STANDARD LABORATORY PROTOCOL ON TESTING MILK SAMPLES FOR QUALITY & SAFETY 1

2 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 3
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Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 5
8 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
 Acknowledgement

We sincerely thank and acknowledge the guidance and support that we received from the
Agriculture Production Commissioner (APC) to the Govt. of Assam; Commissioner & Secretary to
the Govt. of Assam, Animal Husbandry & Veterinary Department (AHVD); State Project Director,
ARIAS Society; Director, Nodal Officer (APART) and other officials of the Dairy Development
(DDD), Assam and concerned officials of the ARIAS Society without which the compilation of
tests protocol would not have been possible to complete.
We also gratefully acknowledge the support received from the local FSSAI officials at different
stages of drafting the tests protocol.
Our sincere thanks also go to those mentioned under reference whose documents helped us in
developing this test protocol.

Team Leader and Resident Consultant, APART-ILRI

International Livestock Research Institute (ILRI)

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 9
 List of Contents
Particulars Pg. No.
Abbreviations
List of tables
1 Background
2 Method of sample collection, labeling, storing, and dispatching
2.1 Milk sampling
2.2 General requirement for sample collection
2.3 Information to be submitted along with the sample
2.4 General considerations in sample collection, handling and storage
2.5 Sample collection, labelling and storing
2.6 Packaging and dispatching of samples to other laboratories
3 Test protocol for milk safety tests
3.1 Physical test
3.1.1 Organoleptic evaluation
3.1.2 Extraneous matter
3.2 Compositional analysis of milk
3.2.1 Determination of fat in milk
3.2.2 Determination of SNF in milk
3.3 Adulteration tests
3.3.1 Salt detection test
3.3.2 Sugar detection test
3.3.3 Maltodextrin detection test: Enzymatic method
3.3.4 Starch detection test
3.3.5 Urea detection test

3.3.6 Neutralizers (Rosalic) detection test

3.3.7 Method of detection of Melamine in milk and milk products
3.3.8 Detection of mineral oil in ghee
3.3.9 BR reading for detection of vegetable fat
3.3.10 Ammonium Sulphate detection test
3.3.11 Synthetic milk detection test
3.3.12 Glucose test
3.3.13 Test for presence of Boric acid and Borates
3.4. Microbiological test procedures
3.4.1 Total plate count or Standard plate count
3.4.2 Yeast and mould counts
3.4.3 Coliform count

10 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
 3.4.4 Escherichia coli (E. coli)
3.4.5 Staphylococcus aureus
3.4.6 Salmonella spp
3.4.7 Listeria monocytogenes
3.4.8 Enterobacter sakazakii
3.4.9 Sulfite reducing clostridia
3.4.10 Bacillus cereus
3.4.11 Milk ring test (MRT)for Brucellosis
3.4.12 California Mastitis Test ((CMT) for Mastitis
3.4.13 MBRT (Methylene Blue Dye Reduction test) for raw milk
and pasteurized milk
3.5 Chemical tests
3.5.1 Determination of pH of milk
3.5.2 Determination of temperature of milk
3.5.3 Determination of Titratable Acidity as Lactic Acid
3.5.4 Clot on boiling test
3.5.5 Phosphatase test for pasteurized milk
3.6 Food safety control
3.6.1 Test for presence of Hydrogen Peroxide (H2O2)
3.6.2 Detection of detergent residues
3.6.3 Rapid test for detection of Aflatoxin M1 in milk
3.6.4 Detection of heavy metal in milk
3.6.5 Detection of insecticide &pesticide residues in milk
3.6.6 Formalin test
3.7 Additional tests
3.7.1 Test for presence of skimmed milk powder in natural milk
3.7.2 Test for Detection of Antibiotic residues
References

List of Tables
Particulars Pg. No
Table-1 List of tests recommended by FSSAI and the tests recommended by
this assessment for state and district laboratories

Table -2 Differentiation of Listeria species

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 11
 Abbreviation

APART : Assam Agribusiness & Rural Transformation Project

BTB : Bromothymol Blue
CFU : Colony Forming Unit
CB : Crossbred
CLR : Corrected Lactometer Reading
CMT : California Mastitis Test
DDD : Directorate Dairy Development
DMAB : Dimethyl Amino Benzaldehyde
DPA : Dipi Colinic Acid
FSSAI : Food Safety and Standards Authority of India
FSSA : Food Safety and Standards Act
GC : Gas Chromatography
HCL : Hydrochloric Acid
LOQ : Limit of Quantification
MBRT : Methylene Blue Dye Reduction Test
MFT : Multiple Fermentation Tube
MRT : Milk Ring Test
PCA : Plate Count Agar
ROSA : Rapid One Step Assay
RSM : Reconstituted Skimmed Milk
SNF : Solids-Not-Fat
TCA :Trichloroacetic Acid
TYMC :Total Yeast and Mold Counts

12 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
1. Introduction

Milk and milk products constitute an important source of nutrition for human. It contains
several nutrients, including carbohydrates, lipids, protein, minerals, vitamins, etc. in a balanced
form for building up and maintaining human and animal body. Milk is a product that can
easily be converted into a number of different milk products or can be used as ingredient for
other food items. However, being a highly perishable product its storage for long duration is
constrained. It is an ideal medium for growth and proliferation of micro-organisms and in liquid
form it is very easily contaminated by microbes. If it contains beneficial group of microbes i.e.
Lactobacillus spp., it forms delicious curd but harmful microbes may cause milk spoilage and
various foodborne diseases.
It is often seen that the milk value chain actors do not follow proper clean and hygienic
measures for milk production, handling, storing and marketing. Besides, some of them also may
adulterate the milk with various, sometimes harmful, substances or with substances with no
nutritional value in lure of earning more profit out of the milk business. Some of them may also
use preservatives for increasing shelf life of milk. Moreover, unknowingly or with the temptation
of avoiding losses milk producers even extract milk from diseased animals/ animals under
treatment. Similarly feed may be contaminated with different substances. In such situations milk
can not only transmit diseases of microbial origin including zoonotic diseases (e.g. tuberculosis,
brucellosis, leptospirosis, Q-fever etc.) but also may pass antimicrobial residue, pesticide residues
etc. to human body. In other words, it may cause a serious hazard to human health.
Milk in the upper part of the udder of a healthy cow generally contains lower microbial load than
the opening of teats. Usually, total bacterial count in milk from a healthy is below 50,000 per ml.
It may go up to several millions because of poor and unhygienic conditions during milking
and milk handling and/or in milk from diseased cow. Milk generally becomes sour 4-6 hours
after keeping it at room temperature post milking. The onset of souring depends on quantity
and quality of contamination and on milk temperature. The higher the number of lactic acid
bacteria in milk, shorter the onset of souring (lowering of pH) and vice versa.
In the wake of potential health hazards through consuming poor quality and unsafe milk, it
is of utmost importance to conduct different tests for assessing physical, microbiological
and chemical quality of milk. Keeping this in view, the Food Safety and Standard Authority
of India (FSSAI) has recommended conducting about 32 milk tests of which some are found
very essential in Assam’s context while some others are not. Also, to conduct some tests,
good laboratory infrastructure and facilities are required while for some other tests minimum
laboratory infrastructure and facilities are required.
To perform each test uniformly and perfectly by all laboratories under DDA, a standard test
protocols required to be followed by each laboratory. Therefore, this protocol has been developed
under the World Bank aided Assam Agribusiness and Rural Transformation Project (APART) to
guide the Lab Manager as well as Lab Technicians in performing the tests and interpreting the
test results.
This test protocol explains the procedure of conducting all the tests as mentioned in Table 1.
For ease of understating of the test procedures, each step of conducting the tests are presented
in the form of a flow chart.

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 13
 Table-1: List of tests to be conducted for milk quality and safety
Milk Food Degree of
Recommended
quality safety difficulties
by FSSAI
relevance relevance to conduct
A. Physical tests
1. Sight and smell test/ Organoleptic √ ++ +
test
2. Extraneous matter √ ++ +
3. Determination of fat in milk √ ++ ++
4. Determination of SNF in milk √ ++ ++
(Validated Method)
5. Salt detection test √ + +
6. Sugar detection test √ + +
7. Maltodextrin detection test √ + +
Enzymatic Method
8. Starch detection test √ + +
9. Urea detection test √ + +
10. Neutralizers (Rosalic) detection test √ + +
11. Method of Detection of Melamine √ + ++++
in Milk and Milk Products
12. Detection of mineral oil in ghee √ +
(Holde’s test)
13. Butyrorefractometer √ ++
(BR) Reading for Detection of
Vegetable Fat
14. Ammonium Sulphate detection test √ +
15. Synthetic milk detection test +
16. Glucose test (Validated Method) +
17. Test for presence of Boric acid and +
Borates (Validated Method)
B. Chemical tests
18. Determination of pH of milk +
19. Determination of Temperature of √ ++ ++ +
milk
20 Determination of Titratable Acidity √ ++ +
as Lactic Acid
21. Clot on boiling test √ + +
22. Phosphatase test for pasteurized √ + ++
milk
C. Food safety controls
23. Test for presence of Hydrogen √ + - +
Peroxide (H2O2)
24. Detection of Detergent residues √ + +

14 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
 Milk Food Degree of
Recommended
quality safety difficulties
by FSSAI
relevance relevance to conduct
25. Rapid test for detection of of √ + ++
aflatoxin M1 in milk
26. Detection of heavy metal in milk √ ++ +
27. Detection of Insecticide & pesticide √ + ++++
residues in milk
28. Formalin test √ + +
D. Additional tests
29. Test for presence of skimmed milk + ++
powder in natural milk
30. Test for Detection of Antibiotic + + +++
residues
E. Microbiological test procedures
31. Total Plate count or Standard plate √ ++ + ++
count
32. Yeast and Mould counts ++++
33. Coliform count √ ++ ++ +++
34. E. coli √ ++ ++ ++++
35. Staphylococcus aureus √ +++ ++++
36. Salmonella √ +++
37. Listeria monocytogenes √ +++ ++++
38. Enterobacter sakazakii ++++
39. Sulfite reducing clostridia ++++
40. Bacillus cereus ++++
42. Milk ring test (MRT) for Brucellosis +++ +
42. California Mastitis Test (CMT) for +++ +
Mastitis
43. MBR (Methylene Blue Dye +
Reduction) test for raw milk and
pasteurized milk
Note:
Degree of relevance in regards to food safety:
Weak relevance= +
Medium relevance= ++
Strong relevance= +++
Degree of difficulties to conduct:
Least difficult=+
Difficult= ++
Fairly difficult= +++
Highly difficult= ++++

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 15
 Milk tests are generally conducted by conventional methods that requires different equipment,
consumables, chemicals, glassware, etc. These conventional methods are labour intensive and
time consuming. Despite the fact, most of the milk testing laboratories follow conventional
methods as per unit cost of the test is relatively lower and it is perceived that results derived
from the conventional methods are more accurate. In this protocol, all milk tests are explained
in conventional method under three different sub-heads as stated below.
• Reagents required
• Test procedure to be followed
• Interpretation of the results
In addition, some of the tests could be conducted by use of compact electronic machine (e.g.
electronic milk analyser) or by rapid test using ready-made kits. These tests are conducted
following the manufacturers’ instructions. These tests give the results quickly. Only disadvantage
is, laboratory becomes more dependent on outside supply of every test kit and cost of
conducting each test may be relatively higher. Under this protocol, these rapid tests have also
been mentioned in appropriate section.
Quality control of laboratory tests
This test protocol will also help in ensuring quality of the lab tests. If all the laboratories, under
DDA follow the same test protocol, it is expected that the test results should be comparable. To
crosscheck the test results from time to time, all the laboratories should use duplicate samples
of the same sample to perform different tests. If all the laboratory shows similar results for each
test, it can be considered that all the laboratories are performing well. If any laboratory shows
significantly diverse result, it can be anticipated that there should be some problem in handling
or performing the tests or interpreting the test result in that particular laboratory. In that case,
the said laboratory should take necessary measures to correct their mistakes with support of
the other laboratories.
Similarly, each Lab Manager can also check the quality of lab test in his/her laboratory by making
duplicate samples of the same sample and can assign different identification no. to the duplicate
samples. The Lab Technicians should be asked to conduct the test of each duplicate samples
without informing him/her about the duplicity. If the results of all the duplicate samples are
almost same, it can be interpreted that the Lab Technicians are performing the test properly.
If any, significant deviation observed among the test results, it can be interpreted that there is
some problem in conducting the test. Lab Manager should explore the reason behind of this
deviation and address the problem.
Further, the Lab Manager should randomly conduct some tests using the same protocol to verify
the test results. If he/she finds the same results with the Lab Technician it can be interpreted
that the Lab Technician has performed the test well. If any deviation is observed between both
the results, both the Lab Manager and Lab Technician should sit together to resolve the issue.
In addition, certain cases, DDA may send some samples outside DDA laboratories, e.g. to FSSAI
laboratory, to verify the test results or may invite any professional agency to evaluate the lab
system and suggest necessary changes.

16 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
2. Method of sample collection, labeling, storing, and dispatching

The first step of lab operation is collection, transportation, handling and storage of milk samples
following a standard process in order to maintain the quality of milk samples that is fit for testing
purpose. In the following sections, a standard process has been explained for the lab and field
staff.
2.1. General requirement for sample collection
Make arrangement of all necessary materials before going to sample collection. Please take the
following items for collection of milk samples.
• Clean, dry, leak-proof, sterile container (mainly plastic) with graduation/calibration on
the body and polythene zip bag;
• Glass Beakers , 100 ml;
• A plunger/ dipper;

Sterile containers in polythene zip bag Marker

Glass Beakers Leak-proof sterile container

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 17
 Face mask Apron

A milk sampling plunger A milk sampling dipper

• A cool box/ thermos flask to carry the sample;

• Required ice/gel packs in cool box to keep the sample cool during transportation;
• Personal protective clothing like apron, gloves, mask, etc.;
• Sticker tags, marker, note pad, mask, sanitizers and biohazard bag;
• A disposal bag for carrying disposable materials like leftover milk, gloves, mask, etc.;
• A hand sanitizer to sanitize hands of the sample collector;
• A small weighing balance to weigh if milk products (e.g. paneer, curd, etc.) are to be
collected;
2.2 Information to be collected along with the sample
The following information need to be collected from the household/source at the time of
sample collection:
• Type of the sample (e.g. milk/curd /cream/others);
• Species: Cow/Buffalo;
• Type of animal: Exotic/ CB/Non descript;
• If milk sample is collected from the cow: Quarter of udder: L (left)/R(right): F(fore)/R(rear);
• If bulk milk is collected: Time of milking;
• Weight/ volume of the sample;

18 STANDARD LABORATORY PROTOCOL ON TESTING MILK SAMPLES FOR QUALITY & SAFETY
 • Place of collection;
• Date and time of collection;
• Name and designation of the collector;
• Purpose of collecting the samples;
• Name, address and thumb impression/signature of the person from whom the sample
has been taken.
• All samples should be marked with a unique sample number
• The above information shall be recorded against the specific sample number allocated
to each sample collected and part of the information shall be supplied with the sample
to the lab.
2.3 General considerations in sample collection, handling and storage
• The samples should never be touched with bare hands. Gloves and mask should always
be used in the process of collection.
• Knife/dippers/plungers, instruments used for cutting, removing and manipulating
samples (e.g. paneer, sweets, etc.) should be sterilized with hot water before and after
use.
• Sample should not be exposed to dirty materials/environment after collection and
should not be mixed with other biological samples.
• Temperature and pH shall be recorded at the collection stage and after transporting to
the laboratory.
• Disinfect the surface of the work area before opening the samples for measuring,
packaging, etc. at the laboratory
• Sample should preferably be measured directly in the sterile container with graduation;
• Gloves, mask and other materials in contact with the sample must be disposed properly.
• The stopper/cover of the container shall be securely fastened to prevent leakage of the
contents in transit.
2.4 Method of milk sample collection from milk container
In order to collect milk sample for testing purpose, following methods should be followed
• Agitate the liquid milk thoroughly before sample is taken in order to make the contents
of a milk container as homogenous as possible for obtaining a representative sample.
• Never agitate too vigorously because air bubbles, if dispersed in milk, will change its
physical properties and disturb the analysis.
• Use a plunger or a dipper having a handle long enough for doing this and immediately
take the sample of required volume into a sample bottle and close it.
• In order to make sure that a sample will well represent the whole contents of milk can
take the half of the required sample from the lower portion and another half from upper
part of the milk can.
• To take sample from a smaller milk container, turn the container upside down few times
before sampling ensuring the container is closed well.
• Agitate the sample carefully again before the sample start to analysis in a laboratory.

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 19
 2.5 Sampling from several containers
If milk needs to be collected from several containers, the following procedure shall be followed -
• Mix the content of each container thoroughly, take equal volume of milk from each
container and pour into a small container.
• Take a sample after mixing the combined sample.
2.6 Sampling from storage tanks and rail and road milk tankers
If milk needs to be collected from a large tanker, the following procedure shall be followed-
• The method of sampling of milk from storage tanks and rail and road tankers is largely
governed by storage/transport conditions.
• In all cases, the milk in the tank/tanker shall be thoroughly mixed by a sufficiently large
plunger, a mechanical agitator or by compressed air; the uniformity of the samples
being determined, when necessary, by mixing till such time as complete agreement is
obtained between samples taken at the manhole and at the outlet cock in respect of fat
and total milk solids.
2.7 Collecting milk sample directly from cow
To collect milk sample directly from a cow, the following procedure shall be followed-
• Ask the owner of the cow to clean the udder and teats of the cow thoroughly with
water.
• Put on the clean gloves, face mask, apron, etc.
Strip two to three streams of milk from each teat in order to flush the teat canal and
thereby to reduce contamination risk.
• Dry teats thoroughly with an individual cloth towel, paying close attention particularly
to the teat end.

• While holding the top of the teat steady, wipe the end of the teat well with an alcohol-
soaked cotton ball. Use as many cotton balls as necessary until the cotton ball still looks
clean after using.

20 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
• Open the milk vial and immediately take the sample, making sure not to touch the
inside of the tube or bottom part of the lid. Hold the milk vial about 3 inches from the
teat end and fill the tube half to three-quarters full of milk. Hold the vial at a 45 degree
angle to prevent dirt from falling into the vial.

• Close the lid immediately and label the top with the date, cow number, and quarter
sampled.

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 21
 • Put the sample in cool box immediately.
Note: Do not place the teat inside the vial when sampling.
2.5 Sample collection, labelling and storing
• About 50-100 ml or gm of milk and milk products should be collected for testing
purpose.
• Separate sterile container should be used for each sample.
• Immediately after collection of each sample, it should be properly labeled stating
sample no., date of collect, time of collection etc.
• If sample is collected for regulatory purpose (as advised by Food safety Officer), a paper
slip of the size that goes round completely from the bottom to top of the container,
bearing the signature of the Designated Officer and number of the sample, shall be
pasted on the wrapper, the signature or thumb impression of the person from whom
the sample has been taken, shall be affixed in such a manner that the paper slip and
the wrapper both carry a part of this signature or the thumb impression. The outer
covering of the packet shall also be marked with the same number of the sample.
• The labeled container should immediately be transferred to the cool box/ thermos
flask filled with ice packs.
• The collected container shall be properly secured and sealed so that no tempering is
possible after collection. To ensure this, signature of the milk producer/trader/sweet
maker and a witness should be taken on the sealed pack.
• All samples should be transported to the laboratory by maintaining cold chain in a cool
box/thermos flask with gel/ice packs.
• After arriving the sample at the laboratory, a separate code should be assigned to each
sample at the reception desk. The sample should be processed in the laboratory and
results should be mentioned against that code only.
• No personal details of the owner of the sample should be supplied to the laboratory
technicians who conduct the tests to avoid any potential pre-judgment by the lab
technicians.
• The sample should be stored at 4°C if milk is tested within a day or two (maximum 96
hours).
• In case of preserving the sample for longer duration, samples need to be stored at
(-)20°C

A deep freezer

22 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
2.6 Packaging and dispatching of samples to other laboratories
• For dispatching the samples to other laboratories, sample with their details should be
put in a thermo-cool box. Adequate quantity of cool pack/gel pack should be put in the
box to keep the sample cool during the time of transportation.

A cool box Gel pack

• The outside of the thermo-cool box should be wrapped up with white paper and address
of ‘From’ and ‘To’ should be clearly written on it preferably in all capital letters.
• A certificate should also be enclosed with the box stating the nature of the materials and
purpose of sending.
• Packages should be marked clearly to provide information about the contents of package
and, nature of the hazard, if any.
• Sample should be sent by the mode of transportation that can deliver the sample at
the quickest possible time in the destination. If the transportation time is more, the ice/
gel pack may come to normal temperature and the sample may get spoiled. In order to
avoid this, adequate no. of ice/ gel pack should be put in the thermos-cool box. There
shall not be empty space inside the box as it will allow movement of samples as well as
allow the gel/ice pack to get melted early.
• The thermo-cool box should be marked with ‘Handle with care’ and an ‘Arrow mark’
showing upside of the box, in order to guide the handlers during handling and
transportation.

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 23
 3. Physical Test

3.1 Sight and smell test (organoleptic test)

The sight and smell test of raw milk and milk products is done using normal senses of sight
and smell in order to observe and record the overall quality. We get an instant result where
and when it is carried out. If used correctly, it is very useful to do rapid screening of physical
quality of milk. It is applicable on farms, during milk collection, at milk reception and at the milk
processing plant. It is the first and basic test for judgement of qualities of milk and various milk
products. This test, of course, should be complemented by further laboratory tests. When milk is
tested by taste to judge the quality of milk there is a risk of disease transmission, and this is not
recommended from a health point of view.
The sight and smell test should be carried out immediately after opening the lid of the milk can/
container by following means-
• Observe the colour, appearance, and cleanliness of milk
• Smell the milk just above the milk surface immediately after removal of the lid.
• Taste of milk is more permanent and easy to define than smell. Before tasting the milk,
ensure that the raw milk is from healthy dairy animal. Do not perform the test using raw
milk if it is not very essential because of risk associated with milk born zoonosis.
The following are the abnormalities that can be detected by organoleptic testing:
• Abnormal colour/consistency/ visible dirt and interpretation
Colour/consistency/ visible dirt Interpretation
Pink colour Contaminated with blood
Yellowish creamy colour Colostrum or late milk
Thin creamy colour Adulterated by adding water
Large clots or flakes Sour milk or milk from cow suffering from mastitis
Small white clots or grains Milk from cow suffering from mastitis or milk
adulterated with flour and skim milk powder. Can
also be early spoiling.
Visible dirt and impurities (fragments of Milk produced under unhygienic conditions
straw, cow dung, etc.)
• Off-flavours from feeds
 Garlic, onion, beets, bad silage, certain plants and pastures can cause off-flavours to
milk
• Absorption of off-flavours from air, milk containers etc.
 It is well known that milk and cream can absorb smelling compounds from the air.
This is caused by the ability of butter fat to absorb, especially after milking when the
milk is warm, strong smells like paint, phenol, cresol, lysol, petroleum, etc. Strongly
smelling paints, disinfectants and other chemicals should not be handled and stored
in places where the dairy animals are kept and milked.

24 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
  Storage of milk together with fruits and fish also causes off-flavours to milk
• Abnormal smell and/or taste and interpretation
Smell and/or taste Interpretation
Souring Lactose fermenting, acid producing bacteria
Bitter Peptonising of milk by Streptococcus liquefaciens
Blue souring unpleasant sweet and sour smell, thin and waterish appearance
caused by bacterial activity and storage in a closed container
without ventilation
Fruit aroma Pseudomonas producing esters
Slimy milk Indicates capsule forming bacteria, e.g. Aerobacter aerogenes and
Alcaligenes viscosus
Bubbles, coagulation and Fermentation by yeast
whey separation
Organoleptic test
In order to conduct organoleptic test, a test panel of milk tasters should be employed. The milk
tasters should have good sense of sight, smell and taste of milk. No equipment is required for
organoleptic testing. The milk tasters should test the milk and make observation on its taste and
smell. Since raw milk testing may pose risk of zoonotic disease to human health, it is advisable
to avoid organoleptic testing of raw milk, if it is not very essential.

3.2 Extraneous matter

Procedure
In order to find out extraneous matter in milk, the following method should be used
• Strain the milk sample;
• Allow the milk to settle in the container and observe at the top fatty layer as well as at
the bottom of the container;
• The milk sample can also be centrifuged for few minutes to get the extraneous matter
Observations and interpretation
• Feed particle in milk: indicates poor management of feed and fodder
• Dung particle in milk :indicates uncleaned cow
• Dust particle in milk: indicates milk got exposed to dirty environment
3.3 Determination of fat in milk {Ref: IS-1224 (Part-I): 1997}
Fat is the most important component of milk. It provides more energy than the energy provided
by carbohydrate (lactose) and proteins taken together. It imparts soft texture and creamy taste
to milk products. It is the source of essential fatty acids and carrier for fat-soluble vitamins.
Due to these reasons the producers and/ or sellers of milk and milk products are paid for their
product on the fat basis. Therefore, determination of fat in milk and its products is an important
exercise. (Agrimoon,2015)

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 25
 Fat in milk is tested by two methods:
A. Conventional method by using Gerber centrifuge
B. Using a Digital milk analyzer
A. Conventional method by using Gerber centrifuge
Apparatus and reagents required
• Sulphuric acid (Specific Gravity 1.807 – 1.812 g/ml at 270C, colourless).
• Amyl alcohol (Specific Gravity 0.810 to 0.812) conforming to grade 1 of IS: 360:1964.
• Butyrometer 10% Scale
• Stoppers and shaker stands for butyrometers made from a suitable grade of rubber or
plastics.
• 10 ml Acid pipette for sulphuric acid (with rubber suction device).
• 10.75 ml pipette for milk.
• 1 ml automatic measure for amyl alcohol
• Centrifuge, electric or hand driven (1400± 70 RPM)
• Water bath at 65 + 20C

A Butyrometer

Procedure
Transfer 10 ml of sulphuric acid into a butyrometer using a 10 ml acid pipette.

Fill the 10.75 ml pipette with milk and deliver the sample into butyrometer.

Add 1 ml of amyl alcohol using a 1 ml pipette and close.

Shake the butyrometer in the shaker stand until no white particles are seen and invert it a few times.

Put the butyrometer in the water bath for 5 min.

Take it out and dry with a cloth, put it in the centrifuge, placing two butyrometers diametrically
opposite, centrifuge at maximum speed for 4 minutes.

Transfer the butyrometers, stoppers downwards into water bath for 3-10 minutes.

Bring lower end of fat column on to a main graduation mark by slightly withdrawing stopper.

26 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
Points to be considered:
• The colour of the fat should be straw yellow;
• The ends of the fat column should be clear and sharply defined;
• The fat column should be free from specks and sediment;
• The water just below the fat column should be perfectly clear;
• The fat should be within the graduation.
Interpretation
Note down the upper and lower scale readings corresponding to the lowest point of fat meniscus
and surface of separation of fat and acid. The difference between the two readings gives the
percentage by mass of fat in milk. The reading has to be done quickly before the milk cools.
Note: The butyrometers should be emptied into a special container for the very corrosive acid-
milk liquid, and the butyrometers should be washed in warm water and dried before the next use.
Fat testing is often carried out on composite or random samples in order to reduce time and costs
involved in testing.
B. Using a Digital milk analyzer Milk fat is
also measured by using a Digital milk analyzer.
Put the milk sample in a container that fits into
the Digital milk analyzer and place it in the
machine. The analyzer will give instant results
for fat percentage. Digital milk analyzer is also
used for assessing SNF%, salt, sugar, added
water, etc. in milk.
Advantage of Digital milk analyzer:
• Gives instantaneous result
• A printed test results can be obtained
in some machines
• Recurring cost is minimal
• Easy to perform the test
A Digital milk analyzer
Disadvantage of Digital milk analyzer:
• Needs to buy a milk analyzer

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 27
 3.4 Determination of SNF in milk
Solids not fat (SNF) are the solid contents present in milk excluding the fat content. Because of
adoption of some unethical practices, like adding water to increase the volume of milk, the SNF
content decreases in milk. So, in order to raise the SNF level in milk, cane sugar, starch, sulphate
salt, etc. are added to milk. (Agrimoon,2016)
SNF in milk is tested by two methods as stated below:
A. Conventional method using Lactometer
B. Using a Digital milk analyzer
A. Conventional method using Lactometer
Apparatus required
• Lactometer
• Lactometer Jar
• Dairy thermometer
Procedure
Mix the milk sample thoroughly.

Pour it into a dry cylinder which enables the lactometer to float without touching the sides.

Let the lactometer into the cylinder.

Allow the lactometer to remain steady in the milk. Take the reading from the lactometer as soon as
it becomes stationary (within about 30 seconds).

Note the corrected lactometer reading (CLR).

Calculation of SNF
CLR
SNF%= ---------------------------- + 0.2F + 0.36
4

Where
CLR = Corrected lactometer reading.
F= Fat percentage in the milk sample.
B. Using a Digital milk analyzer
Procedure
Same as stated in estimation of fat % in milk at 3.3.B

28 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
3.5 Salt detection test (Ref: DGHS Manual)
Salt or sugar is used to mask extraneous water added to milk or to elevate total solids in milk. So
it is important to detect presence of salt in milk.
Presence of salt in milk can be detected by two methods as stated below-
A. Conventional method
B. By using Milk Adulterants Kit 
A. Conventional method
Apparatus and reagents required
• Test tubes
• 5% potassium chromate
• 0.1N silver nitrate
• Milk sample
Procedure
Take 2.0 ml of milk in a test tube

Add 1.0 ml of 5% potassium chromate to the milk

Add 2.0 ml of 0.1N silver nitrate to the test tube

Inference
Appearance of red precipitate indicates the absence of dissolved chloride in milk
Appearance of yellow colour indicates presence of dissolved chloride
B. By using Milk adulterants kit 
These kits are commercially available in the market
Procedure
As instructed by the manufacturer.
Advantage of using Milk adulterants kit:
• It gives an instant result
• Easy to perform

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 29
 3.6 Sugar detection test
The common sugar present in milk is lactose. Sometimes, table sugar like sucrose is added to
the milk to increase the carbohydrate content of the milk and allow adulteration with water
without detection during the lactometer test. 
(vlab.amrita.edu, 2011).
Presence of sugar in milk can be detected by two methods as stated below:
A. Conventional method
B. By using Milk adulterants kit
A. Conventional method
Apparatus and reagents required
• Test tubes
• Concentrated hydrochloric acid
• Resorcinol
• Hot water bath
• Dilute HCl
• Milk sample
Under conventional method, there are two procedures to follow
Procedure -1: Concentrated hydrochloric acid method:

• Take 15ml of milk in a test tube and 1 ml of concentrated hydrochloric acid and 0.1 gm of
resorcinol and mix.
• Place the tube in boiling water-bath for five minutes.
Inference
Brick red colour formation indicates sugar test positive
Procedure -2: Dilute hydrochloric acid method:

• Take 3 ml of milk in a test tube and add 5 ml dilute HCl (1:2) containing resorcinol (0.1 gm.
resorcinol dissolved in 100 ml dilute HCl).
• Mix well and keep the test tube in boiling water for 5 minutes.
Inference
Brick red colour formation indicates sugar test positive
B. By using Milk adulterants kit 
These kits are commercially available in the market.
Procedure
As instructed by the manufacturer.
Advantage of using Milk adulterants kit:
• It gives an instant result
• Easy to perform
30 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
3.7 Maltodextrin detection test:
Maltodextrin is a polysaccharide that is used to increase the volume of the foods like yoghurts,
sauces, puddings, ice-cream, milk powders, cheeses, and also in indigenous milk products such
as Burfi. Maltodextrin can cause several health problems. It is emphasized that milk should
be tested for the presence of various adulterants including maltodextrin for the safety of the
consumers Presence of Maltodextrin in milk can be detected by employing three different
methods as stated below:
A. By using Conventional method (Enzymatic method),
B. By using Mid-infrared and ultrasound milk analyzer
C. By using reagent strips
A. Conventional Method (Enzymatic Method)
Apparatus and reagents required
• Glass beaker
• Lactic acid solution-1 ml (10% w/v to adjust the pH at 4 to 4.5).
• 1.0 ml of enzyme (0.20%w/v-200 mg/100 ml solution)
• Hot water bath
• Milk sample
Procedure

Take 20 ml milk in a beaker. Add lactic acid solution-1 ml (10% w/v).

Add 1.0 ml of enzyme (0.20%w/v-200 mg/100 ml solution) and keep for 5 min at 620C.

Immerse diastic in the curdled milk for 1 to 2 seconds and wipe of excess milk from the strip.

Wait for 30 seconds and compare reagent area with colour chart and report the
result as present or absent.

Inference :
Change in color of glucose strip indicating +ve (+, ++, +++ and ++++ as depicted on glucose
testing strip bottle) after enzyme treatment, indicates presence of maltodextrin
B. By using Mid-infrared and ultrasound milk analyzer
Procedure: As instructed by the manufacturer
Advantage of using Mid-infrared and ultrasound milk analyzer:
• It gives instant result
• Easy to perform
C. By using reagent strips.
These strips are commercially available in the market.

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 31
 Procedure
As instructed by the manufacturer
Interpretation
Visual observation of change in colour in the test strip as indicated by the manufacturer
Advantage of using Reagent strips:
• Reagent strips start working rapidly when dipped and gives result within 5-10 min.
• Easy to perform

32 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
3.8 Starch detection test: (IS-1479 (Part-I): 1997)
Starch is sometimes added to adulterate milk. Starch being cheaper, is sometimes added in the
milk by adulterators to raise the S.N.F of milk.
(vlab.amrita.edu,2011).
Presence of Starch in milk can be detected by employing two different methods as stated below:
A. By using Conventional method
B. By using Mid-infrared and ultrasound milk analyzer
A. Conventional Method
Apparatus and reagents required
• Test tube
• 1% Iodine solution
• Milk sample
Procedure

Take 3 ml milk in a test tube, boil and cool under tap water.

Add a drop of 1% Iodine solution.

Inference
Presence of starch is indicated by the appearance of a blue colour, which disappears when the
sample is boiled and re-appears on cooling.
B. By using Mid-infrared and ultrasound milk analyzer
This milk analyzer is commercially available in the market.
Procedure
As instructed by the manufacturer
Advantage of using Mid-infrared and ultrasound milk analyzer:
• It gives an instant result
• Easy to perform

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 33
 3.9 Urea detection test
Many unscrupulous vendors adulterate milk with urea which have harmful effect on human
health like- indigestion, malfunctions of kidney, even cancers, etc. Urea is added in raw milk to
enhance the nitrogen content resulting in a false appearance of a higher level of protein. The
most widely practiced approach of adulterating milk is to add water in it and subsequently
adding urea to raise SNF%. In addition, a small amount of urea is generally added into pure
milk in order to increase shelf life of milk. In India, the addition of external urea to the milk is not
permitted legally under the FSSA (2006).
(Researchgate,2000)
Presence of Urea in milk can be detected by employing three different methods as stated below:
A. By using conventional method
B. By using Milk adulterants kit
C. By using reagent strips
A. Conventional Method
Apparatus and reagents required
• Test tube
• Dimethyl aminobenzaldehyde (DMAB) solution
• Milk sample
Procedure

• Take 2 ml milk in a test tube, add 2 ml Dimethyl aminobenzaldehyde (DMAB) solution and
mix the contents.
Inference:
Appearance of yellow colour indicates the presence of urea making the milk unacceptable
B. By using Milk adulterants kit
Procedure
As instructed by the manufacturer.
Advantage of using Milk adulterants kit:
• It gives an instant result
• Easy to perform
C. By using reagent strips
These reagent strips are commercially available in the market.
Procedure
As instructed by the manufacturer
Interpretation
Visual observation of change in colour in the test strip as indicated by the manufacturer
Advantage of using reagent strips:

34 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
• Reagent strips start working rapidly when dipped and gives result within 7-8 min.
Development of pink color indicates the presence urea.
• Easy to perform

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 35
 3.10 Neutralizers (Rosalic) detection test (IS-1479 (Part-I): 1997)
There is development of acidity in milk due to microbial growth, which increases under
unhygienic conditions, poor milk quality, lack of proper facilities to transport milk from
production centres to the processing plants soon after milking and non availability of cold
storage ,etc. which reduces the shelf life of milk. Informal milk market actors add neutralizers
like sodium carbonate, sodium bicarbonate, sodium hydroxide, calcium hydroxide, etc. which
improve the shelf life of milk by neutralizing the developed acidity. Addition of neutralizers can
cause increased mineral concentration in body fluids and soft organs leading to kidney stone
development and commercial preparation of neutralizers might even be contaminated with
heavy metals like arsenic, lead, etc. Continuous use of such milk and milk product may cause
serious health hazards.
(J Food Sci Technol. 2015 )
Presence of Neutralizers in milk can be detected by three methods as stated below:
A. Conventional method
B. By using Milk adulterants kit
C. By using reagent strips
A. Conventional method
Apparatus and reagents required
• Test tube
• Alcohol
• 1% (w/v) alcoholic solution of rosalic acid
• Rosalic acid solution (0.05% in 60:40 ethyl alcohol and distilled water, sp. gr. 0.91 g/ml)
• Milk sample
Procedure-1

Take 5 ml of milk in a test tube.

Add 5 ml of alcohol into it.

Add few drops of 1 % (w/v) alcoholic solution of rosalic acid and mix.

Procedure-2

Take 2 ml rosalic acid solution (0.05% in 60:40 ethyl alcohol and distilled water,
sp. gr. 0.91 g/ml) in a test tube.

Add 2 ml of milk.

Inference
If neutralizer is present, a rose red colour appears whereas pure milk shows only a brownish
colouration

36 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
B. By using Milk adulterants kit
These strips are commercially available in the market.
Procedure
As instructed by the manufacturer
Advantage of using Milk adulterants kit:
• It gives an instant result
• Easy to perform
C. By using reagent strips
These strips are commercially available in the market.
Procedure
As instructed by the manufacturer
Interpretation
Visual observation of change in colour in the test strip as indicated by the manufacturer
Advantage of using reagent strips:
• Reagent strips start working rapidly when dipped and gives result within 5-10 min.
• Easy to perform

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 37
 3.11 Method of Detection of Melamine in Milk and Milk Products
Melamine is a toxic triazine compound used as an adulterant in milk & milk related products to
increase the protein content. Milk adulteration may lead to series of health problem like kidney
failure and even death in many cases.
ISSN : 0975-9492 Vol 6 No 02 Feb 2015)
Presence of Melamine in milk can be detected by two methods as stated below:
A. Conventional method
B. By using rapid test kit for melamine detection
A. Conventional method
This method should only be used after adequate training in an institute with experience on the
machine.
Chemicals and reagents required
• Melamine (MEL). CAS #: 108-78-1.
• Acetonitrile (ACN.) LC grade.
• Formic acid. Reagent grade >95%.
• Water. LC grade, or purified by Millipore Milli-Q system to>18 Mohm resistivity, or
equivalent.
• Ammonium Formate. Purity> 97%.
Preparation of solutions
• 0.1% Formic acid in water. 1ml formic acid is transferred to 1L graduated flask and diluted
to volume with LC water.
• Mobile Phase A. 0.1% Formic acid in Acetonitrile (5:95 v/v). Mix 50 ml of 0.1% formic acid
in water with 950 ml ACN in a 1 L solvent bottle.
• Mobile Phase B. 20 mM Ammonium Formate in Acetonitrile (50:50 v/v). Mix 500 ml of 20
mM ammonium formate and 500 ml of acetonitrile in a 1 L solvent bottle.
• 2.5% Formic acid in water. 25 ml formic acid is transferred to 1 L volumetric flask and
diluted to volume with LC grade water.
• 20mM Ammonium formate. 0.63 gm of ammonium formate is weighed and dissolved in
0.5 L LC grade water.
Equipments required
• Liquid chromatograph. Binary LC pump is recommended for accurate mixing at low flow
rate and rapid response to mobile phase gradient.
• Liquid chromatography column. ZIC-HILIC, 2.1 X 150mm, 5μm, 200 A
• Mass Spectrometer. Triple quadrupole capable of meeting system suitability.
• Centrifuge. Capable of 4000 RPM with 50 ml tubes.
• Microcentrifuge. Capable of 13,000 RPM with 1.5 or 2 ml tubes. (vi) Mixers and shakers.
Single and multi tube vortex mixers (VWR), platform shaker.

38 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
 • Utrasonic bath. Including timer and heater
• Centrifuge tubes. 50ml disposable polypropylene with caps, with graducations from 5
to 50 ml and 1.5 ml microcentrifuge tubes. (ix) Syringe Filters. Polyvinylidene fluoride
(PVDF), 13mm, 0.22um (x) Syringes. Three ml polypropylene.
Procedure
i. Standard preparation

Individual stock solutions, melamine, approximately 100 μg/ml. Weigh approximately 10 mg of

standard using a weigh boat to nearest 0.1 mg and transfer to a 100 ml glass volumetric flask.

Add 70 ml 0.1% formic acid in water and sonicate for 10 minutes. Maintain the volume as 100ml
with 0.1% formic acid in water and mix thoroughly

Calculate exact concentration, correcting for purity

Standard mixture dilution, 50 μg/ml is used for fortification and matrix calibration standards.
Transfer 5.00 ml of each stock standard into a 20 ml glass scintillation vial.

ii. Sample preparation

Sample powder (2.0 ± 0.1gm) is weighed in a 50 mL polypropylene centrifuge tube

Pre-fortify control and matrix calibration standards

14 mL of 2.5% Formic acid in water is added to samples. Tube is tightly sealed. Dissolve sample by
shaking for 15-30 seconds (vortex as needed), then sonicate in ultrasonic bath and mix on multi
vortex mixer for 30 minutes each.

Centrifuge at 4000 rpm for 10 minutes at room temperature.

Approximately 1.4 mL of the supernatant is transferred into a 1.5 mL micro centrifuge tube

Centrifuge at 13,200 rpm (16100 gm) for 30 minutes

Load aqueous extract into a plastic 3 ml syringe and force through a 13mm, 0.22um PVDF filter into a micro
centrifuge tube. Possible stopping point: aqueous extracts can be stored at 5-10°C for future dilutions

Vortex mix for 30 seconds and centrifuge at 13200 rpm (16100gm) for 30 minutes

Supernatant is transferred to a 2 mL autosampler vial, avoiding the precipitate

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 39
 Instrumental analysis
• The column is equilibrated in Mobile Phase A at 0.4 ml/min for 30-60 min.
• It is necessary to evaluate system suitability. To do this, solvent blank (1x) and mixed
standard are injected at 7.0 ng/ml (3-4x).
• Data should meet the signal-to-noise and ion ratio criteria before continuing.
• It is recommended to inject the standards and sample in following sequence: (i) solvent
blank (Mobile Phase A), (ii) extracted matrix standards from 0.25 to 5 μg/g, (iii) solvent
blank, (iv) control extracts, (v) post-fortified extracts and solvent standards for calculation
of recoveries and matrix effects, (vi) solvent blank, (vii) unknown samples, and (viii)
continuous calibration standards (an extracted matrix standard as well as solvent
standard at 7 ng/ml), to verify that instrument response was maintained during the run.
Calculations
• Use external standard calibration. The calibration curve should not include the origin,
but does include a matrix blank with a concentration of 0.
• Export the processed data into Microsoft Excel or equivalent spreadsheet program for
further calculations:
• Recovery (%) = calculated from extracted calibration curve
• Matrix effect (%) = 100 x Post-fortified sample / solvent standard (same cone)
• The limit of quantification (LOQ) for each analyte is defined as the concentration of the
lowest calibration standard used, or the lowest calibration standard which shows > 10-
fold higher response than background signals in negative control sample.
Calculations for Confirmatory Analysis
Calculate ion ratios as percent relative abundances. The melamine ion ratio is m/z 68/85.
B. By using rapid test kit for melamine detection
This kit is commercially available in the market.
Procedure
As instructed by the manufacturer
Advantage of using melamine rapid test Kit:
• It gives an instant result
• Easy to perform

40 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
3.12 Detection of mineral oil in ghee (Holde’s test)
Milk fat is a highly valuable and costlier product consumed throughout the world as it imparts
good sensory and nutritional properties and also adds economy to the milk and other milk
products. Therefore it is highly prone to adulteration with cheaper and inedible mineral oils etc.
Apparatus required Flat-bottom flask: 250 ml capacity fitted with reflux condenser
Reagents required
Alcoholic potassium hydroxide (4%):
Dissolve 4 g of potassium hydroxide in approximately 10 ml water and make the final
volume to 100 ml with ethanol (95%, v/v). The solution should be colourless or very pale
yellow. Keep in a dark place.
Procedure

Take 2 ml rosalic acid solution (0.05% in 60:40 ethyl alcohol and distilled water,
sp. gr. 0.91 g/ml) in a test tube.

Cool and add 100 ml hot (just boiled) distilled water.

Inference
Appearance of turbidity indicates the presence of mineral oil

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 41
 3.13 BR reading for detection of vegetable fat IS: 1479(part–1): 1997
Milk fat is a highly valuable and costlier product consumed throughout the world as it imparts
good sensory and nutritional properties and also adds more value to milk and milk products.
Therefore it is highly prone to adulteration with cheaper oils/fats such as vegetable oils, animal
depot fats, hydrogenated fats and inter esterified fats.
Presence of vegetable fat in milk can be detected by two methods as stated below:
A. Conventional method
B. By using Milk adulterants kit 
A. Conventional method
Apparatus and reagent required
• Butyrorefractometer
• Hot water bath
• Milk sample
Procedure
Extract ghee from milk sample

Put few drops of melted ghee on cleaned prism of butyrorefractometer connected to the
circulating water bath at 400C and close it gently

Allow it to stand for one minute

Note down the reading at 40°C

If the Butyro-refractometer reading of ghee (extracted from milk) exceeds 43.0 or less than 40,
indicates the presence of vegetable oils / foreign fat

After use clean the prism with rectified sprit and tissue paper

If B.R. reading taken from the Butyrometer fat after Gerber test, apply the following formula:
Corrected BR = Observed BR + 0.08*Observed BR (40°C)
Note:
For homogenized milk, fat has to be extracted by the Gerber method using open-ended
butyrometer. Observed B.R reading is to be corrected as follows to nullify hydrolytic effect of
H2SO4 on the fat.
Corrected B.R. = Observed BR + 0.08 * observed BR
B. By using Milk adulterants kit
This kit is commercially available in the market.
Procedure
As instructed by the manufacturer
Advantage of using Milk adulterants kit:
• It gives an instant result.
• Easy to perform.
42 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
3.14 Ammonium sulphate detection test
Bovine milk contains around 80% casein and 20% whey proteins of high biological value. In
general, fraudulent producers and traders of milk tries to increase the volume produced and
delivered to the market by adding water, which alters its composition and reduces its nutritional
quality. The reduction in protein concentration is one of the most significant effects. As a
consequence, unethical producers and traders add nitrogen-rich compounds like ammonium
sulphate to correct the apparent milk protein content. This practice is very dangerous for
consumers leading to serious health hazards.
Presence of ammonium sulphate in milk can be detected by two methods as stated below:
A. By using barium chloride
B. By using Milk adulterants kit
A. By using barium chloride
Reagents and apparatus required:
• Barium chloride (BaCl2.2H2O) solution: 5% (w/v, aq.)
• Trichloroacetic acid (TCA): 24% (w/v, aq.).
• Milk Sample
• 50 ml stoppered test tube
• Whatman filter paper Grade 42
Procedure:
Take 10 ml of milk in a 50 ml stoppered test tube and add 10 ml of TCA solution

Filter the coagulated milk through Whatman filter paper Grade 42

Take 5 ml of clear filtrate and add 5 ml of barium chloride solution.

Inference:
Formation of milky-white precipitates indicates the presence of added sulfates like ammonium
sulfate, sodium sulfate, zinc sulfate, magnesium sulphate, etc. to milk.
B. By using Milk adulterants kit
This kit is commercially available in the market.
Procedure
As instructed by the manufacturer
Advantage of using Milk adulterants kit:
• It gives an instant result
• Easy to perform

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 43
 3.15 Synthetic milk detection test
Some milkmen remove milk fat and replace it with vegetable oil. However, vegetable oil is not
miscible with milk, so they put detergent in it to make it miscible. The milk thus prepared is
called synthetic milk.
Conventional method:
Reagents required
• One test tube
• Urease (20 mg. per ml)
• 0.5% BTB solution (bromothymol blue)
Procedure
Take 5 ml milk in a test tube and add 0.2ml urease (20 mg. per ml)

Shake well and then add 0.1 ml of BTB solution (0.5%)

The methods for detection of urea and synthetic milk are same; the only difference is appearance
of dark blue colour in case of synthetic milk and yellow colour in case of urea.

Inference
Appearance of dark blue colour indicates the presence of synthetic milk

44 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
3.16 Glucose test (Validated Method)
Presence of Glucose in milk can be detected by three methods as stated below:
A. Conventional method
B. By using Milk Adulterants Kit 
C. By using reagent strips
A. Conventional method
Procedure

Immerse diastic (Bayer Diagnostic Ltd) in milk for 1 to 2 seconds

Wipe off excess milk from the strip

Wait for 30 seconds

Compare reagent area with colour chart and report the result as present or absent.

B. By using Milk adulterants kit

This kit is commercially available in the market.
Procedure
As instructed by the manufacturer
Advantage of using Milk adulterants kit:
• It gives an instant result
• Easy to perform
C. By using reagent strips
These strips are commercially available in the market.
Procedure
As instructed by the manufacturer
Interpretation
Reagent strips start working rapidly when dipped in milk and gives a result within 3-4 min.
Visual observation of change in colour in the test strip as indicated by the manufacturer
Advantage of using reagent strips:
• It gives an instant result
• Easy to perform

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 45
 3.17 Test for presence of boric acid and borates (Validated Method)
Presence of boric acid and borates can be detected in milk by conventional method.
Apparatus and reagents required
• 50 ml beaker
• Two test tubes
• 1% phenolphthalein indicator
• N/9 NaOH solution
• Distilled water
• 50% Glycerol
• Milk sample
Procedure (Glycerol method)
Take 10 ml milk in a 50 ml beaker

Add 1 ml 1% phenolphthalein indicator

Titrate against N/9 NaOH soln until pink colour persist for 0-15 seconds

Add 3-4 drops of N/9 NaOH. The milk will be pinkish in colour

Divide the sample in two parts in test tubes

In one test tube, add some volume (~2.0 ml) of distilled water and in another test tube,
add 50% Glycerol (~2.0 ml)

Compare the colour of Glycerol added test tube with the water added test tube

Inference
The test tube added with glycerol will be faded or pink colour will disappear indicates the
presence of boric acid i.e. boric acid Positive
Detection Level: 0.02% (200 ppm)

46 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
4. Chemical Tests
4.1 Determination of pH in milk
The measurement of pH in milk is important in testing for impurities, spoilage, and signs of
mastitis infection. Milk is slightly acidic or close to neutral pH. For all species, milk with colostrum
has a lower pH and mastitic milk has a higher pH. Fresh milk has a pH value of 6.7. When the
pH value of the milk falls below pH 6.7, it typically indicates spoilage by bacterial degradation.
Eventually, when the milk reaches an acidic enough pH, coagulation or curdling will occur along
with the characteristic smell and taste of “sour ” milk. Mastitis is an ever-present challenge with
dairy milking cows.  A pH measurement offers a quick way to screen for infection of the udder
Determination of pH of milk can be me done by two methods as stated below:
A. By using indicator strips
B. By using pH meter
A. By using indicator strips
Procedure
Indicator paper strips are made by soaking strips of absorbent paper in
a suitable indicator and drying them

A rough estimate of pH is obtained by dipping a strip of the prepared paper

in milk and observing the colour

Bromocresol purple (pH range 5.2 to 6.8 – colour changes from yellow to purple) and
bromothymol blue (pH range 6.0 to 7.6 – colour changes from straw-yellow to bluish-green)
are commonly used as indicators

Both narrow and wide range ready-made indicator papers are available
commercially over the pH range 2.0 to 10.5

Note: Indicator paper strips shall always be kept in a closed glass bottle and in a dry condition. These
strips are commercially available in the market.
B. By using pH meter
Take 50 ml of milk sample at 30°C in a 100 ml glass beaker

Measure the pH of milk with help of a calibrated pH meter (calibrated with a standard buffer of
known pH value i.e. pH 7.0 or 9.2) by dipping the electrode in the beaker

Read the pH of the milk after 30 sec

Inference
• On an average cow milk gives a pH of 6.7and buffalo milk 6.8.
• Milk of pH below 6.7 should be considered suspected as an indication of some diseases
of the udder or of late lactation milk.
• Alkaline pH of the milk may also indicate neutralization of milk by NaOH, Na2CO3, and
NaHCO3 etc.
Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 47
 4.2 Determination of temperature of milk
Materials required
• Standard calibrated thermometer
• Milk sample
Procedure
Determine the temperature of milk with a standard calibrated thermometer. Bulk raw milk,
when received from a chilling centre in the dairy factory shall not have temperature above 7°C.
4.3 Determination of titratable acidity as lactic acid (Ref: 79 (Part-I): 1997)
The titratable acidity test is employed to ascertain if milk is of such a high acidity so as to
reduce its keeping quality and heat stability. Generally the acidity of milk means the total acidity
(Natural + developed) or titratable acidity. It is determined by titrating a known volume of milk
with a standard alkali .
Determination of titratable acidity of milk as Lactic Acid can be me done by two methods as
stated below:
A. Conventional method
B. By using paper strip test with color comparator
A. Conventional method Apparatus and materials required
• 100 ml conical flask
• Distilled water
• Phenolphthalein indicator
• N/10 NaOH
• Milk sample
Procedure
Take 10 ml milk in 100 ml conical flask, add 10 ml distilled water

Add 1ml phenolphthalein indicator and titrate against N/10 NaOH till a faint pink colour appears

Calculate the acidity % as volume of NaOH used×0.09

Calculation
Titratable acidity % (as lactic acid) = 9V1N/ V2
Where
• V1 = Volume in ml of the standard sodium hydroxide required for titration,
• N = Normality of the standard sodium hydroxide solution, and
• V2 = Volume in ml of milk taken for the test
B. By using paper strip test with color comparator
These strips are commercially available in the market.

48 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
Procedure: As per the instruction given within the kit
Advantage of using Milk Adulterant Kit :
• It gives an instant result.
• Easy to perform.

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 49
 4.4 Clot on boiling test (IS-1479 (Part-I): 1997) *
This test is done for assessment of keeping quality of milk. Formation of clot means milk is no
longer marketable.
Apparatus and materials required
• Test tube
• Water bath
• Spirit lamp
• Milk sample
Procedure

Transfer 5 ml of the sample to the test tube and smell for any acidic flavour

Place the tube in a boiling water bath and hold for about 5 minutes,
and smell again for any acidic flavour

Remove the tube from the water bath and rotate it in an almost horizontal position
and examine the film of milk or side of the test tube for any precipitated particles

Alternately, take 5 ml of milk in a test tube, boil on the flame of a spirit lamp and
examine the film of milk or side of the test tube for any precipitated particles
Inference
Formation of clots in the test tube indicates COB positive milk and is unacceptable

50 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
4.5 Phosphatase test for pasteurized milk IS: 1479 (Part II): 1961
Pasteurisation is an essential process of making milk safe and free from pathogens. Alkaline
phosphatase is an enzyme which is naturally present in milk, but is destroyed at a temperature
just near to the pasteurization temperature. Alkaline phosphatase test is used to indicate
whether milk has been adequately pasteurised or whether it has been contaminated with
raw milk after pasteurisation. The test is not applicable to sour milk and milk preserved with
chemical preservatives.
Apparatus required
• Lovibond All-Purpose Comparator with stand
• Standard Discs - giving 0, 6, 10, 18, 42 or 0, 6, 10, 14, 18, 25, 42 readings.
• Fused Glass cells - 25 mm.
• Test-Tubes - 15 x 1.9 cm, fitted with rubber stoppers.
Reagents required
• Buffer solution - 3-5 g of sodium carbonate analytical reagent grade (see IS: 296-1951),
and 1.5 g of sodium bicarbonate analytical reagent grade (see IS: 491-1954) dissolved in
1ltr of water.
• Substrate - disodium p-nitrophenylphosphate not less than 95 percent pure.
• Buffer substrate - Transfer 0.15 g of the substrate into a 100 ml measuring cylinder
or stoppered graduated flask and make up to the mark with the buffer solution. The
solution should not be stored for long periods but may normally be kept in a refrigerator
for up to one week.
The solution is practically colourless; when viewed through a 25 mm cell in the all-purpose
comparator, it should give a reading of less than 10 on the disc.
Procedure
Take 10 ml of the buffer substrate solution into test-tubes marked at
10 ml and bring to 37 to 38°C in a water-bath

Add 2 ml (one ml if 5 ml of buffer substrate are used) of the milk to be tested,

close the tubes with rubber stoppers and invert to mix

Prepare in the same way a blank from a boiled milk of the same type as that under test.
Incubate all the tubes at 37-38°C

Read the yellow colour after 30 minutes, return to the bath, and take a second reading after
incubation for a further 90 minutes

The yellow colour is read in a Lovibond all-purpose comparator on a resazurin stand,

fitted with the disc calibrated in microgram p-nitrophenol

Blank should be placed on the left of the stand and the sample on the right

Readings are taken by looking down on to the two apertures with the comparator facing
a good source of north daylight; the disc is revolved until the sample is matched;
readings falling between two standards are recorded to the nearest reading

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 51
 Interpretation of results
Disc reading after 30 minutes incubation Interpretation
0 or trace Properly pasteurized
6 Doubtful
10 or over Under pasteurized
Disc reading after 2 hours incubation
0 to 10 Properly pasteurized
Over 10 Under pasteurized
The 30-minute test will reveal any serious fault in pasteurization, but to enable minor errors to
be detected, readings shall be taken after further incubation for 90 minutes.

52 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
5. Food Safety Control Tests

5.1 Test for presence of hydrogen peroxide (H2O2)

(Ref: A.O.A.C 17th edn, 2000 Official Method 957.08 Hydrogen Peroxide in milk)
Hydrogen peroxide is used as a sanitizing of milk handling equipment. The presence of hydrogen
peroxide can contaminate the milk. The purpose of the test is to detect any traces inside the
product. This test also allows to check the possible addition of hydrogen peroxide in raw milk,
before pasteurization, to increase its shelf-life.
Presence of hydrogen peroxide in milk can be detected by three methods as stated below:
A. Rapid test for detection of presence of hydrogen peroxide (H2O2)
B. By using potassium iodide and starch method
C. By using reagent strips
A. Rapid test for detection of presence of hydrogen peroxide (H2O2)
Apparatus and reagents required
• Beaker
• Venedium pentoxide (V2O5)
• 6% H2SO4
Procedure

To 10 ml of sample add
Dissolve 1 gm V2O5 in
10- 20 drops of reagent
100 ml of 6% H2SO4)
and mix

Inference
The development of pink or red colour indicates presence of H2O2
B. By using potassium iodide and starch method
Preparation of milk sample for analysis
Reagents required
• Potassium iodide solution: Weigh 20 g of potassium iodide and dissolve it in distilled
water to obtain a 100 ml solution.
• Starch solution: Take 1 g starch powder and dissolve it in distilled water by heating and
make up the volume to 100 ml.
• Potassium iodide–starch reagent: Mix equal volumes of 20% potassium iodide solution
and 1% starch solution.

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 53
 Procedure
Take 1 ml of milk sample in a test tube

Add 1 ml of the potassium iodide-starch reagent and mix well

Observe the color of the solution in the tube

Inference
Blue color will be developed in the presence of H2O2, whereas pure milk sample remain white
in color
C. By using reagent strip
These strips are commercially available in the market.
Procedure
As instructed by the manufacturer
Interpretation
Reagent strips start working rapidly when dipped in milk and gives a result within 30-60 seconds.
Visual observation of change in colour in the test strip as indicated by the manufacturer
Advantage of using reagent strips:
• It gives an instant result
• Easy to perform

54 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
5.2 Detection of detergent residues
Detergents are used for a different purpose. Some milk value chain actors remove milk fat
and replace it with vegetable oil. However, vegetable oil is not miscible with milk, so they put
detergent in it to make it miscible. The milk thus prepared is called synthetic milk. Detergents
are used most widely in such milk-like preparations due to their low cost and ease in availability.
Presence of detergent in milk can be detected by two methods as stated below:
A. Conventional method employing methylene blue test
B. By using detergent detector
A. Conventional method employing methylene blue test
Reagents required:
• Methylene blue dye
• Chloroform
Reagent(s) preparation:
• Methylene blue dye - 12.5 mg is dissolved in 100 ml of distilled water. Protect the solution
against direct sunlight.
• Chloroform (Inflammable and toxic on inhalation. Mouth pipetting is not recommended).
Procedure

Pipette 1 ml of suspected milk sample into a 15 ml test tube

Add 1 ml of dye solution followed by addition of 2 ml chloroform

Vortex the contents for about 15 sec and centrifuge at about 1100 rpm for 3 min

Note the intensity of blue color in lower and upper layer

Inference
Relatively, more intense blue color in lower layer indicates presence of detergent in milk.
Relatively more intense blue color in upper layer indicates absence of detergent in milk (as
mentioned in below Fig.).
Limits of detection:
The method can detect presence of 12.5 mg of laboratory grade detergent (labolene) in 100 ml
of milk sample. Thus limit of detection of method is 0.0125% labolene in milk.

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 55
 Fig. : Comparison of developed color in presence and absence of detergent in milk.
Relatively more blue color in lower layer (right side tube) than upper layer indicates
presence of detergent in milk.
Note: The method is capable of detecting all the commonly available detergents in different brand
names such as Ezee, Safe Wash, Super Nirma, Rin Advanced, Rin Shakti, Rin (Powder), Tide (Powder),
Nip (Powder), Clinic Plus, Sunsilk, Pentene, Head & Shoulder etc. The efficacy of the method is not
affected in presence of other additives viz., urea, sucrose, glucose, starch, formalin, hydrogen peroxide
and neutralizers etc. In presence of high concentration of neutralizers, lower layer may appear
pinkish. It has been observed that defatted milk provides better clarity in results. Defatted milk can
be prepared by centrifuging milk at 15°C for 10 minute followed by removal of fat accumulated at
top surface of milk present in centrifuge tube. In the literature, bromocresole purple test has been
mentioned. However, the test has not worked in authors’ laboratory even in the presence of 1%
detergent in milk.
B. By using detergent detector
This detector is commercially available in the market.
Procedure
As instructed by the manufacturer
Advantage :
• It gives an instant result
• Easy to perform

56 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
5.3 Rapid test for detection of aflatoxin M1 in milk
Aflatoxin is a kind of toxins produced by certain fungi (Aspergillus spp.) which are generally
found in agricultural crops and by products like maize, wheat bran, rice bran, rice polish,
hay, paddy straw, etc., mainly when feeds and fodder are stored in a damp condition and
gets mouldy. When dairy animals are fed with these crops, crop residues and by-products
contaminated with aflatoxins, the toxin is secreted through milk, eventually affecting the
consumers of this contaminated milk. The toxin is carcinogenic in nature, which means they
can cause cancer. Very high doses of aflatoxins cause acute aflatoxicosis, which leads to acute
liver failure, jaundice, lethargy and nausea, eventually leading to death, according to a World
Health Organization (WHO) study.
Presence of Aflatoxin in milk can be detected by multiple methods, of which the three easiest
methods are:
A. By using Charm EZ platform
B. By using Rapid One Step Assay (ROSA)
C. By using rapid test kit
A. Using Charm EZ platform
Charm EZ is very small portable machine and it can give quantitative results of presence of
aflatoxins within 10-60 min depending on types of aflatoxins present in the milk sample.
This machine is a complete simultaneous incubator and analyzer system specially designed for
use in the new ultra-rapid EZ tests, and is also compatible with most Charm 3 and Charm Rosa
test strips. Results are interpreted based on a numerical scale and are stored in the Charm EZ
memory. Data can be printed, displayed on the Charm EZ screen or, downloaded to computer
system in a real time or post analysis mode.
It is a platform connecting with a customized printer in which printed form of results can be
obtained instantly. Manufacturer/Supplier can send their technical person to train the laboratory
staff on how to operate the machine.
Requirement
• Charm EZ platform
• Calibrator
• Aflatoxin M1 specific Strips (It is a readymade one and needs to be procured from the
manufacturer). No other consumables are required for the test.
Precaution
• Sample must be maintained at 2-80 C
• Milk fat in the sample must be kept at below 5%
• Strips should be brought to room temperature before use
• Calibration check must be done
• Look for spillage of milk in the compartment where strip is put
Calibration of the machine
Calibration to be done before conducting any test

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 57
 Calibration check
Daily performance monitoring is to verify that the equipment is performing properly.
Procedure

Step 1:

Go to the Press the Perf Mon

main menu button

Step 2:

Insert the calibrator strip

with the Low calibrator Press the OK button on
Close the door
(Dark Pink) side facing the screen
down

Step 3:
After evaluating the
strip, the Charm EZ will Press the OK button
report the result with when ready to proceed
the high and low limits

Step 4:

Insert the calibrator strip

with the high calibrator Press the OK button on
Close the door
side (Light Pink) side the screen
facing down

Step 5:
After evaluating the
strip, the Charm EZ will Press the OK button
report result with the when ready to proceed
high and low limits

Step 6:
When completed, Press the OK button when
ready to proceed

Step 7:
If calibrator strip fails to read within specified
range, see “Cleaning the Reader Lens” section
or contact the Technical Services Staff at Charm
Sciences.

58 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
Temp adjustment
The temperature requirement is specific to aflatoxin M1 residue, hence the temperature must
be adjusted prior to conducting the test .
Actual Procedure
Before starting the test, put any one of the strips in the machine without closing the door

Check the incubation temperature

Orange colour indicator turns to green after sometime and show the incubation temperature in green

Then, start the work by putting water in the strip

This is required to check the incubation temperature , especially , if it is different from the each
other as the machine needs to be adjusted with the specific temperature required for aflatoxin M1

Results
The results will be displayed on the monitor of the machine. As it can be connected to the
printer and the print out can be taken out for record keeping.
B. By using Rapid One Step Assay (ROSA)
Apparatus and Reagents
• Rosa incubator
• ROSA strip
• Dilution buffer
• Milk sample
Test procedure

Place the strip in the incubator and the peel off the tape

For testing of aflatoxin M1 in milk, mix equal amount of milk sample and
dilution buffer and kept at 4 oC

Add 300 µl of milk sample carefully into Sample loading space and release the tape

Close the lid and tighten the latch; incubate the sample for 8 minutes

Remove the strip after incubation and inspect the C (control) line visually for
even development and afterwards , read the strips on respective channel

Quantify antibiotic residues in milk sample based on the standard curve

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 59
 C. By using rapid test kit
This kit is commercially available in the market.
Procedure
As instructed by the manufacturer
Advantage of using rapid test kit:
• It gives an instant result.
• Easy to perform.

60 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
5.4 Detection of heavy metal in milk
Heavy metal pollution is very much a concern because of their toxicity for animal and human
beings and their lack of biodegradability. Heavy metal toxicity is linked with a number of
diseases, mainly due to chronic exposures. Heavy metals in milk, which is the basic food item of
vulnerable age group of people is of particular concern. Contamination of environment leads
to contamination of food chain, which is the main route of entry of heavy metal in the animal
body, which ultimately causes contamination of milk and animal originated food.
Testing is done by conventional method, and lab technicians needs adequate training on the
instrument.
Acid digestion of milk
Apparatus and reagents required
• Test tubes
• 10% HNO3 solution
• Distilled water
• Hydrogen peroxide (H2O2)
• Hot plate
• Flame Atomic Absorption Spectrophotometer
Procedure
All glassware shall be first cleaned with 10% HNO3 solution and
then further washed with the distilled water

Digest 10 mL milk with 1:3 of H2O2 and HNO3 on a hot plate

Heat the samples on hot plate until their volume reduces to 2 ml

Dilute this 2 mL with 20 mL of distilled water and make a clear solution of

it and brought to the required volume with distilled water

Examine by Flame Atomic Absorption Spectrophotometer

The method covers Fe, Ni, Cu, Cd, Zn, Mn, Cr and Pb.
3 different concentrations, 1.0 ppm, 1.5 ppm and 2.0 ppm, shall be prepared to calibrate
the Flame AAS.
The resultant calibration curve of well-prepared standard concentrations results in a linear curve
by Atomic Absorption Spectrophotometric Analysis
Data analysis
Data was gathered and ordered in tables. The concentrations of milk samples in ppm yielding
positive results for the occurrence of heavy metals were transformed into concentration in mg/
kg (ppm).

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 61
 5.5 Detection of insecticide & pesticide residues in milk
A pesticide is a substance used for killing pests (insects, fungi, herbs and nematodes) harmful
to cultivated crops.  Residues of pesticides and their metabolites can be found in milk due to
contamination of water, use of pesticides in the control of ectoparasites directly in the animal,
consumption of contaminated pastures and/or ration with pesticides and these cause a wide
range of toxic effects in human beings. The presence of these compounds in milk may pose a
potential threat to public health.
Paper strip for rapid detection of pesticide residues in milk (Patent Reg. no 2213/DEL/2014)
A. Extraction of pesticide from milk
Extraction of pesticide from milk: Pesticide are extracted from spiked reconstituted skimmed
milk (RSM) / natural milk sample as per following protocol:
Procedure
Mix equal quantity of milk sample and organic solvent, vortex and
centrifuge @ 10000 rpm for 5 min at 37 oC

Mix supernatant with clean up reagent (I), vortex and centrifuge @ 10000 rpm for 5 min at 37°C

Transfer solvent layer carefully to a tube containing cleanup reagent (II), vortex and centrifuge @
10000 rpm for 5 min at 37°C

Separate out upper organic solvent layer and filter through specific filter tips

Evaporate filtrate using block heater at 80°C for 40 min

The tube containing pesticide residue (Tube-2) is used to carry out paper-strip assay.

Stepwise extraction procedure for pesticide residues in milk

62 STANDARD LABORATORY PROTOCOL ON TESTING MILK SAMPLES FOR QUALITY & SAFETY
B. Paper-strip protocol
Enzyme pesticide interaction: Transfer reconstituted spores from Tube-1 to Tube-2 containing
evaporated pesticide residues from extracts of spiked / natural milk sample and incubate in dry
block heater at 37 °C for 40 min and vortex for 25 sec

Addition of paper strip: Add paper-strip functionalized with chromogen to test and control tube
and incubate in dry block heater at 37°C for 15-20 min. After incubation, paper strips were
air dried for 5 min after color development in control tubes

Result interpretation
Development of sky blue color on paper strip, indicates absence of pesticide and no blue color
indicates presence of pesticide in milk as depicted in below mentioned figure.

Fig: Stepwise assay procedure of paper strip assay for detection of pesticide residues in milk

GC procedure for confirmation / quantification of pesticide residues in milk

Sample preparation protocol for GC (Gas Chromatography)
This method should only be used after adequate training in an institute with experience on the machine.
5mL of milk sample was taken in dry mortar, add 20g of silica gel and 15g of
anhydrous sodium sulphate and mix using pestle to make free flowing powder

Prepare a glass column by plugging glass wool and add 40mL of Dichloromethane (DCM)

Transfer the prepared content from step-1 into glass column. Allow to stand for 90 min

Elute DCM drop wise @ 20 drops/ min. Again eluate the column using 150ml
of acetone and DCM in the ratio of 2:1 (v/v)

Elute the solvent again and evaporate by keeping in fume board till the volume get concentrated

Dissolve the concentrate obtained using n-hexane by thorough shaking and again evaporate by
keeping in fume board to ensure complete removal of DCM

The obtained extract or concentrate is reconstituted using 5mL of n-hexane

and acetone in the ratio of 1:1 and used for further GC analysis

STANDARD LABORATORY PROTOCOL ON TESTING MILK SAMPLES FOR QUALITY & SAFETY 63
 Fig: Sample preparation for Gas chromatography analysis of milk samples

Equipment and Conditions for GC

• Gas chromatographic (GC) methods are suitable for the separation and quantitative
determination of compounds which are volatile or semi-volatile and thermally stable
at the temperature of the measurement. Estimation of pesticide residues is done using
GC equipped with electron capture detector (ECD) and flame thermionic detector (FTD),
like Shimadzu 2010 Plus.
• The GC oven temperature for ECD was programmed for an initial temperature of 170°C
with holding time of 13 min, and then increased to 270°C at a rate of 3°C/min with the
hold time of 20 min.
• Detection was carried out by electron capture at 310°C.The chromatographic conditions
were: injector, 280°C; sample volume 1 ml; carrier gas, nitrogen at a pressure of 117.4 kPa
and a flow-rate of 1 ml/min.
• Whereas for FTD oven temperature was programmed for an initial temperature of 180°C
with holding time for 2 min, then increased to 270°C at a rate of 10°C/min with holding
time of 3 min and finally to 280°C at a rate of 5°C/min with holding time of 5 min.
• The injection port temperature was kept at 280°C and the detectors temperature at
310°C.

64 STANDARD LABORATORY PROTOCOL ON TESTING MILK SAMPLES FOR QUALITY & SAFETY
5.6 Formalin test
Formalin is not permitted to add in any edible food products. It is also not allowed in milk
meant for regular use, but it is the only legally permitted preservative for milk and milk products
samples meant for analytical purposes in India.
Test for presence of formalin in milk by Hehner’s Test
Apparatus and reagents required
• Test tube
• 90 percent H2SO4 containing traces of ferric chloride
• Formaldehyde free milk
• Milk sample
Procedure

Take 2 ml of milk sample in a test tube

Add 2 ml of 90 percent H2SO4 containing traces of ferric chloride

from the side of the test tube slowly

If sucrose is present, distil the milk sample (25 ml) and then carry out the test on the distillate by
taking 2-3 ml of distillate and adding 2 ml of formaldehyde free milk

Inference
Formation of purple ring at the junction indicates formaldehyde is present in milk. The violet
coloration does not appear usually when relatively large quantities of formaldehyde are present.
The milk supplies the tryptophane that must be present for the test to operate.

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 65
 6. Additional Tests

6.1 Test for presence of skimmed milk powder in natural milk

By conventional method: (Journal of Food Science and Technology, 1985)
Apparatus and Reagents Required
• Centrifuge machine with centrifuge tube
• Acetic acid:4%.
• Phosphomolybdic acid: 1% solution in water
• Distilled water
• Milk sample
Procedure
Take 50 ml of milk in a 60 ml centrifuge tube

Place the tube in the centrifuge and balance it properly

Centrifuge at 5000 rpm for 15 minutes. Decant the supernatant creamy layer carefully

Add 0.5 ml of 4% acetic acid to skim milk portion for coagulation of protein

Centrifuge the tubes at 5000 rpm for 5 min. Decant the supernatant and
wash the precipitate with distilled water twice. Discard the washings

Then, add 2 ml of 1% phosphomolybdic acid to the washed precipitates

Mix the contents thoroughly and heat in a water bath at boiling temperature
for 15 minutes and then cool
Inference:
The curd obtained from pure milk shall be greenish in colour whereas the curd of sample
containing skimmed milk powder shall be bluish in colour
The intensity of bluish colour depends on the amount of the skimmed milk powder
present in the sample

66 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
6.2 Test for detection of antibiotic residues
Presence of Antibiotic residues in milk can be detected by three methods as stated below:
A. Rapid test for detection of antimicrobial residue in milk by using Charm EZ platform
B. By employing DPA (dipicolinic acid) based test
C. By employing paper strip based test
D. By employing Charm assay for detection of antibiotic residues in milk
E. By employing Rapid One Step Assay (ROSA)
F. By using broad spectrum screening test kit
A. Rapid test for detection of antimicrobial residue in milk by using Charm EZ platform
Charm EZ is very small portable machine and it can give results of presence of antimicrobial
residues within 10-60 min depending on types of antimicrobials present in the milk sample.
This machine is a complete simultaneous incubator and analyzer system specially designed for
use in the new ultra-rapid EZ tests, and is also compatible with most Charm 3 and Charm Rosa
test strips. Results are interpreted based on a numerical scale and are stored in the Charm EZ
memory. Data can be printed, displayed on the Charm EZ screen or, downloaded to computer
system in a real time or post analysis mode.
It is a platform connecting with a customized printer in which printed form of results can be
obtained instantly.
Manufacturer/supplier sends their technical person to train the laboratory staff on how to
operate the machine.
Requirement
• Charm EZ platform
• Calibrator
• Antimicrobial specific strips (These are antibiotic specific and each needs to be procured
from the manufacturer). No other consumables are required for the test.
Precaution
• Sample must be maintained at 2-8°C
• Milk fat in the sample must be kept at below 5%
• Strips should be brought to room temperature before use
• Calibration check must be done
• Look for spillage of milk in the compartment where strip is put
Calibration of the machine
Calibration to be done before conducting any test
Calibration check
Daily performance monitoring is to verify that the equipment is performing properly.

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 67
 Step 1:

Go to the Press the Perf Mon

main menu button

Step 2:

Insert the calibrator strip

with the Low calibrator Press the OK button on
Close the door
(Dark Pink) side facing the screen
down

Step 3:
After evaluating the
strip, the Charm EZ will Press the OK button
report the result with when ready to proceed
the high and low limits

Step 4:

Insert the calibrator strip

with the high calibrator Press the OK button on
Close the door
side (Light Pink) side the screen
facing down

Step 5:
After evaluating the
strip, the Charm EZ will Press the OK button
report result with the when ready to proceed
high and low limits

Step 6:
When completed, Press the OK button when
ready to proceed

Step 7:
If calibrator strip fails to read within specified
range, see “Cleaning the Reader Lens” section
or contact the Technical Services Staff at Charm
Sciences.

Temp adjustment
The temperature requirement is specific to particular antimicrobial residue, hence the
temperature must be adjusted prior to each test as required by the particular antimicrobial
residue test.

68 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
Actual Procedure
Before starting the test, put any one of the strips in the machine without closing the door

Check the incubation temperature

Orange colour indicator turns to green after sometime and show the incubation temperature in green

Then, start the work by putting water in the strip

This is required to check the incubation temperature, especially , if it is different from the each
other as the machine needs to be adjusted with the specific temperature required for each type of
antimicrobials. Say, for Chloramphenicol-39.60 C, for Quinolone-59.0 C, and so on

Results
The results will be displayed on the monitor of the machine. As it can be connected to the
printer and the print out can be taken out for record keeping.
B. By employing DPA (dipicolinic acid) based test (patent No. 264145)
Stepwise procedure:
• Instrument settings: Calibration with known standards provided with the kits
• Analytical methods: The following analytical methods were used for detection of
antibiotic residues in milk samples procured during 2018-19 from Rajasthan, Punjab,
and Haryana.

Pipette out 100 μl of milk sample to be tested and transfer to labeled tubes slowly straight to the
agar medium. Negative reconstituted skimmed milk (RSM) is also kept as negative control along
with test sample

Incubate negative / test samples in a pre-set incubator at 64±0.5°C for 3:00 hours

After incubation, Observe for color change from purple to yellow

within time period as specified above
Results Interpretation
• Yellow color indicates the complete absence of antibiotic Residues in milk sample.
• A Purple color indicates the presence of antibiotic Residues in milk sample at
MRL or >MRL level

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 69
 C. By employing paper strip based test
Stepwise procedure:
Results Interpretation
Appearance of blue color on paper strip indicates absence of antibiotic
Add 250 µl of distilled water to the tube containing a nutrient disc to
hydrate it and vortex for 25 seconds

Impregnate the paper strip with spore, dip chromogenic substrate into the milk sample
and insert into the tube containing rehydrated nutrient media

Incubate the preparation at 64°C for 1 hr. and 15 min in dry block heater,
then observe for sky blue color development

70 STANDARD LABORATORY PROTOCOL ON TESTING MILK SAMPLES FOR QUALITY & SAFETY
residues in milk while no color development indicates presence of antibiotic residues in milk at
or above MRL as depicted in below figure
Step-wise working procedure of paper strip test
D. By employing Charm assay for detection of antibiotic residues in milk
Test procedure
Same as in case of 5.3 Rapid test for detection of aflatoxin M1 in milk
E. By employing Rapid One Step Assay (ROSA):
Test procedure
Same as in case of 5.3 Rapid test for detection of aflatoxin M1 in milk
F. By using broad Spectrum screening test kit .
Procedure
As per the instruction given within the kit.
It covers 15 groups of antibiotics and detect more than 40 antibiotic residues. It complies with
the detection limit of antibiotic residues in different types of milk at MRL (maximum residue limit)
levels recommended by the Codex / EU limits/FSSAI. Kit can be used for routine monitoring of
raw milk, pasteurized milk and dried milk products. It is suitable to implement in the system at
each segment of milk supply value chain to monitor drug residues in milk for routine monitoring
as well as for regulatory compliance of requirements set by FSSAI.
Advantage: A cost effective and affordable way of testing for antibiotic residues.

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 71
 7. Microbiological Test Procedures

Milk , being a good medium for growth and proliferation of a variety of microorganisms such as
–bacteriamoulds & yeast and their toxins, needs to be stringently screened before use in order
to prevent transmission of these microbes to consumers through milk. The most commonly
used tests recommended by FSSAI and the test recommended by this assessment for state and
district laboratories are given below:
7.1 Total plate count or Standard plate count
IS 5402:2012 (RA-2018)/ISO 4833:2003.
Total plate count results reflect the number of colonies that can emerge under the given physical
and chemical conditions (atmosphere, temperature, pH, available nutrients, and presence of
growth inhibitory compounds). Colonies are aggregates of living microbial cells, and hence,
the results cannot be compared with those from direct counts. Plate counts underestimate
the presence of microorganisms, as quiescent, viable but not culturable, and non-culturable
microorganisms are omitted from the count
[Rebhun’s Diseases of Dairy Cattle (Second Edition),2008]
Materials Required
• Diluent: 0.1% Peptone or Phosphate buffer (90 or 99 ml, 9 ml)
• Media: Plate count agar (PCA) medium (pH 7.0 at 25º C); autoclaved at 121º C for 15
minutes
• Pestle & mortar, Petri dishes, pipettes, incubator (35º C) & Marker
Procedure
Sampling: Collect the sample randomly from entire lot. According to FSSAI sampling 5 sachets
(500 ml) of samples will be collected from entire lot. Sample will be stored at refrigeration
temperature (2-7oC) until analysis.
Preparation of the test sample: 100-150 ml of the sample will be poured into the sterile sample
bottle from each sachet. Opened sachets will be sealed and kept at refrigeration temperature
(2-7oC).

72 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
Serial dilution and plating

Mark the tubes and petri dishes for batch no., sample no., parameter, dilution etc.

Pipette out 10 or 11 mL of sample from sample bottle into 90 or 99 mL

of diluent bottle (1:10 dilution)

Pipette out 1 mL of diluted sample from 1:10 dilution bottle into 9 mL of diluent tube

Subsequently go for further dilutions if required by pipetting out from previous

dilution into 9 mL of diluent tube

Pipette out 1 mL from each dilution into respectively marked dilution plates

Pour the PCA (50oC) medium into the plates and allow it to solidify

Incubate the plates at 35 ± 2oC for 48–72 hrs

Fig: Typical colonies on plate count agar

Counting & Calculation
After incubation, retain dishes containing not more than 300 colonies at two consecutive
dilutions. It is necessary that one of these dishes contains at least 15 colonies. Calculate the
number N of micro-organisms per millilitre or per gram of product, depending on the case,
using the following equation:

Where
∑C is the sum of colonies counted on all the dishes retained; n1 is the number of dishes retained
in the first dilution; n2 is the number of dishes retained in the second dilution; d is the dilution

STANDARD LABORATORY PROTOCOL ON TESTING MILK SAMPLES FOR QUALITY & SAFETY 73
 factor corresponding to the first dilution. Round the result calculated to two significant figures.
Take as the result the number of micro-organisms per millilitre or per gram of product, expressed
as a number between 1.0 and 9.9 multiplied by 10 x where x is the appropriate power of 10.
7.2 Yeast and mould counts
( IS 5403:1999 (RA 2013) /| ISO 7954:1987)
Total Yeast and Mold Counts (TYMC) are used to detect and quantify the amount of fungal
growth and allow for identification of viable yeast and mold species present. The amount of
fungi is reported as the number of colony forming units (CFUs).
Materials Required:
• Diluent: 0.1% peptone or Phosphate buffer (90 or 99 ml, 9 ml)
• Media: Yeast Extract-Dextrose Chloramphenicol (YEDC) Agar (pH 5.4); Autoclaved at
121ºC for 15 minutes
• 10% tartaric acid solution/ 1% lactic acid
• 0.25 % of sterile sodium propionate solution
• Pestle & mortar, Petri dishes, Glass pipettes, Incubator (25ºC) and Marker
Procedure
Sampling: Collect 100 – 150 gm. of milk and milk product from the entire lot randomly and
store the sample at refrigeration temperature (27ºC) until analysis.

74 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
Serial dilution & plating

Weigh 10 of milk and milk product sample aseptically and add into pestle containing 90 ml
potassium phosphate buffer (pH 7.0) or 0.1% peptone water

Triturate the sample in pestle by using mortar along with diluent for few minutes and pour it back
into the dilution bottle (1:10 dilution/ 1st Dilution) for further dilution

Pipette out 1 ml of diluted sample from 1st Dilution into 9 ml of diluent and it is treated as
(1:100 or 2nd dilution).

At the same time pipette out 1 ml diluted sample from respective dilution 1:10/ 1: 100)
dilution into respectively marked petri dishes

If required you can go for further serial dilution and plating

Pour about 15 ml of the YEDCA medium, previously melted and maintained at

45±1oC in a water-bath, from a culture bottle into each petri dish.

The time elapsing between the end of the preparation of the initial suspension (or of the 10-1
dilution if the product is liquid) and the moment when the medium is poured into the dishes shall
not exceed 15 min. At the time of addition the pH of the medium should be reduced to 3.5 using
10% tartaric acid or 1% lactic acid

Carefully mix the inoculum with the medium and allow the mixture to solidify by leaving
the petri dishes to stand on a cool - horizontal surface. Prepare a control plate, with 15 ml
of the medium, to check its sterility

Invert the plates and place them in the incubator at 25 ± 1°C

Make a separate count of the yeast colonies, which usually will be characterized as smooth, moist,
elevated or surface colonies.

After counting the typical yeast colonies, count the mould colonies. Mould colonies are
easily recognized by their profuse growth of hyphae. If only yeast counts are required,
add 0.25 % of sterile sodium propionate solution to the plate at the time of
pouring to inhibit the growth of moulds

If necessary, carry out a microscopic examination in order to distinguish, according to their

morphology, the colonies of yeasts and moulds from colonies of bacteria. Generally, it is
desirable to differentiate between moulds and yeasts. It is advisable to examine the plates at
the end of three days for yeast colonies as they are likely to be overgrown by mould growth.

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 75
 Fig: Plates showing yeast and mould colonies

Interpretation
Count the colonies on each plate after 3, 4 and 5 days of incubation. After 5 days, retain those
plates containing fewer than 150 colonies. If parts of the plates are overgrown with moulds, or if
it is difficult to count well-isolated colonies, retain the counts obtained after 4 or even 3 days of
incubation. In this event, record the incubation period of 3 or 4 days in the test report.
Expression of results
Use counts from plates containing fewer than 150 colonies. The number of yeasts and moulds
per gram or per millilitre is calculated according to the equation described in D.16. If there are
no colonies on plates from the initial suspension, if the initial product is solid, the number of
yeasts and moulds per gram of product should be reported as fewer than 10. If there are no
colonies on plates from the test sample. If the initial product is liquid, the number of yeasts and
moulds per millilitre of product should be reported as less than 1.

76 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
7.3 Coliform count (IS 5401: 2002/ ISO 4833: 1991 (RA-2012)
Coliforms are a group of Gram-negative rod-shaped bacteria that have similar biochemical
characteristics and are not a single species of microorganism. They are used to monitor the
quality of milk being able to ferment lactose with the production of acid and gas within 48 hr at
35°C and grow with or without oxygen. These are usually present in small number in raw milk. It
is a simple test and easy to conduct.
Absence of coliforms in 1:100 dilutions in raw milk and in 1:10 dilution of pasteurized milk is
accepted as a satisfactory quality.  The presence of  E. coli is a proof that contamination from
excreta has occurred.
Materials required
A-1 Medium, EC medium, m-FC medium, Membrane filtration unit; Test tubes/ Sampling bottle;
Incubator (37oC and 45oC)
Procedure-1: Single-step procedure

Dilutions of the sample are inoculated into fermentation tubes of A-1 medium

The tubes are first incubated for 3 hrs at 35ºC and then transferred to a 44.5ºC water bath for an
additional 21 hrs of incubation

Tubes showing growth plus gas are considered positive for fecal coliforms

Procedure-2: Multiple Fermentation Tube (MFT) Technique

Cultures from positive tubes from the Presumptive (total) coliform test are
inoculated into fermentation tubes of E. coli medium

Incubated at 44.5ºC for 24 hrs

Tubes showing growth with gas production are

considered confirmed positives

Procedure-3: Membrane Filtration (MF) technique

Samples are filtered onto membranes as in the total coliform test; the membranes are placed
onto plates of m-FC medium, sealed in water-tight plastic bags, and submerged in a 44.5ºC
water bath incubator for 24 hrs. Colonies with a characteristic faecal coliform appearance are
then counted and faecal coliform density is computed.

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 77
 Fig: Typical colonies ion VRBL Agar

Flow chart for E. coli and coliforms

78 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
7.4 Escherichia coli (E. coli)
IS 5887 Part I 1976 (RA-2013)
Conventional method for the enumeration of E. coli
Media and reagents :
• Lauryl tryptose (LST) broth
• Levine’s eosin-methylene blue (L-EMB) agar
• MR-VP broth
• Butterfield’s phosphate-buffered water
• Kovacs’ reagent, Voges-Proskauer (VP) reagents
• Methyl red indicator ,Violet red bile agar (VRBA)
• Peptone Diluents, 0.1% & Brilliant Green Lactose Bile Broth
Materials
• Test sample
• 1% peptone
• MacConkey broth medium
• MacConkey agar medium
• Eosin methylene blue lactose agar
• Tergitol-7 agar
• Kovac’s reagent
• Methyl red
• alpha-naphthol
• Potassium hydroxide
• Incubator
• Pipette

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 79
 Procedure
Blend the sample in a sterile blender jar for 2 minutes or macerate with sterile sand in a sterile
mortar using approximately 200 mL of diluting fluid per 25 g of the sample

The diluting fluid for preparing the homogenate should be at 1% peptone

(ISO 6887 solution in water

Inoculate 1 mL of the blended or macerated sample into 10 mL of single strength MacConkey broth
medium. If the numbers of organisms are assumed to be very small, inoculate 10 mL of double
strength MacConkey broth medium.

Also streak loop full on to MacConkey agar medium, eosin methylene blue lactose agar,
and if available Tergitol-7 agar

Incubate all the inoculated media at 37°C overnight

If there is growth with fermentation of lactose in the MacConkey broth medium streak
out a loop full on to each of the solid media, and incubate at 37°C overnight

Test for Identification: Pick out and mark as many suspected colonies from the solid media as
possible, but not less than 5, to investigate. The suspect colonies are smooth and are lactose
fermenting on Mac Conkey agar and on eosin ethylene blue lactose agar, and are yellow colonies
surrounded by yellow zones on Tergitol-7 agar medium.
(1) Test for Indole - Inoculate peptone water medium as in with a loopfull of 24 hour growth in
nutrient broth and incubate at 37°C for 48 hours. Add 0.5 ml of Kovac’s reagent, prepared by
dissolving 10 g p-dimethyl-aminobenzaeldehyde in 150 ml amyl alcohol or isoamyl alcohol
and to which 50 ml of concentrated hydrochloric acid is slowly added. Prepare the reagent
in small quantities and store in refrigerator. After adding Kovac’s reagent, shake the tube
gently, the appearance of a red colour indicates the presence of indole.
(2) Test with Methyl Red- Inoculate the glucose peptone water 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.
(3) Test for Voges-Proskauer Reaction - Inoculate the glucose peptone water 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 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.
(4) Test for Citrate Utilization- Inoculate the strain on to Simmion’s Citrate Agar 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.

80 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
Procedure for enumeration:
Preparation of Sample

Blend at 8 000 to 10000 rev/min for 2

minutes. Alternatively macerate with
Take 25 to 50 g of the sample in a, sterile
the diluting fluid in a sterile mortar with
blender jar and to this add diluting fluid
sterile sand, Make serial ten-fold dilutions
(0.1% Peptone) to have dilution of 10-1
with the diluting fluid, in duplicate series,
up to 10-6

Plate Count:

Enumerate the colonies of E. coli, which

Spread out 0.1 mL from each dilution
are yellow in colour surrounded by a
tube, evenly on to Tergitol-7 agar, and
yellow zone, and confirm these as being
incubate at 37°C for 24 hours
Escherichia coli by IMViC tests

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 the sample. If Tergitol-7agar is not in use,
then MacConkey agar plates or eosin methylene blue lactose agar plates may be used.

Fig: E. coli on MacConkey Agar, Tergitol 7 Agar and EMB Agar

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 81
 7.5 Staphylococcus aureus
Highly pathogenic strains of  Staphylococcus aureus  can cause disease in both humans and
animals. In animal species, including ruminants,  S. aureus  may cause severe or sub-clinical
mastitis. Dairy animals with mastitis frequently shed S. aureus into the milk supply which can
lead to food poisoning in humans.
Materials required
• Incubator,
• Drying cabinet or incubator,
• Water bath,
• Test tubes,
• Flasks or bottles with screw caps,
• Petri dishes, straight wire and Pasteur pipette,
• Total-delivery graduated pipettes,
• Spreaders,
• pH Meter.
Test portion, initial suspension and dilutions:
Agitate the sample thoroughly by inverting the sample container 25 times

Pipette out 1mL or 10 mL of the sample with a sterile pipette and add to
9mL or 90 mL mL of diluents

Shake this primary dilution with a movement of about 300 mm for 7s manually or using a
mechanical agitator to obtain a 10-1 dilution
Inoculation:
Transfer 0.1 ml of the test sample if liquid, or 0.1 ml of the initial suspension
(10-1 dilution) in the case of other products, to each of two Baird Parker agar (BPA) plates.

Repeat the procedure for the 10-2 dilution and for further decimal dilutions, if necessary.

If, for certain products, it is desirable to count low numbers of coagulase-positive staphylococci,
the limits of detection can be raised by a factor of 10 by inoculating 1.0 ml of the test sample if
liquid, or 1.0 ml of the initial suspension for other products, either on the surface of one large
agar plate (140 mm) or on the surface of three small agar plates (90 mm). In both cases, prepare
duplicates by using two large plates or six small ones.

Carefully spread the inoculum as quickly as possible over the surface of the agar plate, trying not
to touch the sides of the dish, using the spreader. Allow the plates to dry with their lids on for
about 15 min at laboratory temperature.

82 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
Incubation
Invert the plates prepared and incubate them for 24 h ± 2 h then re-incubate for a further 24 h
± 2 h in the incubator at 35ºC or 37ºC).

Fig: Staph. aureus on Baird Parker Agar

Selection of plates and interpretation

 After incubation for 24 h ±2 h, mark on the bottom of the plates the positions of any
typical colonies present
 Re-incubate all plates at 35ºC or 37ºC for a further 24 h ± 2 h, and mark any new typical
colonies. Also mark any atypical colonies present. Take for enumeration only those plates
that contain at the maximum 300 colonies with 150 typical and/or atypical colonies at
two successive dilutions. One of the plates shall contain at least 15 colonies.
 Select for confirmation a given number A (in general 5 typical colonies if there are only
typical colonies, or 5 atypical colonies if there are only atypical colonies, or 5 typical
colonies and 5 atypical colonies if both types are present, from each plate).

Fig: Catalase test (+ve indication)

Catalase reaction: Take an isolated colony and suspend it in a drop of hydrogen peroxide
solution (3%) on a slide. The immediate formation of gas bubbles indicates a positive reaction.

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 83
 Confirmation (coagulase test)
From the surface of each selected colony, remove an inoculum with a sterile wire and transfer it to
a tube or bottle of brain-heart infusion broth

Incubate at 35ºC or 37ºC for 24 h ± 2 h. aseptically add 0.1 mL of each culture to 0.3 mL of the
rabbit plasma (unless other amounts are specified by the manufacturer) in sterile haemoliysis
tubes or bottles, and incubate at 35ºC or 37ºC).

By tilting the tube, examine for clotting of the plasma after 4 h to 6 h of incubation and, if the test
is negative, re-examine at 24 h of incubation, or examine at the incubation times specified by the
manufacturer

Consider the coagulase test to be positive if the volume of clot occupies more than half of the
original volume of the liquid. As a negative control, for each batch of plasma,
add 0.1 mL of sterile brain-heart infusion broth to the recommended quantity of
rabbit plasma and incubate without inoculation

For the test to be valid, the control plasma shall show no signs of clotting

Fig: Interpretation of Coagulase test

84 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
7.6 Salmonella spp IS 5887 Part 3: 1999 (RA 2013)
Contamination of raw milk and products with Salmonella spp. is mostly due to infected persons,
fecal contamination from the cow, and contamination of the environment, since natural
infections of the udder are rare. Deficient hygiene in dairies, especially those from developing
countries, has often been considered as one of the major reasons for contamination of milk with
both spoilage and pathogenic bacteria. Raw milk and milk products are increasingly becoming
important sources of human infection.
Pre-enrichment
Twenty-five (25) grams or ml of sample is added to 225 ml of buffered peptone water and
incubated at 37°C. In case of Salmonella 2 ml of brilliant green (0.1% sol.) is added and incubation
is done for 24 hours.
Selective Enrichment:

Transfer 1ml portion from pre-enrichment step to each 10ml of serenity cosine broth &
tetrathionate broth and incubated at 37°C for overnight

After required incubation time, contents of both tubes are mixed and a loopful from the mixed
contents is streaked on to the Xylose lysine deoxycolate agar (XLD), and Bismuth Sulfite Agar
(BSA) plate and hektoen enteric agar

These plates are incubated at 37°C for 24 hrs. The incubation may be continued up to 72 hrs before
report as nil.

Observation:

Pink and black color colony of Salmonella on XLDA and BGA

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 85
 Black color colony of Salmonella on BS agar Blue-green to bluish colony of Salmonella on HE agar

Red color colony of Salmonella on Rambach agar (Chromogenic media)

Biochemical test for detection of Salmonella

Materials required:
• Lysinedecarboxylase broth,
• Phenol red dulcitol broth,
• SI test,
• Tryptone broth,
• Potassium cyanide (KCN) broth,

86 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
 • Malonate broth,
• Indole test, P
• henol red sucrose broth or Purple sucrose broth,
• MR-VP broth etc.
Observation:

Indole test Urease Test

Methyl Red Test Triple Sugar Iron Test

Observation for TSI slants

Reaction Fermentation
• Acid butt (yellow), alkaline Glucose
• Glucose fermented
fermented Slant (red)
• Acid throughout medium, butt Lactose or
• Lactose or Sucrose or both fermented
sucrose or and slant yellow both fermented
• Gas bubbles in butt, medium sometimes split • Aerogenic culture
• Blackening of the butt • Hydrogen sulfide Produced
• Alkaline slant and butt(Medium entirely red • None of the three sugars fermented

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 87
 Observation for biochemical tests
Result Salmonella
Test or substrate species
Positive Negative reaction
Glucose (TSI)
 Yellow Butt Red Butt +
Lysine decarboxylase (LIA) Purple butt
 Yellow butt +
H2S (TSI)
 Blackening No blackening +
Urease
 Purple-red coor No color change -
Lysine decarboxylase broth Purple color
 Yellow color +
Phenol red dulcitol broth
 Yellow color and/or gas No gas; no color change +
KCN broth
 Growth No growth -
Malonate broth
 Blue color No color change -
Indole test
 Violet color at surface Yellow color at surface -
Phenol red lactose broth
 Yellow color and/or gas No gas; no color change -
Phenol red sucrose broth
 Yellow color and/or gas No gas; no color change -
Voges-Proskauer test
 Pink-to-red color No color change -
Methyl red test
 Diffuse red color Diffuse yellow color +

88 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
7.7 Listeria monocytogenes
Listeria monocytogenes is an omnipresent bacterium which causes listeriosis in humans, a serious
infectious disease that occurs on consumption of milk contaminated with this pathogen. Listeria
monocytogenes most commonly occurs as a consequence of post-pasteurization contamination.
L. monocytogenes has the ability to multiply and grow at low temperatures (4 ° C) and to survive
even on freezing temperatures, and as such poses risk for health of consumers, if found in milk,
cheese, ice-cream and other dairy products.
Materials required:
• Test sample
• Half Fraser broth
• Fraser broth
• PALCAM agar
• Incubator
• Pipette
Procedure:
Test portion and initial suspension: In general, to prepare the initial suspension, add a test
portion of x g or x ml to 9x ml or 9x g of the selective primary enrichment medium (half Frazer
broth), to obtain a ratio of test portion to selective primary enrichment medium of 1/10 (mass
to volume or volume to volume).
Primary enrichment: A, food samples of 25 g are homogenized in 225 ml, respectively, of half
Fraser broth and incubated for 24 h at 30ºC.
Secondary enrichment: A volume of 0.1 ml of the primary-enriched sample is used to inoculate
10 ml of Fraser broth and incubated 24 h at 37ºC.
Plating out and identification:
• From the primary enrichment culture incubated for 24 h ± 3 h at 30°C, take, by means of
a loop or glass rod, a portion of the culture and inoculate the surface of the first selective
plating medium, Agar Listeria according to Ottaviani and Agosti (ALOA), so that well-
separated colonies are obtained. Proceed in the same way with the second selective
plating-out medium
• From the secondary enrichment medium incubated for 48 h ± 2 h at 35°C or 37°C, repeat
the procedure with the two selective plating-out media.
• Invert the dishes and place them in an incubator set at 37°C for ALOA and at the appropriate
temperature for the second selective medium (PALCAM Agar). If a commercial medium
is used for the second selective medium, follow the manufacturer’s instructions.
• After incubation for 24 h ± 3 h (and for an additional 24 h ± 3 h if the growth is weak
of if no colony is observed after 24 h incubation) for ALOA or for the appropriate time
(second selective agar), examine the dishes for the presence of colonies presumed to be
Listeria spp.”
a. ALOA: Consider as L. monocytogenes the green-blue colonies surrounded by an
opaque halo (typical colonies). If growth is slight, or if no colony is observed, or if

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 89
 no typical colony is present after 24 h ± 3 h of incubation, re-incubate the plates for
further 24 h ± 3 h.
b. Second selective medium: Examine after the appropriate time to check for the
presence of colonies which, from their characteristics, are considered to be
presumptive Listeria spp. or monocytogenes, depending on the type of medium
used.”
Confirmation of Listeria spp.
Selection of colonies for confirmation:
• Take from each plate of each selective medium, select up to five colonies presumed to
be Listeria spp.
• Streak the selected colonies onto the surface of pre-dried plates of TSYEA (tryptone soya
yeast extract agar ).
• Place the plates in the incubator set at 35°C or 37°C for 18 h to 24 h or until growth is
satisfactory.
• Typical colonies are 1 mm to 2 mm in diameter, convex, colorless and opaque with an
entire edge
Catalase reaction: Take an isolated colony and suspend it in a drop of hydrogen peroxide
solution (3%) on a slide. The immediate formation of gas bubbles indicates a positive reaction.
Gram staining: Perform the Gram stain on a colony separated Listeria spp. are revealed as
Gram-positive slim, short rods.
Motility test:
• Take an isolated colony and suspend it in a tube containing TSYEB. Incubate in the
incubator set at 25°C for 8 h to 24 h until a cloudy medium is observed.
• Deposit a drop of the above culture using a loop onto a clean glass microscope slide.
Place a coverslip on top and examine it with the microscope.
• Listeria spp. appears as slim, short rods with tumbling motility.
• Cultures grown above 25°C may fail to exhibit this motion.
Carbohydrate utilization:
• Inoculate using a loop each of the carbohydrate utilization broths with a culture from
TSYEB. Incubate at 35°C or 37°C for up to 5 days.
• Positive reactions (acid formation) are indicated by a yellow colour and occur mostly
within 24 h to 48 h.
Table -1 Differentiation of Listeria species
Species β-Hemolysis Mannitol Rhamnose Xylose Virulence
L. monocytogenes + - + - +
L. ivanovii + - - + +
L. innocua - - Vd - -
L. welshimeri - - Vd + -
L. seeligeri + - - + -
L. grayi - + Vd - -

90 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
7.8 Enterobacter sakazakii
Enterobacter sakazakii causes infections in mainly neonates leading to development of
bacteraemia, necrotizing enterocolitis (NEC) and infant meningitis. It is an emerging disease.
Pre-enrichment in non-selective liquid medium: The pre-enrichment medium i.e. buffered
peptone water is inoculated with the test portion and incubated at 37°C±1°C for 16- 20 h.
Enrichment in selective liquid medium: The selective enrichment medium is inoculated
with the culture obtained in Pre-enrichment in non-selective liquid medium and incubated at
44°C±0.5°C for 22-26 hrs.
Plating out and identification: A chromogenic agar is inoculated with the enrichment culture
and incubated at 44°C±1°C for 22-26 hrs.
Confirmation: Typical colonies are selected from the chromogenic agar, and isolates producing
a yellow pigment on tryptone soya agar are biochemically characterized.
Materials required
Diluent: Buffered peptone water
Media for growth
• Modified lauryl sulfate tryptose broth (mlST)/vancomycin medium
• Enterobacter sakazakii isolation agar (ESIATM)
• Tryptone soya agar (TSA)
• L-Lysine decarboxylation medium
• L-Ornithine decarboxylation medium
• L-Arginine dihydrolation medium and
• Simmons citrate medium
Media for fermentation of carbohydrates
• Peptone water with phenol red, D-sorbitol, L-rhamnose, D-sucrose, D-melibiose and
amygdaline); Carbohydrate solutions (D-sorbitol, L-rhamnose, D-sucrose, D-melibiose
or amygdaline), 80 mg/ ml
• Oxidase test reagent: N,N,N’,N’-Tetramethyl-p-phenylenediamine dihydrochloride
(C10H16N2·2HCl)
Equipment and other requirements
• Total delivery pipettes, having a nominal capacity of 1 ml;
• Water bath, capable of being maintained at 44°C±0.5°C.
• Petri dishes made of glass or plastic, of diameter 90 mm to 100 mm.
• Incubators, capable of operating at 25°C±1°C, 30°C±1°C and 44°C±1°C, respectively.
• Loop, made of platinum-iridium or nickel chromium, of diameter approximately 3 mm,
or disposable loops.
• Test tubes, of diameter 18 mm and length 160 mm (plugged or with screw caps).
• pH meter, accurate to 0.1 pH unit at 25°C±1°C.

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 91
 Procedure
Test portion
To prepare the primary dilution, add x g of the test sample to 9 times x ml of pre-enrichment
medium, which is the ratio of test sample to pre-enrichment medium specified in this method.
Allow dry samples to disperse in the liquid without stirring. If a sample has not been dissolved
completely after 30 min, then mix it gently with the medium.
Pre-enrichment: Incubate the inoculated pre-enrichment medium at 37°C±1°C for 18 ± 2 hrs.
Selective enrichment
After incubation of the inoculated pre-enrichment medium, transfer 0.1 ml of the obtained
culture into 10 ml of mlST/ vancomycin medium.
Incubate at 44°C±0.5°C for 24±2 hrs. It is recommended to use either a water bath or a forced-air
incubator to ensure that the maximum temperature (44.5°C) is not exceeded.
Isolation of presumptive E. sakazakii: After incubation of the inoculated mlST/ vancomycin
medium, streak a loopful (ca. 10 μL) onto the surface of the Enterobacter sakazakii isolation agar
plate. Incubate the plate at 44°C±1°C for 24±2 hrs. After incubation, examine the chromogenic
plate for the presence of typical colonies of presumptive E. sakazakii.
NOTE Typical colonies are small to medium sized (1 mm to 3 mm) green to blue-green colonies.
Non-typical colonies are often slightly transparent and violet colored.
Confirmation: Production of a yellow pigment
Selection of colonies: Select one to five of the typical colonies of presumptive E. sakazakii
examined on the incubated chromogenic plate.
Incubation:
• Streak the selected colonies onto the surface of the TSA plate so that after incubation
separate colonies can be observed.
• Incubate the plate at 25°C±1°C for 44-48 hrs. After incubation, examine the TSA plates
for the presence of yellow-pigmented colonies.
• When only one colony is selected and transferred to the TSA plate and after incubation
no yellow pigmented colonies can be seen, select four more typical colonies.
• If there are fewer than five typical colonies, select all of them.
Biochemical confirmation: Miniaturized biochemical identification kits, currently available
commercially and permitting the identification of Enterobacter sakazakii, may be used.
Selection of colonies: Select one yellow pigmented colony from each tryptone soya agar plate
for following biochemical characterization.
Oxidase disposable
• Using a glass rod or inoculation needle, take a portion of each selected characteristic
colony.
• Streak the taken portion on a filter paper moistened with the oxidase reagent or on a
commercially available disc.

92 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
 • Do not use a nickel/ chromium loop or wire. Consider the test to be negative when the
color of the filter paper has not changed to mauve, violet or deep blue within 10 s.
L-Lysine decarboxylase
• Using a loop, wire or glass rod, inoculate the L-lysine decarboxylation medium with each
of the selected colonies just below the surface of the liquid medium.
• Incubate the tubes at 30°C±1°C for 24±2 hrs.
• A violet color after incubation indicates a positive reaction. A yellow color indicates a
negative reaction.
L-Ornithine decarboxylase
• Using a loop, wire or glass rod, inoculate the L-ornithine decarboxylation medium with
each of the selected colonies just below the surface of the liquid medium.
• Incubate the tubes at 30°C±1°C for 24±2 hrs.
• A violet color after incubation indicates a positive reaction. A yellow color indicates a
negative reaction.
L-Arginine dihydrolase
• Using a loop, wire or glass rod, inoculate the L-arginine dihydrolation medium with each
of the selected colonies just below the surface of the liquid medium.
• Incubate the tubes at 30°C±1°C for 24±2 hrs.
• A violet color after incubation indicates a positive reaction. A yellow color indicates a
negative reaction.
Fermentation of various sugars
• Using a loop, wire or glass rod, inoculate each carbohydrate fermentation medium with
each of the selected colonies just below the surface of the liquid medium.
• Incubate the tubes at 30°C±1°C for 24±2 hrs.
• A yellow color after incubation indicates a positive reaction. A red color indicates a
negative reaction.
Utilization of citrate
• Using a loop, wire or glass rod, streak the selected colonies onto the slant surface of
Simmons citrate medium.
• Incubate the tubes at 30°C±1°C for 24±2 hrs.
• The reaction is positive if the medium turns blue.
Interpretation
Confirmatory test +/ - ve reaction Observation
Production of a yellow pigment +
Oxidase –
L-Lysine decarboxylase -
L-Ornithine decarboxylase +
Arginine dihydrolase +

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 93
 Acid from +/ - ve reaction Observation
Fermentation of D-sorbitol -
Fermentation of L-rhamnose +
Fermentation of D-sucrose +
Fermentation of D-melibiose +
Fermentation of amygdaline +
Hydrolysis of citrate +

94 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
7.9 Sulfite reducing clostridia
Clostridia spores are omnipresent in the farming environment. It has been recommended to
use sulphite reducing clostridia as a hygiene indicator for dairy plant systems.
Materials required
• Culture medium and Saline peptone diluent
• Plate count medium: Iron sulfite agar
• Homogenization equipment, for samples of solid food
• Water bath, capable of being maintained at between 44oC & 47oC
• Anaerobic jars, with equipment for generating an anaerobic atmosphere, and including
a system to check the anaerobic conditions;
• Incubator, capable of being maintained at 37 oC ± 1oC and, if necessary, at 50oC ± 1oC;
• Test tubes, of dimensions 16 mm × 160 mm, and flasks or bottles of capacity 500ml.
Procedure
Sampling: Collect 100 – 150 gm. of milk and milk product sample from the entire lot randomly
and store the sample at refrigeration temperature (2-70C) until analysis

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 95
 Serial dilution & plating

Weigh 10 gm. of sample aseptically and add into pestle containing 90 mL

potassium phosphate buffer (pH 7.0)

Triturate the sample in pestle by using mortar along with diluent for few minutes and
pour it back into the dilution bottle (1:10 dilution/ 1st Dilution)

Heat treatment of the initial suspension may be necessary to eliminate vegetative

forms of spore-forming bacteria and/or non-sporeforming bacteria.

Temperatures and heating times vary according to the actual need, from combinations producing
a definite pasteurization effect at a moderate heat activation effect (e.g. 75o C for 20min),
to boiling for several minutes. In this case, results could be given as number of spores of sulfite
reducing bacteria growing under anaerobic conditions

Pipette out 1 mL of diluted sample from 1st Dilution into 9 mL of diluent and it is
treated as 1:100 or 2nd dilution

At the same time pipette out 1 mL diluted sample from respective dilution 1:10/ 1: 100) dilution
into respectively marked petri dishes

For the test to be valid, the control plasma shall show no signs of clotting

Pour into each Petri dish approximately of iron sulfite agar which has been cooled to 44oC - 47oC in
the water bath. The time elapsing between inoculation of the Petri dishes and addition of the agar
should not exceed 15 min. carefully mix the inoculum with the medium by horizontal movements
and allow the medium to solidify

After the medium has solidified, pour 10 to 15 mL of the same

medium into the dish as an overlay

If tubes are used, inoculate a 1 mL volume from each dilution into each of two tubes of medium
kept at to 44oC to 47oC. Mix gently without forming bubbles, and leave the medium to solidify in
a cold water bath. After the medium has has solidified, pour to of the same medium into each tube
as an overlaysolidified, pour to of the same medium into each tube as an overlay

After solidification, incubate the Petri dishes in anaerobic jars at 37oC ± 1oC for 24-48 hrs. If
thermophilic bacteria are suspected, prepare a second set of Petri dishes. Incubate this set at 50oC
± 1oC. In the case of tubes, incubation in anaerobic jars is not necessary

Counting of the colonies

• Read the results after 24 and 48 hrs, depending on the degree of black color and the
growth rate of the microorganisms. Black colonies, possibly surrounded by a black zone,
are counted as sulfite-reducing bacteria.

96 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
Note 1: Diffuse, unspecific blackening of the medium may occur, especially when inoculation is
performed in agar tubes instead of Petri dishes.
• The growth of anaerobic bacteria, which only produce hydrogen (not H2S), may also
reduce the sulfite present and lead to a general blackening of the medium.
• Count colonies of sulfite-reducing bacteria in each dish containing less than 150 typical
colonies and less than 300 total colonies.
• When the number of colonies is high, some tubes may be unreadable. In this case, only
tubes where the colonies are clearly separate should be considered for counting.
Note 2: This International Standard may be used to enumerate only Clostridium. After obtaining
characteristic colonies, pick five of them from each dish, and confirm the genus Clostridium with
confirmation tests (e.g. respiratory tests, spore forming tests).
Expression of results
Calculate the number N of mesophilic lactic acid bacteria present in the test sample, as the
weighted mean from two successive dilutions, using the equation:

Where
• ∑C is the sum of the colonies counted on all the dishes from two successive dilutions, at
least one of which contains at least 15 colonies; V is the volume of inoculum applied to
each dish, in milliliters; n1 is the number of dishes retained in the first dilution; n2 is the
number of dishes retained in the second dilution; d is the dilution factor corresponding
to the first dilution.
• Round the result calculated to two significant figures. Take as the result the number of
mesophilic lactic acid bacteria per milliliter (liquid products) or per gram (other products),
expressed as a number between 1.0 and 9.9 multiplied by the appropriate power of 10.

STANDARD LABORATORY PROTOCOL ON TESTING MILK SAMPLES FOR QUALITY & SAFETY 97
 7.10 Bacillus cereus
Bacillus cereus causes of two types of food borne disease: (i). an emetic intoxication and (ii).
diarrhoeal infection. The enterotoxins are heat-labile and sensitive to acid conditions or
proteolysis. They are destroyed during cooking or gastro-intestinal digestion, however the
spores are highly resistant. Milk containing too many cells will are not destroyed by digestion,
but instead colonize the gut of the host and produce sufficient enterotoxin to cause disease.
Materials required
• Drying cabinet or incubator (37ºC ± 1ºC and 55ºC ± 1ºC),
• Incubator, capable of operating at 30ºC±1ºC,
• Water baths, capable of being maintained at 45ºC±0.5ºC and 50 ºC±1ºC.
• Loops, made of platinum/iridium or nickel/chromium (3mm),
• pH-meter, Test tubes, (18 mm diameter and length 180 mm, and culture flasks, Petri
dishes (90 mm to 100 mm),
• Graduated pipettes.
Test portion, initial suspension and dilutions
• Agitate the sample thoroughly by inverting the sample container 25 times.
• Pipette out 10 ml of the sample with a sterile pipette and add to 90 ml of diluents.
• Shake this primary dilution with a movement of about 300mm for 7s manually or using
a mechanical agitator to obtain a 10-1 dilution.
Inoculation and incubation

Transfer 0.l ml of the test sample, if the product is liquid, or of the initial suspension in the case of
other products, to each of two agar plates (MYP Agar). Repeat the procedure using further decimal
dilutions if necessary

Carefully spread the inoculum as quickly as possible over the surface of the agar plate without
touching the sides of the dish with the spreader

Use a fresh sterile spreader for each plate. Leave the plates with the lids on for about 15 mm at
ambient temperature for the inoculum to be absorbed into the agar

Invert the prepared plates and incubate them for 18 h to 24 h in an incubator) set at 30ºC. If
colonies are not clearly visible, incubate the plates for an additional 24 h before counting

Counting of the colonies

• After the period of incubation, select plates, preferably at two successive dilutions,
containing less than 150 colonies.
• Count the presumptive B. cereus colonies on each plate. The presumptive colonies
are large, pink (indicating that mannitol fermentation has not occurred, and generally
surrounded by a zone of precipitation (indicating the production of lecithinase).
• If there are less than 15 characteristic colonies on plates inoculated with the liquid
product or the lowest dilution for other products.

98 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
 • Calculate the arithmetic mean m of the colonies of B. cereus on both dishes.
Confirmation
Select five presumptive colonies from each plate selected. If there are less than five colonies on
the plate, take all presumptive colonies present. Confirm these colonies by glucose utilization,
VP test and nitrate reduction test. If the plates are overcrowded and it is not possible to select
well-isolated colonies, streak five presumptive colonies on plates with complete medium.
Incubate in an incubator set at 30ºC for 18 h to 24 h.
Biochemical confirmation
Glucose utilization
• Inoculate selected colonies, using a stab inoculation wire, centrally into tubes containing
freshly heated glucose agar. Incubate for 24 h in an incubator set at 30ºC.
• A yellow colour throughout the whole tube indicates a positive reaction.
Voges-Proskauer test
• Inoculate selected colonies into tubes containing VP medium. Incubate for 24 h in an
incubator set at 30ºC.
• Transfer from each tube 1 ml of the culture to a clean tube to test for acetylmethylcarbinol.
• Add 0.2 ml of potassium hydroxide solution, 0.6 ml of α-naphthol solution and a few
crystals of creatine. Shake vigorously and leave to stand for 1 h.
• Formation of an eosin pink colour indicates a positive reaction.
Nitrate Reduction Test
• Inoculate selected colonies into tubes containing nitrate medium. Incubate for 24 h in
an incubator set at 30ºC.
• Test for the reduction of nitrate to nitrite by adding 0.2 ml to 0.5 ml of nitrite reagent to
each tube with a pipette equipped with a rubber bulb.
• In all other cases, calculate the number of B. cereus from the percentage of B. cereus
obtained after counting which are confirmed. Formation of a red colour indicates the
reduction of nitrate to nitrite.
• If no red colour is formed within 15 min, add a small amount of zinc dust and leave for
10 min. If after the addition of zinc dust a red colour is formed, the confirmatory test is
negative.
• If no red colour is formed after the addition of zinc dust, then the test is positive.

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 99
 7.11 Milk ring test for Brucellosis
Brucellosis is a communicable disease of zoonotic importance which causes mainly abortion in
cattle and undulant fever and sterility in humans. Milk is a good medium for transmission of this
organism from animal to humans
Materials Required
• 5 ml glass tubes
• B. abortus Bang Ring Antigen (hematoxylin-stained antigen manufactured by the State
Biological Laboratory, Institute of Veterinary Preventive Medicine, Ranipet, India).
Procedure
Take sufficient quantity of milk samples so as achieve approximately 25 mm height

Add 30 μl (0.03 ml) of B. abortus Bang Ring Antigen carefully

Incubate the preparation at 37°C for 1 hr

Also run negative and positive controls simultaneously

Observation
• A strongly positive reaction is indicated by formation of a dark blue ring above a white
milk column.
• The test is considered negative if the color of the underlying milk exceeded that of the
cream layer and when the cream layer is normal.
• Samples are read as negative, 1+, 2+, 3+ and 4+ depending on the intensity of color in
the cream layer.

Milk ring test for detection of brucellosis in milk samples

Inference
The criteria of reactions are as follows:
• Negative reaction (-): cream ring white, skim milk fraction blue white;
• Suspicious reaction (1+): cream ring pale blue but less colored than the skim milk fraction;
• Suspicious reaction (2+): the pink color of the cream ring equal to that of the skim milk
fraction;
• Positive reaction (3+): color of cream ring deeper blue than that of the skim milk fraction;
• Positive reaction (4+): cream ring pink, skim milk fraction white.

100 STANDARD LABORATORY PROTOCOL ON TESTING MILK SAMPLES FOR QUALITY & SAFETY
7.12 California Mastitis Test for mastitis
Milk is a very good medium for growth and proliferation of the microbial organisms. Milk, when
milked from a cow infected with subclinical mastitis, seems to be quite normal in appearance
but it harbours millions of pathogenic germs which quickly multiply and may cause diseases
in humans on consumption of the same. It is a rapid and easy test for detection of subclinical
mastitis.
Materials Required
• Stripping cup
• CMT paddle
• Reagent
• Gloves
Procedure

After discarding the first stream of milk, draw the next milk into the shallow cups on the paddle,
keeping the quarters separate

Drain Excess milk: The ideal amount of milk is that which remains in the cup when the paddle is
tilted to an almost vertical position

Add an equal amount of the reacting solution

Form pools of milk in cups, as shown, by tilting paddle

Squirt test solution over milk. Avoid making bubbles

Proportion of solution to milk should be at least one to one

Mix the reagent and milk

Gently rotate the paddle in horizontal plane, swirling the mixture for 10-30 seconds

Positive reactions occur and can be graded during this rotary motion

Always assume the same position when holding the paddle under the udder to keep track of the
quarters when interpreting results

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 101
 Leukocyte count per Test appearance CMT score Description Percent
milliliter infected
Below 200,000 Mixture liquid, no precipitate negative Negative 25
Slight precipitate, tends
150,000 to 500,000 to disappear with paddle T Trace 50
movement
Distinct precipitate but
400,000 to 1,500,000 does not gel with paddle 1 Weak positive 75
movement
Distinct
800,000 to 5,000,000 Distinct gel formation 2 90
positive
Strong gel formation that
Over 5,000,000 tends to adhere to paddle. 3 Strong positive 95-100
Forms distinct central peak

102 STANDARD LABORATORY PROTOCOL ON TESTING MILK SAMPLES FOR QUALITY & SAFETY
7.13 MBR (Methylene Blue Dye Reduction) test for raw milk and pasteurized milk
Methylene Blue Dye Reduction Test, commonly known as MBR test is used as a quick method
to assess the microbiological quality of raw and pasteurized milk. This test is based on the fact
that the blue colour of the dye solution added to the milk get decolourized when the oxygen
present in the milk get exhausted due to microbial activity. The sooner the decolourization, more
inferior is the bacteriological quality of milk assumed to be. This test is widely used at the dairy
reception dock, processing units and milk chilling centres where it is followed as acceptance/
rejection criteria for the raw and processed milk
Materials and Reagents Required
• One sterilized test tube
• MBR dye solution
• Sterilized cork
• Serological water bath
• Milk sample
Procedure
The test has to be done under sterile conditions.

Take 10 ml milk sample in sterile MBRT test tube

Add 1 ml MBRT dye solution (dye concentration 0.005%)

Stopper the tubes with sterilized rubber stopper and invert the tube to mix contents properly

Carefully place them in a test tube stand dipped in a serological water bath maintained at 37±1⁰C

Record this time as the beginning of the incubation period

Decolorization is considered complete when only a faint blue ring (about 5mm) persists at the top.

Results of MBR Test

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 103
 Recording of Results
During incubation, observe colour changes as follows:
• If any sample is decolourized on incubation for 30 minutes, record the reduction time as
MBRT - 30 minutes.
• Record such readings as, reduction times in whole hours. For example, if the colour
disappears between 0.5 and 1.5 hour readings, record the result as MBRT - 1 hour;
similarly, if between l.5 and 2.5 hours as MBRT - 2 hour and so on.
• Immediately after each, reading, remove and record all the decolourized samples and
then gently invert the remaining tubes if the decolourization has not yet begun.

104 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
References:
Ananthakumar, T., Suresh, A.J. & Niraimathi, V. (2015). Detection of Melamine residue in raw milk
and milk related products by UV Spectrophotometry, International Journal of Pharma Science
and Research, Vol. 6, No. 22
Bowman, B. & Stone, A. (2017). Collecting samples for microbiological analysis – publication
3124 (POD-09-17), Mississippi State University.
Devrani, M. & Pal, M. (2018). How to detect adulteration of Maltodextrin in milk, Retrieved from
www.foodnbeveragesprocessing.com on April 10, 2020
Fricker (2003). Microbiological Analysis, Elsevier Science
FSSAI (2016). Manual on general guidelines on sampling, Food Safety and Standard Authority of
India, Ministry of Health and Family Welfare, Govt. of India
Iversen, C. & Forsythe, S. (2003). Risk profile of Enterobacter Sakazukii, an emergent pathogen
associated with infant milk formula, Tends in Food Science and Technology, Vol. 14, Pp. 443-454
Kasalica, A., Vukovic, V., Vranjes, A., Memisi, N. (2011). Listeria Monocytogenes in milk and dairy
products, Biotechnology in Animal Husbandry, Vol. 27, No. 1067-1082
McMillan, K., Moore, S.C., McAuley, C.M., Fegan, N. and Fox, E.M. (2016). Characterization of
staphylococcus aureas isolates from raw milk sources in Victoria, Australia, BMC Microbiology,
Vol. 16, 169
Mhone, T.A., Matope, G., and Saidi, P.T. (2012). Detection of Salmonella spp., Candida Albicans,
Aspergillus spp., and antimicrobial residues in raw and processed cow milk from selected farms
of Zimbabwe, Veterinary Medicine International, Pp. 1-5
Paradhkar, M.M., Singhal, R.S. & Kulkarni, P.R. (2000), An approach to the detection of synthetic
milk in dairy milk 2: Detection of detergents, International Journal of Dairy Technology, Vol. 53,
No. 3, Pp. 92-95
Sharma, V., Lal, D. & Aparnathi, K.D. (undated). Chemical Quality Assurance, retrieved from www.
agrimoon.com on 05 June, 2020
Siirtola, Teuvo V.A. (2000), Quality control manual: Raw milk and milk products
Sowmya R., Indumathi, K.P., Arora, S., Sharma, V. & Singh, A.K. (2015). Detection of calcium based
neutralizers in milk and milk products by AAS, Journal of Food Science Technology, Vol. 52, No. 2,
Pp. 1188-1193
vlab.amrita.edu (2011). Detection of Adulteration in Milk

Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety 105
 URLs:
https://aurum-labs.com/2018/10/12/microbial-testing-understanding-total-yeast-and-mold-counts/
http://ecoursesonline.iasri.res.in/mod/resource/view.php?id=101517
https://milktest.co.nz/Our-Services/Milk-Quality-Testing/Sulphite-Reducing-Clostridia

106 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety
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108 Standard Laboratory Protocol on Testing Milk Samples for Quality & Safety