Mediaa Typee Function Incubation Control strains Criteria Characteristic reactions
organisms numbersc media control

Bolton L Campylobacter Productiv- Campylobacter jejuni 00156b

ity 00005
Preston
+ Escherichia colid 00012
or 00013
+ Proteus mirabilis 00023 > 10 colonies Small, flat or convex colonies with a
— Qualitative
(44 ± 4) h/ on mCCDA glossy surface
(37 ± 1) °C Campylobacter coli 00004b
microaerobic 00012
atmosphere + Escherichia coli d
or 00013
+ Proteus mirabilis 00023b
Selectivity 00012 Total inhibi-
Escherichia colid
or 00013 — Qualitative tion on TSA —
Proteus mirabilis 00023 (0)

MUG/ECg L Escherichia Productiv-

48 h/ Details for method of control and quality criteria of MUG/EC medium
coli / coliform ity Escherichia coli 00179
bacteria (44 ± 0,5) °C

MUD/SFh L Intestinal Productiv- Enterococcus faecalis 00176

enterococci ity
Enterococcus hirae 00089
Enterococcus faecium 00178
(44 ± 4) h/ Details for method of control and quality criteria of MUD/SF medium
Selectivity (44 ± 0,5) °C Aerococcus viridans 00061
Lactococcus lactis 00016
Staphylococcus
epidermidis 00132
 Annexure II —
(continued)

Micro- WDCM Reference Method of

Mediaa Typee Function Incubation Control strains Criteria Characteristic reactions
organisms numbersc media control

RVS L Salmonella Productiv- Salmonella

ity Enteritidisd,i 00030
Salmonella 00031
Typhimuriumd,i > 10 colonies
on XLD or Characteristic colonies according to
— Qualitative
00012 other medium each medium (see standard)
+ Escherichia colid
or 00013 of choice
(24 ± 3) h/ + Pseudomonas
aeruginosa 00025
(41,5 ± 1) °C
Selectivity Partial inhibi-
00012 tion ≤ 100
Escherichia colid — Qualitative —
or 00013 colonies on
TSA
Enterococcus 00009 < 10 colonies
— Qualitative —
faecalisd or 00087 on TSA

Micro- WDCM Reference Method of

Mediaa Typee Function Incubation Control strains Criteria Characteristic reactions
organisms numbersc media control

DRCM L Sulfite- Productiv- Clostridium 00007b Turbidity

reducing anaer- ity (44 ± 4) h/ — Qualitative Blackening
(36 ± 1) °C perfringens 00080 (1-2)j
obes (clostridia)
anaerobic
Specificity atmosphere 00012 Turbidity
Escherichia colid — Qualitative No blackening
or 00013 (0-1)j
Saline L Dilution Diluent 00012
Escherichia colid
solution liquids or 00013
Peptone
diluent
Peptone +/- 30 %
salt
45 min – 1 h/ Quantita- colonies/
solution TSA —
T0 (+/- 30 %
20 °C – 25 °C Staphylococcus tive
Ringer’s 00034b of original
solution aureus count)
(1/4
strength)
Phosphate
buffer
solution
 Annexure II —
(continued)

Micro- WDCM Reference Method of

Mediaa Typee Function Incubation Control strains Criteria Characteristic reactions
organisms numbersc media control

mCCDA S Campylobacter Productiv- 00156b

Campylobacter jejuni Good growth Small, flat or convex colonies with a
ity 00005 — Qualitative
(2) glossy surface
Campylobacter coli 00004
(44 ± 4) h/
Selectivity (41,5 ± 1) °C 00012 Total or par-
Escherichia or 00013 tial inhibition
microaerobic — Qualitative No characteristic colonies
atmosphere colid or 00179
or 00090 (0−1)

Staphylococcus 00032 Total inhibi-

— Qualitative —
aureusd or 00034 tion (0)
XLD S Salmonella Productiv- Salmonella Colonies with black centre and a
00031
ity Typhimuriumd,i Good growth lightly transparent zone of reddish
— Qualitative
Salmonella (2) colour due to the colour change of the
00030 medium
Enteritidisd,i
Selectivity (24 ± 3) h/ Growth or
(36 ± 2) °C 00012 partial inhibi-
Escherichia colid — Qualitative tion Yellow colonies
or 00013
(0−1)
00009 Total inhibi-
Enterococcus faecalisd — Qualitative —
or 00087 tion (0)

Micro- WDCM Reference Method of

Mediaa Typee Function Incubation Control strains Criteria Characteristic reactions
organisms numbersc media control

BPWk L Diluent for Dilution 00012 +/- 30 %

Escherichia colid
enumerations or 00013 colonies/T0
45 min – 1 h/ Quantita-
of all TSA (+/- 30 % —
20 °C to 25 °C Staphylococcus tive
microorganisms 00034 of original
aureus count)
Pre-enrichment Productiv- Salmonella
00031
for ity (18 ± 2) h/ Typhimuriumd,i Turbidity
Salmonella — Qualitative —
(36 ± 2) °C Salmonella (1-2)j
detection 00030
Enteritidisd,i
 Annexure II —
(continued)

Reference media for enumeration of microorganisms

Micro- WDCM Reference Method of

Mediaa Typee Function Incubation Control strains Criteria Characteristic reactions
organisms numbersc media control

BCYE S Colony count Productiv- Media batch Quantita- PR ≥ 0,7 White-grey-blue-purple colonies
ity 2-5 days / Legionella BCYE tive with an entire edge and exhibit- ing
00107b
(36 ± 2) °C pneumophila already a characteristic ground-glass
validated appearance
TSAl S Colony count Productiv- 00012
ity Escherichia colid 00013
00090
As specified in 00179
the method Media
Clostridium batch TSA Quantita- Characteristic colony according to
perfringens 00007 PR ≥ 0,7
in which TSA is already tive each species
used as refer- validated
Pseudomonas
ence medium 00024
aeruginosa
Enterococcus
faecalis 00087

a Full names of media abbreviated terms are given.

b Strains to be used as a minimum.
c Make reference to the reference strain catalogue available on http://www.wfcc.info for information on culture collection strain numbers and contact details.
d Strain free of choice; one of the strains has to be used as a minimum.
e L: liquid medium, S: solid medium, SS: semi-solid medium.
f More details for quality control of Legionella media including storage of the control strains are given
g More details for quality control and quality criteria of MUG/EC medium
h More details for quality control and quality criteria of MUD/SF medium are given.
i Some national restrictions and directions may require the use of a different serovar. Make reference to national requirements relating to the choice of Salmonella serovars.
j Growth/turbidity is categorized as: 0 — no growth/turbidity; 1 — weak growth/turbidity; 2 — good growth/turbidity
k If BPW is used for two of these different applications: perform the Salmonella enrichment test as a minimum (if laboratory tests for this organism).
l Choose the strain(s) according to the method for which TSA is used as a reference medium.
 Annexure II — Abbreviated terms for media used

Media abbreviated
Full name of the media
term
BCYE Buffered charcoal yeast extract agar medium
Bolton Bolton broth

BPW Buffered peptone water

DRCM Differential reinforced clostridial medium

Buffered charcoal yeast extract agar with glycine, van- comycin, polymyxin B,
GVPC
cycloheximide
Lactose triphenyltetrazolium chloride agar with sodium heptadecylsulfate
Lactose TTC

mCCDA Modified charcoal cefoperazone deoxycholate agar

mCP Membrane clostridium perfringens agar

MUD/SF 4-methylumbelliferyl-α-D glucoside /SF medium

MUG/EC 4-methylumbelliferyl-β-D glucuronide /EC medium
Preston Preston broth
Pseudomonas CN Pseudomonas cetrimide nalidixic acid agar
RVS Rappaport-Vassiliadis soya peptone broth
Slanetz and Bartley Slanetz and Bartley medium
Sulfite Iron Iron Sulfite agar
Tryptose Sulfite (TS) Tryptose sulphite agar
TSA Tryptone soya agar
TSC Tryptose sulphite cycloserine agar (without egg yolk)
XLD Xylose lysine deoxycholate agar
YEA Yeast extract agar