Guide for CMP
Chapter – IV
Adulteration in milk
We have learnt about the definition, importance and relevance of clean milk
production in earlier chapter. Any extraneous matters present or added in the
milk that effect the composition of milk adverse and make the milk un hygienic
and also cause serous health problem in human being are called adulterants.
When consumers buy milk they have a right to assume that it will be pure and
unadulterated. Hence, there is an obligation on the dairy industry to provide
adequate quality control and assurance system. Milk may be adulterated on
purpose in order to defraud or accidentally during production, handling or
processing. Even after knowing the adverse effect, few milk handlers use
adulterants to gain marginal additional income or to hide their weakness. There
are many potential adulterants:
a) Extraneous water
b) Detergents accidentally finding access to milk during production
c) Udder salves
d) Neutralizers used to mask developed acidity
e) Salt or sugar used to mask extraneous water or to elevate total solids
f) Skim milk powder used to elevate milk solids
g) Preservatives such as formalin, hydrogen peroxide, used to mask poor
hygienic quality
h) Foreign fat etc.
The detection of very common adulterants is described below:
A. Detection of adulterants (Cane Sugar)
Testing Procedure
1. Take 15 ml of milk in a test tube.
2. Add 1 ml of concentrated hydrochloric acid and mix.
3. Add 0.1 gm of resorcinol powder and mix thoroughly.
4. Place the tube in a boiling water bath for 5 minutes and observe the colour.
Standards
The test should be negative
Interpretation
Red colour obtained indicates the presence of cane sugar.
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Guide for CMP
Frequency
Suspected samples.
Critical Factor
Note: This test is generally carried out for raw milk and this can be used
for processed milk also.
B. Detection of Adulterants (Starch and other Cereal Flours)
Testing Procedure
1. Take about 3 ml of well-mixed milk in a test tube.
2. Boil the milk.
3. Cool to room temperature and add one drop of 1% iodine
solution and observe the colour.
Standards
The test should be negative
Interpretation
Blue colour that disappears on boiling and re-appears on cooling indicates
the presence of starch / cereal flours.
Frequency
Suspected samples
Critical Factor
Iodine solution should be prepared fresh.
Note: This test is carried out for raw milk and this can be used for
processed milk also.
C. Detection of adulterants (Cellulose)
Testing procedure
1. Take 10 ml of milk in a 100 ml beaker.
2. Add 50 ml hot water and stir for 2 minutes.
3. Filter through a fine mesh nylon cloth.
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Guide for CMP
4. Wash residue on cloth with 50 ml hot water twice.
5. Stain part of residue on cloth with iodine-zinc chloride reagent
and another part with iodine solution.
Standards
The Test should be negative.
Interpretation
Development of blue colour with iodine-zinc reagent and absence of blue
colour with iodine solution confirms presence of cellulose.
Frequency
Suspected samples.
Critical Factor
1. Nylon cloth should be fine enough to retain the residues.
2. Cotton cloth should never be used.
Note: This test is used for raw milk during milk reception.
D. Detection of Preservatives (Boric Acid and Borates)
Testing Procedure
1. Take about 10 ml of milk in a test tube and add about 1 ml of concentrated
hydrochloric acid and mix well.
2. Dip a strip of turmeric paper in the acidified milk.
3. Dry the paper immediately.
Standards
The test result should be negative.
Interpretation
Turning to red colour that turns to dark green colour upon addition of
ammonium hydroxide and can be restored by addition of hydrochloric acid
indicates the presence of boric acid.
Frequency
Suspected samples.
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Guide for CMP
Critical Factor
Note: This is carried out for raw milk and can also be used for processed
milk.
E. Detection of Preservatives (Formalin – Hehner Test)
Testing Procedure
1. Take about 10 ml of milk in a test tube and add 0.5 ml of 10%
ferric chloride solution.
2. Add carefully about 5 ml of concentrated sulphuric acid down
the side of the test tube in such a way that it forms a separate layer at the
bottom without mixing with the milk.
Standards
The test result should be negative.
Interpretation
Observe the colour of the ring formed at the junction of the two liquids.
Development of violet coloured ring indicates presence of formaldehyde
as preservative.
Frequency
Suspected samples.
Critical Factor
1. The test is given only in presence of traces of ferric chloride or
other oxidizing agents.
2. The test is sensitive to one part in thousand.
Note: This test is carried out for raw milk and can also be used for
processed milk.
F. Detection of Preservatives (Hydrogen Peroxide)
Testing Procedure
1. Take 10 ml of a sample of milk in a test tube and add 2 drops of
2% paraphenylenediamine hydrochoride (freshly prepared).
2. Mix thoroughly and observe the colour.
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Guide for CMP
Standards
The test result should negative.
Interpretation
Development of an intense blue colour shows the presence of hydrogen
peroxide.
Frequency
Suspected samples.
Critical Factor
1. Addition of a very small quantity of hydrogen peroxide (less than 0.1%)
cannot be detected by the above test after a period of 24 hours.
2. Hydrogen peroxide is destroyed when milk is heated or stored for long
period.
Note: This test is carried out for raw milk.
G. Detection of Neutralizers (Carbonates and Bicarbonates)
Testing Procedure
1. Take about 5 ml of milk in the test tube.
2. Add 5 ml of alcohol and shake well.
3. Add three drops of 1% (w/v in a alcohol) solution of rosalic acid
and mix well.
Standards
The test result should be negative.
Interpretation
1. Appearance of rose red colour indicates the presence of
carbonate or bicarbonate.
2. Pure milk indicates brownish colour.
Frequency
Suspected samples.
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Guide for CMP
Critical Factor
Note: This test is carried out for raw milk and can also be used for
processed milk.
H. Detection of Neutralizers (Alkalinity of Ash)
Testing Procedure
1. Take 20 ml of milk in a silica dish and ash it well.
2. Disperse the ash in 10 ml of water.
3. Titrate against 0.1 N hydrochloric acid.
Standards
The test result should be negative.
Interpretation
If neutralizers have been used, the hydrochloric acid requirement shall
exceed 1.2 ml.
Frequency
Suspected samples.
Critical Factor
Note: This test is carried out for raw milk and can also be used for
processed milk.
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Guide for CMP
Chapter – V
Detection of Bacteria
Several methodologies are used to evaluate the bacterial content of milk. The
most common procedure is called the Standard Plate Count (SPC). The most of
the co-operative milk unions use this method to detect the bacterial count in milk
and milk products.
Why bacterial detection?
It became necessary to judge the shelf life of milk & milk products, to prevent
health hazards in human being, to fulfill the statutory requirement, to know the
status of sanitary condition, to prevent probable spoilage & to protect consumers.
Microbiological testing methods for detecting bacteria load
a) Direct Methods (DM)
b) Direct Microscopic Count (DMC)
c) Standard Plate Count (SPC)
d) Bactaslydes
e) Methylene blue reduction test (MBRT)
Procedures for detecting bacteria load
a) Direct Microscopic Count (DMC)
The microscopic examination of a sample provides a rapid indication of the
quality of liquid milk products and, at the same time, supplies a permanent record
of the quality. But it does impose considerable eye-strain on the operator. A small
amount (0.01 ml) of the sample is spread over an outlined area of 1 cm 2 of a fat-
free slide using a metal rake. After fixing and staining the smear is placed under
the oil immersion lens of a microscope of which the microscopic factor (MF) is
known:
10,000 x 4 1273
MF = -------------- = -------
31416d2 d2
where d = diameter of the microscopic field in mm.
The average number of organisms per field multiplied by the MF. Yields the
number of organisms per ml of the milk products or the dilution thereof. The
number of fields to be counted depends on the average number of organisms per
filed, for instance:
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Guide for CMP
No. of organisms per field No. of fields to be counted
0–3 64
4–6 32
7 – 12 16
13 – 25 8
By the microscopic method, clump counts or individual counts can be
determined. All bacterial cells, which are not further than 5 µm from another, are
considered to belong to one clump. A single cell, which is further than 5 µm from
another cell, is also a ‘clump’. When determining the individual count, all bacterial
cells within clumps or in isolation are counted. Dead bacteria can also be stained.
However, bacteria killed by heat usually disintegrate soon after, or lose their
stainability. In addition, there are stain solutions, which permit the recognition of
dead cells, and then the microscopic count can be applied to heated milk, or
reconstituted samples of powdered milk, to furnish information concerning the
past history of the product.
The microscopic method can also be used for counting somatic cells. For milk
from individual quarters of cows in normal lactation, a somatic cell counts of
500 000 cells ml –1 has been accepted. For bulk milk, cell counts ranging from
less than 300 000 to 1000 000 ml –1 were considered acceptable in various
countries (International Dairy Federation, 1967). The recommended methods for
somatic cell counting, including the microscopic cell count, have been published
by the International Dairy Federation (1979).
b) Standard Plate Count (SPC)
Testing Procedure
1. Melt the required quantity of Plate Count medium by boiling and cool 44 –
45 ºC.
2. Add 1 ml of thoroughly mixed appropriate dilution of sample aseptically in
sterile labeled petri dish in duplicate, near flame.
3. Gently lift the cover of the petri dish near a flame and pour 12 to 15 ml of
liquified agar medium. Wipe dry the bottom of the medium bottle.
4. Sterilize the lips of medium container by exposing it on the flame,
immediately before pouring, periodically during pouring and when pouring
is completed for each batch plates.
5. Rotate and tilt the dish without splashing the mixture over the edge and
spread the mixture evenly over the bottom of the plate.
6. after solidification invert the plate and promptly place in the incubator.
7. Run agar medium blank as well as dilution water blank.
8. Incubate plates at 37 ± 0.5º C for 48 hours.
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Guide for CMP
Standards
Standards Type of milk Coliform count Quality of milk
Up to 2,00,000 / ml Very good
2,00,000 – 10,00,000 / ml Good
Raw milk
10,00,000 – 50,00,000 / ml Fair
BIS
Above 50,00,000 / ml poor
Pasteurized Not more than 30,000 / ml
Milk
Interpretation
Count the colonies and compute the bacterial colony count per ml.
Frequency
Every day
Critical Factor
1. All glassware, media, dilution blanks should be sterilized.
2. Open the petridish only partly near a flame for pouring diluted
sample milk and agar.
3. Incubate the petridish only after proper cooling.
4. Maintain incubation temperature and period as specified.
5. Temperature of melted medium should not be more than 45º C
tolerable to the back of the palm but the medium still liquid without lumps)
while pouring in the petridishes.
6. Use only the standards media.
7. Do all the work aseptically.
8. Dispose agar with growth only after autoclaving or suitably
destroying the growth by other means.
Note: This test is carried out for raw milk and can also be used for
processed milk.
c) Bactaslydes
Detection of bacterial count by SPC method requires well-equipped
laboratory, experienced skilled manpower and also consumed longer time.
Bacterial detection through bactaslydes has been developed, which does
not requires modern laboratory, skill manpower and also consume less
time. It is also suitable to field condition, which is an added advantage
over SPC method. The following procedure should be adopted to detect
bacterial count by bactaslyde.
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Guide for CMP
Removal of slide: Cut open polybag.
Unscrew the cap and remove the slide from
the tube. Take care not to touch the media
surfaces.
Cut open polybag
Labeling
Fill in the label, entering the date, time and place of sampling. Stick the
label on the tube, making sure, it is stuck right from the bottom rim of the
tube, the ‘arrow side’ down
Sampling
Dip for 20 seconds. Use a Or if a clean container is not
clean container to hold available, the outer tube can
the test material be used to hold the test material.
i) Liquids: Plunge the slide into the test liquid ensuring the media
surfaces are absolutely immersed in the liquid. Hold the slide
vertically in the liquid for 20 – 25 seconds.
The slide should then be shaken 3 – 4 times gently to remove the excess
water. Put the slide back into the tube and close it tightly.
ii) Solld Surfaces: Hold the slide by it’s cap and cover
both the media surfaces with the material to be
tested simultaneously, i.e. hold the slide between
two layers of the test material like a sandwich, for 20
–25 seconds. Put the slide back in the tube and
close it tightly.
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Guide for CMP
Incubation
Place BACTASLYDE in an upright position and incubate
at a suitable temperature. If an incubator (for 37° C.) is
not available, keep the slide in a warm place, e.g.
wrapped in a blanket or behind electrical appliances like
refrigerators, ovens, etc.
Incubate (in warm place)
Normally bacteria take around 18 to 24 hours to produce colonies which
can be seen by the naked eye. With the BACTASLYDE this range can be
reduced to 12 – 18 hours.
But the manifestation of colonies also depends upon the bacterial species,
their growth phase, etc. Therefore, at times the growth may be slower and
may require 24 hours.
As far as yeasts & fungi are concerned the manifestation of growth seen
by the naked eye would take around 72 to 96 hours with a maximum of
120 hours.
Reading & Interpretation of Results
After incubation compare the density of the
colonies grown on the media surfaces with the
charts provided. There is no actual need for
counting the colonies.
Match with density chart
i) TOTAL BACTERIAL COUNT (TBC)
Almost all species of aerobic bacteria will grow on this BACTASLYDE.
Almost all the colonies would be red in color. If there are any colorless
colonies, they should also be accounted for. Here the size of the colonies
should not be much importance, as it is the density, which is being
measures.
In some cases, when the bacterial growth is very high, the media surface
of the slide would be uniformly red with a thick confluent growth. Such
slides should be discarded as a negative result. The sample could be
diluted and tested afresh. (Vide “Note”).
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Guide for CMP
Even one colony (spot) means 102. No colony (spot) means less than 102
ii) PSEUDOMONS
Only Pseudomonas species will grow on this BACTASLYDE. The colonies
would be white, off white, red or green. The interpretation of the results,
would depend on the type of industry and specific standards. For instance
“Bottled water” should not have any Pseudomonas, while “Cooling Water”
could possibly not face any problems if the Pseudomonas count would be
around 104 /ml.
Even one colony (spot) means 102. No colony (spot) means less than 102
iii) ESCHERICHIA COLI (E. Coli)
On this media the colonies of E. coli would be yellow in color. Since [Link]
is the indicator of faecal contamination even a 102 count is alarming, as all
standards, both National and International for this organism are “Zero”.
Even one colony (spot) means 102. No colony (spot) means less than 102.
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Guide for CMP
IV) YEASTS & FUNGI
Almost all species of yeasts &fungi will grow on this BACTASLYDE. The
growth may consist either purely of yeasts or purely of fungi or may be
mixed. Yeasts will give rise to either ball shaped & puffed up colonies or
flat & dry colonies. The yeasts colony charts are read as the bacterial
charts. For fungi it is not possible to be quantitative, as growth originates
from mycelia or spores. The chart therefore shows slight, moderate or
heavy infection. All bacteria would be inhibited and would not grow on this
medium.
Even one colony (spot) means 102. No colony (spot) means less than 102.
Storage
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Guide for CMP
BACTASLYDE is best kept at room temperature, protected from heat, light
and draught. If stored this way the slides will keep for six months from the
date of manufacture.
BACTASLYDE SHOULD NOT BE REFRIGERATED OR FROZEN. Check
the media surfaces for any kind of contamination and/or shrinkage of
media due to dehydration. Such slides are to be discarded.
Disposal of Used Slides
Used BACTASLYDE should be handled carefully, as it contains live
microorganisms. These slides can be best disposal of either by
incinerating, or by immersing the whole slide, container and all, in a
disinfecting solution overnight or by autoclaving them after losening the
cap. An autoclave is not essential, a pressure cooker will suffice.
Note:
If the sample requires dilution, there is a provision in BACTASLYDE to
dilute the sample 10 times. There is an arrow marked on the bottom of the
label. After sticking the label, (making sure that it is stuck right from the
bottom rim of the tube), fill the original sample upto the arrow marked on
the bottom of the label. Add Sterile water (or water boiled for 20 minutes &
subsequently cooled) to the top of the label. Insert the slide and tightly
screw the cap on. Mix by inverting the tube a couple of times. Hold the
slide in for 20 seconds, before discarding the fluid. Follow the earlier
procedure for the rest of the operation.
D) Methylene Blue Reduction Test (MBRT)
Testing Procedure
1. Thoroughly mix the milk samples and transfer 10 ml of each sample into
separately identified sterile test tubes.
2. Add 1 ml of the bench methylene blue solution to the milk in each of the
test tubes to get a concentration of 1 part of dye to 300000 parts of milk.
Replace the cotton plugs with sterile rubber bungs (using sterile forceps).
3. Mix the dye and the milk by inverting the tubes twice.
4. Place the tubes in the water bath, maintained at 37± 0.5ºC.
5. The tubes shall be observed and inverted initially at half an hour and
hourly intervals thereafter. If the milk begins to decoloyrise, the tubes shall
not be inverted. Tubes shall be incubated till milk is completely
decolourised or decolourised up to 5 mm from the surface.
6. if any sample is decolourised on incubation for 30 minutes, record the
reduction time as MBRT – 30 minutes.
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Guide for CMP
7. Record such readings as reduction times in whole hours. For example, if
the colour disappears between 0.5 and 1.5 hour readings, record as
MBRT – 1 hour, similarly, if between 1.5 and 2.5 hours as MBRT – 2
hours and so on.
Standards
BIS : Raw milk
S. No. MBR Time (hour) Quality of milk
1 5 hours & above Very good
2 3 – 5 hours Good
3 1 – 2 hours Fair
4 ½ hour & below Poor
BIS: Pasteurized milk shall not decolourize in 4 hours.
Interpretation
Match the reduction time with standards.
Frequency
1. For pasteurized milk the sample should be drawn from storage
tank after standardization.
2. For packed milk every hour.
3. Before despatch of milk through tanker.
Critical Factor
1. The methylene blue solution should be stored in dark.
2. The pipette should not come in contact with the side of the tube.
3. Five seconds should be allowed for the pipette to drain.
4. The water level in the water bath should be higher than that of the milk in
the tubes, and the tubes should not be exposed to direct sunlight.
Note: This test is carried out for raw milk and processed milk.
e) Coliform Count (Presumptive Test with Liquid Media)
Testing Procedure
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Guide for CMP
1. Inoculate a series of Mac-Conkey’s broth tubes each containing an
inverted Durham’s tube with appropriate volumes (such as 10 ml, 1 ml,
etc.) of the appropriately diluted sample in triplicate.
2. Incubate tubes at 37.0 ± 0.5º C for 48 hours.
Standards
BIS : Raw milk: Absence of coliform in 1:100 dilution of raw milk is
considered satisfactory. Pasteurized milk: Coliform should be absent in
first dilution (1:10).
Interpretation
Acid and gas formation within 48 hours in at least two tubes out of three
constitute a positive presumptive test.
Frequency
Every day
Critical Factor
1. Sterile tubes should be used.
2. Incubation temperature should be maintained at 37º C.
3. Standard media should be used.
4. Do all the work aseptically.
5. Dispose broth with growth only after autoclaving or suitably destroying the
growth by other means.
Note: This test is carried out for raw milk and can also be used for
processed milk.
Coliform Count (Presumptive Test with Solid Media)
Testing Procedure
1. Introduce 1 ml of the sample or decimal volumes thereof into sterile plates.
2. Add to each plate 10 – 15 ml of violet red bile agar, previously melted and
cooled at 42 – 44º C.
3. Mix the contents thoroughly by tilting and rotating the dish.
4. After the mixture has solidified, distribute additional 3 – 4 ml of the
medium completely over the surface of the solidified medium for inhibiting
surface colony formation. Allow to solidify.
5. Invert and incubate plates at 37.0 ± 0.5º C for 18 – 24 hours.
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Guide for CMP
Standards
BIS: Raw milk: Absence of coliform in 1:100 dilution of raw milk is
considered satisfactory. Pasteurized milk: Coliform should be absent in
first dilution (1:10).
Interpretation
1. Presence of dark pink / red colonies measuring at least 0.5 mm in
diameter constitutes a positive presumptive test.
2. Count such colonies only and record the result as the number of coliform
colonies per ml.
Frequency
Every day
Critical Factor
1. All glassware, media, dilution water should be sterilized.
2. Open the petridish only partly near a flame for pouring diluted sample and
agar.
3. Incubate the petridish only after proper cooling.
4. Maintain incubation temperature and period as specified.
5. Temperature of melted medium should not be more than 45 ºC (tolerable
to the back of the palm but the medium still liquid without lumps) while
pouring in the petri dish.
6. Use only the standard media.
7. In case of milk containinng added sugar, the results of the presumptive
coliform test should be interpreted with caution because of the possible
risk of false positives.
8. Do all the work aseptically.
9. Dispose agar with growth only after autoclaving or suitably destroying the
growth by other means.
Note: This test is carried out for raw milk and can also be used for
processed milk.
Coliform Count (Confirmatory Test )
Testing Procedure
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Guide for CMP
1. Pick from the colonies developed on violet red bile agar in the presumptive
test and transfer to violet red bile broth in tubes having inverted Durham’s
tubes.
2. Incubate at 37 ± 0.5º C for 24 hours.
Interpretation
Production of acid and gas after incubation indicate the presence of
coliform in the sample.
Frequency
Critical Factor
1. Do all the work aseptically.
2. Dispose broth with growth only after autoclaving or suitably destroying the
growth by other means.
Note: It is not advisable to perform enumeration of pathogenic bacteria
like staphylococcus, salmonella, shigella etc. in the dairy premises.
Arrangements should be made to get the samples analysed in a reliable
laboratory to ensure compliance with specifications, if any.
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