CFD 20203LMPRACTICAL

1.
MALAYSIAN INSTITUTE OF CHEMICAL AND BIOENGINEERING
TECHNOLOGY.

FOOD MICROBIOLOGY (CFD 20203)

UNIKL MICET
DOC. NO. : JAN 07
FDTLM2033 LAB MANUAL (REV. NO. 1)

PRACTICAL 7: ENUMERATION OF COLIFORM BACTERIA (MPN

METHOD)

OBJECTIVE:

• To familiarize with the enumeration techniques employed for coliform bacteria

• To determine coliform bacteria count by using Most Probable Number (MPN) method.

KEYWORDS

Enumeration, Coliform Bacteria, Escherichia coli, MPN method

1. INTRODUCTION

Coliform bacteria are defined as rod-shaped Gram-negative organisms which

ferment lactose with the production of acid and gas when incubated at 35 °C.
Coliforms are abundant in the feces of warm-blooded animals, but can also be
found in the aquatic environment, in soil and on vegetation. In most instances,
coliforms themselves are not the cause of sickness, but they are easy to culture and
their presence is used to indicate that other pathogenic organisms of fecal origin
may be present. Fecal pathogens include bacteria, viruses, protozoa or parasites.

Escherichia coli (E. coli), a member of the coliform group, can be distinguished
from most other coliforms by its ability to ferment lactose at 44°C, and by its
growth and colour reaction on certain types of culture media. Unlike the general
coliform group, E. coli are almost exclusively of fecal origin and their presence is
thus an effective confirmation of fecal contamination. Typically, E. coli are about
10% of the coliforms in human feces.

Serial dilution tests measure the concentration of a target microbe in a sample with
an estimate called the most probable number (MPN). The MPN is particularly
useful for low concentrations of organisms (<100/g), especially in milk and water,
and for those foods whose particulate matter may interfere with accurate colony
counts.

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2. MATERIALS AND INSTRUMENTS

2.1 Lake water, prawn meat as food sample and cultures of appropriate
microorganisms supplied or as advised
2.2 Violet red bile agar (VRBA)
2.3 Brilliant green lactose bile (BGLB) broth, 2%
2.4 Peptone water as diluent for food preparation in analysis (Sterilised)
2.5 Numerous Sterilised MacCartney bottles filled up with 9 ml of
Peptone water for serial dilution.
2.6 Stomacher, blender or equivalent
2.7 Compound microscope, slides and coverslips
2.8 Colony counting device (optional)
2.9 Incubator capable of maintaining 35° C
2.10 Autoclave for sterilization
2.11 Sterile utensils
2.12 Durham tubes
2.13 Screw cap test tubes

3. PROCEDURE

3.1 Sample Handling

All sample units are refrigerated (0-5° C) until needed, with exception of
shelf-stable products. The sample units of frozen products shall be kept frozen.
Thawing of frozen samples must be cautiously undertaken under time and
conditions which prevent microbial growth or death. Analyze the sample units as
soon as possible after receipt at the laboratory.

3.2 Preparation for Analysis

Preparation of the violet red bile agar (VRBA) for analysis must be carried out prior
to the analysis according to the manufacturer's instructions. Since the pour plate
technique will be used, molten VRBA tempered to 45 - 48° C will be required.
Clean surface of working station with suitable disinfectant. Mark clearly the
duplicate/triplicate Petri dishes identifying the sample, sample unit, dilution or
date of inoculation as required for identification where appropriate.

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3.3 Preparation of Sample

Prepare Peptone water as diluent (It may be provided.) Conduct serial dilution up to
10-6 dilution factor for each sample.

3.4 Plating

The pour plate technique will be used.

Molten VRBA tempered to 45 - 48° C is prepared.

Cool it to recommended temperature before pouring it into the plates.

Transfer 1 ml of each dilution into Petri dishes aseptically.

Pour 10 ml VRBA into the plates and swirl plates to mix.

In order to prevent surface growth and spreading of the colonies, overlay

with 5 ml VRBA and allow solidifying.

3.5 Incubation

Invert solidified plates and incubates for 18 - 24 hour at 35° C. If dairy products
being analyzed, incubate at 32° C.

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3.6 Counting of Colonies and Growth Examination

Examine plates under illumination and use of magnification glass may be required
in the process. Count the colonies that have purple-red coloration with diameter of
0.5 mm or larger, and are surrounded by zone of bile acids deposition or
precipitation. Plates should score around 25 - 250 colonies.

Plate Count

1. Choose a plate that appears to have between 25 and 250 colonies.

For colonies more than 250, report as TNTC (too numerous too count)

For colonies less than 25, report as TSTC (too small to count) or <30

For plate which appears no growth, express as NC (no colony)

If half of the plate surface is covered with spreader , report as SPR
(Spreader)

If the higher dilution factor gives the higher number of colonies than the
lower dilution factor, report as LA (laboratory accident)

If ALL of the plates have more than 300 colonies, choose a plate that near to
the 300 colonies or from the highest dilution factor. Report as Estimated
cell number.

If no colonies appear in ALL plates, even though no antimicrobial agent has
been used, report the cell number as <1 multiply with the lowest dilution
factor.

2. Count the exact number of colonies on that plate using the colony counter (as
demonstrated by your instructor). Then record your result as in Table 1

Table 1 : total number of colonies

Microorganism:

Dilution No of colonies

factor 10-1 10-2 10-3 10-4 10-5 10-6 10-7

Plate 1

Plate 2

Average

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3. Calculate the number of CFUs per ml of original sample as follows:

The number of CFUs per ml of sample =

The number of colonies (25-250 plate) X

The dilution factor of the plate counted X

1/ amount of sample in the plate

____________ = Number of colonies

____________ = Dilution factor of plate counted

____________ = Number of CFUs per ml

In the case of the example above, 150 x 1,000,000 = 150,000,000 CFUs per ml.

For a more accurate count it is advisable to plate each dilution in duplicate or

triplicate and then find an average count.

4. If there are more than one plate has total colonies in the range of 30 – 300, the
following rules should be followed:

a) If the highest number of cells from the countable plate is two times or less than
two times from the lowest number of cells, report the total average of all plates as
the amount of cells in the sample.

b) If the highest number of cells from the countable plate is more than two times
the lowest number of cells, choose the LOWEST counts. The lowest count
represents the number of bacterial cells in the undiluted original sample.

5. Record your results .

If the test organisms are detected at counts of 100 or higher per gram, report with
one figure before and one figure after the decimal point expressed to the power of
10 in the form of :

a x 10b cfu/ g or mL

where a is never less than 1.0 or greater than 9.9 and b represents the appropriate
power of ten.

Round counts up if the last figure is 5 or more and down if the last figure is 4 or less

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e.g. 11,300 cfu/g reported as 1.1 X 104 cfu/g

235,000 cfu/g reported as 2.4 X 105 cfu/g

3.7 Most Probable Number (MPN)

The sample should be diluted by serial dilution manner up to 10-6 dilution factor.
Then transfer each to a tube of Brilliant green lactose bile (BGLB) broth that comes
with inverted Durham tube and incubate the tubes at 35° C. Examine the tubes at 24
and 48 hour for gas production. Label the tubes for positive sign if gas production
occurs in the tube, while negative sign should be labeled to the tubes with no gas
production as in Table 2. By using 3 – tubes MPN table, we can determine the
estimated coliform number in our sample. (Please refer to Appendix 1).

3.8 Record of Results and Discussion

You are required to compute the coliform bacteria count and present your results in
colony forming units (cfu) per gram or ml of product. Round off counts to TWO(2)
significant figures, to the nearest whole number.

You should compare the result total plate count of coliform by using two different
kinds of method (pour plate technique and MPN technique). Discuss the factors that
may affect the result.

Table 2 : Observation of gas production in BGLB broth after 48 hours

Microorganism:

Positive/negative (+/-) of gas production

Dilution factor
10-1 10-2 10-3 10-4 10-5

Tube 1

Tube 2

Tube 3

Number of positive tube(s)

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4. REFERENCE
1. Bell C., Neaves P. and Williams A.P. (2005). Food Microbiology and Laboratory
Practice, Wiley-Blackwell, Oxford.
2. Garg N. and Garg K.L. (2010). Laboratory Manual of Food Microbiology, IK
International Publishing House Pvt. Ltd.

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2.
SEM Plate of a Coliform bacteria

Durham tube

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APPENDIX 1- MOST PROBABLE NUMBER (MPN) METHOD FOR

COLIFORMS ENUMERATION

To envision the theoretical basis for the Most Probable Number (MPN) Method, think of a
ten-fold dilution series being made of a water sample with one ml of each dilution being
inoculated into a separate tube of an all-purpose broth medium.

After incubation, the broth tubes are observed for the presence or absence of growth.
Theoretically, if at least one organism had been present in any of the inocula, visible
growth should be seen for that particular tube. If the broth inoculated from the 10–3 dilution
shows growth, but the broth from the 10–4 does not, it is then possible to say that there were
greater than 1X103 organisms per ml of the original sample but less than 1X104 per ml.

Bacteria are rarely, if ever, distributed evenly in a sample. For example, if a 10 ml sample
contains a total of 300 organisms, not every one ml aliquot will contain 30 organisms;
some will contain more or fewer, but the average of all ten aliquots in the entire 10 ml
sample will be 30. This also applies to any of the dilution tubes from which inocula are
taken.

To increase the statistical accuracy of this type of test, more than one broth tube can be
inoculated from each dilution. Standard MPN procedures use a minimum of 3 dilutions and
3, 5 or 10 tubes per dilution. The statistical variability of bacterial distribution is better
estimated by using as many tubes as possible or practical. After incubation, the pattern of
positive and negative tubes is noted, and a standardized MPN Table is consulted to
determine the most probable number of organisms (causing the positive results) per unit
volume of the original sample.

In the following example, a set of 3 tubes of an all purpose broth medium is inoculated
from each of the ten-fold dilutions, with each tube being inoculated with one ml.

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After incubation, the number of tubes showing growth is recorded. As the succeeding
dilutions were made, the organisms were diluted to such an extent that none were in the
inocula of seven of the tubes (marked negative). In order to estimate the number of
organisms per ml of the sample which would cause this kind of growth response, we locate
the three sets of tubes which show dilution of the organisms "to extinction" – i.e., those
tubes which were inoculated from the 10–2, 10–3 and 10–4 dilutions.

3-tube MPN Table as below will be used to determine the coliform numbers in the sample.
The heading of the last column tells us that this combination of results (in order: 3-2-0)
suggests an average of 0.93 organisms being inoculated into each of the tubes of the middle
set (of the three sets of tubes chosen) – i.e., those inoculated with one ml of a 10–3 dilution.
Therefore, the most-probable number of organisms per one ml of the original, undiluted
sample would be 0.93 X 103 or 9.3 X 102.

This method, with the associated table, only works if there is a succession of 1/10 dilutions
being tested (in an appropriate medium) for growth, and the tubes chosen to compare with
the table are in consecutive order of increasing dilution.

Some things to keep in mind:

• Other amounts than one ml can be inoculated into the broth tubes. For example,
inoculating 0.1 ml of a 10–3 dilution is equivalent to inoculating 1 ml of a 10–4
dilution; the "plated dilution" (10–4) is the same in each case.
• It doesn't matter what amount of medium there is in the tubes being inoculated. For
example, the same growth response should be evident if we doubled the amount of
broth in each tube.
• Also, we don't have to inoculate our tubes from dilutions. For example, one can set
up a series of tubes where 10 grams are inoculated into each tube in the first set, 1
gram is inoculated into each tube in the second set, and 0.1 gram is inoculated into
each tube in the third set. The sequence of decimally-decreasing amounts is
maintained. (Recall how the so-called "plated dilution" represents the actual
amount of sample being inoculated.)

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3-tubes MPN Table

No. of Tubes Positive in MPN in the

2 0 0 0.091
inoculum of the
first middle last middle set of 2 0 1 0.14
set set set tubes 2 0 2 0.20
0 0 0 ‹0.03 2 0 3 0.26
0 0 1 0.03 2 1 0 0.15
0 0 2 0.06 2 1 1 0.20
0 0 3 0.09 2 1 2 0.27
0 1 0 0.03 2 1 3 0.34
0 1 1 0.061 2 2 0 0.21
0 1 2 0.092 2 2 1 0.28
0 1 3 0.12 2 2 2 0.35
0 2 0 0.062 2 2 3 0.42
0 2 1 0.093 2 3 0 0.29
0 2 2 0.12 2 3 1 0.36
0 2 3 0.16 2 3 2 0.44
0 3 0 0.094 2 3 3 0.53
0 3 1 0.13 3 0 0 0.23
0 3 2 0.16 3 0 1 0.39
0 3 3 0.19 3 0 2 0.64
1 0 0 0.036 3 0 3 0.95
1 0 1 0.072 3 1 0 0.43
1 0 2 0.11 3 1 1 0.75
1 0 3 0.15 3 1 2 1.2
1 1 0 0.073 3 1 3 1.6
1 1 1 0.11 3 2 0 0.93
1 1 2 0.15 3 2 1 1.5
1 1 3 0.19 3 2 2 2.1
1 2 0 0.11 3 2 3 2.9
1 2 1 0.15 3 3 0 2.4
1 2 2 0.20 3 3 1 4.6
1 2 3 0.24 3 3 2 11
1 3 0 0.16 3 3 3 ›24
1 3 1 0.20
1 3 2 0.24
1 3 3 0.29

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