Milk Hygiene Practical GUIDE - 24-5-22

GUIDELINES FOR MILK HYGIENE PRACTICAL
VPM 4500: MILK HYGIENE PRACTICAL MANUAL
A. MILK PLATFORM TESTS

Milk Platform or milk reception tests are tests carried at the point of milk collection by the persons responsible for
raw milk collection. These tests are rapid and important quality control tests that help in detecting milk of inferior
or questionable quality before it is mixed with bulk milk. This is important because one single lot of poor quality
milk can spoil the rest of the milk in the tank. Milk that does not pass the quality control tests are rejected.
The following are some of the most common milk platform tests carried out at the milk collection centres:
a. CLOT-ON-BOILING TEST ( COB TEST)

This test determines suitability of milk for heat processing. If the milk is acidic, heating will precipitate the
protein in milk thereby resulting in clotting.
Materials Required
I. Test tube (20ml capacity)
II. Hot water bath (100°C)
III. Test tube stand
IV. Milk sample
Test procedure
1. Take 5 ml of the milk sample in a test tube.
2. Insert the test tube containing the milk sample in a boiling water bath for 5 minutes.
3. Remove the tube from water bath without shaking.
4. Note any acid smell or precipitated particles on the sides of the test tube.
Judgement
Any milk sample showing precipitates, clots or flakes is recorded as positive for COB test and rejected on
the platform. On the other hand, milk sample that did not show precipitates or coagulates is passed for heat
processing.
b. ALCOHOL TEST
This test also determines suitability of milk for heat processing. If the milk is acidic and not suitable for heat
treatment, adding 70% alcohol will precipitate the protein in milk thereby resulting in clotting.
Materials Required
I. Test tubes
II. Alcohol (70%)
III. Test tube stand
IV. Milk sample
Test procedure
1 Take 2ml of the milk sample in a test tube.
2 Add 2ml of 70% alcohol, shake gently and observe for 5 minutes.
3 Note any acid smell or precipitated particles on the sides of the test tube.
Judgement
Any milk sample showing precipitates, flakes or particles is recorded as positive for alcohol test and is
rejected on the platform. On the other hand, milk sample that did not show clots, flakes or coagulates is
passed for heat processing.
B. MILK QUALITY CONTROL TEST

These are tests aimed at to ensuring application of approved practices, standards and regulations concerning
milk and milk products. This also ensure that milk producers, processors and marketing agencies adhere to
accepted codes of practices.
The tests include the following:
a. METHYLENE BLUE REDUCTASE TEST
1
This test detects the presence of reductase enzyme which affects the keeping quality of milk. The test is
based on the principle that methylene blue (an oxidation-reduction dye or indicator) which is blue in its
oxidized state is reduced to colourless compound as a result of the metabolic reaction by microbes in milk.
Milk samples of poor quality with high bacterial counts decolourizes the milk – dye mixture rapidly, while milk
samples of good quality with low bacterial counts will take several hours to decolourize. The rate of dye
decolourization is a reflection of the degree of bacterial enzyme reductase activity exhibited in the milk
sample.
Materials Required
I. Methylene blue dye (MB)
II. Sterile test tubes- without rim preferably with marking at 10ml and stoppers to fit
III. Water bath set at 37ºC
IV. Clock, watch, or an interval timer
V. Milk sample
Procedure
1. Measure 10mls of milk sample in sterile test tube and add 1ml of Methylene Blue dye (1%) and mix
2. Incubate the tubes containing the milk-dye mixture in water bath at 37ºC and observed at regular
intervals (say 30 minutes) for 5 hours for colour changes.
3. The time taken for complete discoloration of the dye is the major concern for the test. Continue the
observation until a complete reduction of the dye occurs or the formation of persistent blue rings
(0.5mm) at the top.
4. Record the time taken for the reduction of methylene blue dye.
Results and interpretation

Result Interpretation
Complete reduction in 15 minutes Very bad milk

Complete reduction in 30-60 minutes Bad milk
Complete reduction in 1½ -2 hours Poor milk
Complete reduction in 3- 4 hours Doubtful milk
Complete reduction in 4½ -6 hours Good milk
Complete reduction after 6 hours Very good milk
The Malaysian Food Regulation provides that the time taken to completely decolorize the methylene blue dye
should not be less than four (4) hours for raw milk and for pasteurize milk the time taken to completely decolorize
the dye should not be less than five (5) hours.
C. MILK DIAGNOSTIC TESTS

The milk diagnostic tests are aim to identify the specific organisms in the milk sample. Qualitative and
quantitative approaches may be used. Serial dilution of the milk sample under consideration are made before
inoculation on culture media. Colony growths on culture are enumerated after incubation for 24 hours using a
colony counter. Colonial morphology on the plate is appraised. The growths are then stained on glass slide for
morphological identification of the organisms using a microscope. Several biochemical and serological tests will
be needed for confirmation of micro-organisms.
Serial Dilutions of Milk Sample

The milk samples are serially diluted before plating on culture media to ease counting of the growth colonies
which otherwise may be too numerous to count (TNTC) in a mastitic sample. Several tubes containing 9mls of
sterile physiological saline solution (PSS) are used. One milliliter of the sample is then added to the first tube
then mixed thoroughly. One milliliter of the diluted tube is again taken into subsequent tube and the exercise is
continued in that order to the maximum dilution desired.
The desired dilution can then be used to inoculate the culture media.
Culture Media for Milk Diagnostic Tests
Several types of media are available for use in milk diagnostic test but the most commonly used include:
2
a. Enriched media-nutrient agar
b. Selective media -MacConkey
c. Differential media- Brilliant green agar (BGA)
For the purpose of this exercise, the following culture media shall be employed:
i. Brilliant green agar
ii. MacConkey
iii. Nutrient agar
iv. Sabauroud dextrose agar (SDA)
Inoculation of Media
Serially diluted milk samples are inoculated onto culture media either by:
a. Pour plating or
b. Surface plating.
Pour Plating technique
In the Pour plating technique, the media is maintained in a liquid state at 55ºC in a water bath. One ml of the
serial dilution of choice is first added to sterile petri dishes followed by the addition of molten media. Both are
immediately mixed gently.
Advantages of pour plating technique
1. Increased chances of detecting the organisms due to higher volume of sample that may be used,
especially in samples with low bacterial counts
2. Both aerobic and anaerobic growths are possible.
Disadvantages
1. Fragile organism may be destroyed due to heat shock on addition of the molten media.
2. It is difficult to count colonies in solid media.
3. Typical colonial morphology is distorted (especially those underneath the surface of the media).
Surface Plating technique

In the surface plating technique, the media is already poured and solidified and plating is done on the surface of
the media. A tenth of a ml of the diluted sample is routinely added to the culture media and evenly spread out
with the sterile spreader.
Advantages
1. Fragile organism can grow
2. Typical colonial appearance can be expressed.
Disadvantages
1. If the degree of contamination of sample is low, the presence of organism cannot be detected.
Incubation
Plated culture media are incubated at 37ºC for 24 hours after which the colonial morphological identification and
enumeration of counts are carried out.
Microscopical identification is done after staining the colonies on glass slides using suitable stains. The
commonly used stains include:
a. Gram stain-for bacteria
b. Lactophenol cotton blue- for fungi and yeast
c. Zeel-nelson stain (occasionally) – for Acid fast bacteria.
Colony counts
Growths on culture media are enumerated using a colony counter and the counts expressed in colony forming
units per ml (CFU/ml) of the diluted milk sample under consideration.
Pour plate method: suppose 1ml of the 102 dilution was employed and 6 colony forming units were counted
following incubation then, the number of organism in the sample is therefore 6.0 x 102 CFU/ml.
By the surface plating method using 0.1ml the dilution of the plate is 10-5(i.e after removing 0.1ml from the tube
with 10 dilution). If 6CFU are counted, the sample therefore contains 6.0 x 105CFU/ml.
Interpretation
Milk sample with counts of over 300 CFU/ml i.e. 3.0 x 102 CFU/ml are usually considered unfit for human
consumption.
3
DETECTION OF ANTIBIOTICS IN MILK
Dairy cows that have just undergone antibiotics therapy are not recommended for milking for human
consumption due to the adverse effects the antibiotics could have on the consumer. Such adverse effects include
the development of antibiotic resistance by micro-organisms in the gut and hypersensitivity and allergic reaction
that could result in susceptible consumers. The standard withdrawal time of 72 hours (3 days) post treatment
must be ensured before milk from such animals could be passed for human consumption.
Detection of microbial inhibitor in milk

Requirements
1. Absorbable paper discs
2. Bacillus subtilis broth- grown over night at 37 ºC.
3. Mueller-Hinton agar
4. Milk sample
Procedure
1. Milk samples A and B supplied are to be screened for the presence of microbial inhibitors or antibiotics.
2. The discs with corresponding letter had earlier been dipped in the milk sample and dried in an oven at
60ºC for 30 minutes.
3. The broth in tube I contains an overnight growth at 37ºC of Bacillus subtilis.
4. Inoculate 0.1ml of B. subtilis broth into each plate of Mueller-Hinton agar. Allow to dry for a few seconds
and then carefully drop the discs evenly distributed on the agar. Ensure that the discs make proper
contacts with the agar by using a flamed glass rod.
5. Incubate overnight at 37ºC.
6. Observe and record the zone sizes around the discs which indicate the degree of inhibition of B. subtilis
by antibiotic or inhibitor.
7. To determine the specific antibiotic present in the milk sample, you may need discs containing the
specific antibiotic or inhibitor. Other using more advanced equipment such a high-performance liquid
chromatography are also available for the analysis.

Milk Hygiene Practical GUIDE - 24-5-22

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GUIDELINES FOR MILK HYGIENE PRACTICAL

VPM 4500: MILK HYGIENE PRACTICAL MANUAL

A. MILK PLATFORM TESTS

a. CLOT-ON-BOILING TEST ( COB TEST)

B. MILK QUALITY CONTROL TEST

a. METHYLENE BLUE REDUCTASE TEST

Results and interpretation

Complete reduction in 15 minutes Very bad milk

C. MILK DIAGNOSTIC TESTS

Serial Dilutions of Milk Sample

Surface Plating technique

Detection of microbial inhibitor in milk

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