Laboratory Manual Quality Control of Milk: Quality Control of Milk

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Laboratory Manual Quality Control of Milk: Quality Control of Milk
Book · August 2015
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MONITORING OF MARKET MILK FOR
NORMAL COMPONENTS, CHEMICAL
ADULTERANTS AND HYGIENIC STATUS
CONTENTS
1. Introduction
2. Monitoring components of normal milk and its quality control
1 Milk sampling
2 Milk sampling microbial testing
3 Preservation of milk sample
4 Labeling and record keeping
5 Milkoscan
6 Quality control tests
7 Organoleptic tests
8 Clot on boiling (C.O.B.) test
9 Alcohol test
10 Alcohol Alizarin test
11 Acidity test
12 The Gerber butterfat test
13 The lactometer test
14 Freezing point determination test
15 Inhibitor test
16 Tests for pasteurized milk
3. Monitoring of chemical adulterants in market milk
a. Cane sugar
b. Urea
c. Formalin

2
d. Hydrogen peroxide
e. Neutralizers/carbonates
f. Starch
g. Detergents/soaps
h. Sodium chloride
i. Hypochlorite and Chloramines
j. Quaternary ammonium compound
4. Monitoring of hygienic status of market milk
a. Resazurin reduction test
b. Methylene reduction test
c. Coliform count
d. Standard plate count
e. Somatic cell count

3
1-INTRODUCTION
Milk is natural balanced diet and is extensively produced in Pakistan. Currently, there are
more than 67 million cattle and buffaloes, 89 million sheep and goats and 0.2 million
camels. Pakistan is blessed with high yielder genetic dairy animals such as Nilli-Ravi
buffaloes, Sahiwal Cow, Kajli Sheep and Beetle Goat. Without application of scientific
tools, dairy farmers are producing more than 33 million tons of milk.
Milk is produced through out the year. However, milk production is extensively reduced
during summer months due to heat stress, scarcity of fodder etc. Milk is transported from
point of production to cities mainly through middle man called dodhies. Such milk is
watered to increase volume. To maintain its composition, starch, flour, urea, cane sugar,
vegetable oil, etc., are added as chemical adulterants. Milk is perishable item so during
summer months, it is likely to be spoiled during transportation. The middle men therefore
add chemical preservatives such as penicillin, strepto-penicillin, formaldehyde, hydrogen
peroxide, sodium bi-carbonate, etc. The adulterants/ preservatives are health hazards for
end consumer particularly vulnerable group such as infants.
Government of Punjab established a Quality Control Laboratory (WTO-QCL) during
2004-2008, in University of Veterinary and Animal Sciences (UVAS), Lahore. All the
required equipments for testing the quality of milk are installed in milk testing laboratory.
Quality testing techniques such as Charm II, spectrophotometer, HPLC, HPTLC, etc.,

4
have been standardized. The laboratory was accredited through Pakistan National
Accreditation Council (PNAC) ISO-1725 during 2008. Keeping in view the health hazard
of adulterated milk, Toxicology Section, WTO-QCL, UVAS, developed a field kit
namely Milk Adulterant Tests (MAT) through a research project (worth 0.7 million)
funded by Higher Education Commission (HEC), Islamabad. Using this kit, inspectors in
food testing laboratories can screen adulterants in milk rapidly.
Department of Microbiology, UVAS, Lahore trains inspectors, researchers,
postgraduates, faculty members, etc., through organized workshops almost every year in
monitoring components. chemical adulterants and hygienic status of market milk.
2- MONITORING COMPONENTS OF NORMAL MILK AND ITS
QUALITY CONTROL
2.1. Milk sampling
Appropriate sampling is the primary and basic requirement for monitoring its normal
components. The milk in the containers must be thoroughly mixed to have uniform
distribution of its milk before a septic collection of the milk sample. There should be
random sampling of the packed milk from shops monitoring its components.
.
2.2 Milk sampling milk for hygienic monitoring
Sampling milk for bacteriological tests require a lot of care. Containers for sampling are
sterilized in an autoclave for 15min at 120° C to avoid environmental contamination. In
the field condition, disinfection of the containers can be done with 70% Alcohol swab
and washing them in boiled water for 1 minute.
2.3 Preservation of milk samples
Milk samples for chemical tests
Milk samples for butterfat testing may be preserved with chemicals like Potassium
dichromate (0.5 ml of 14% solution in 250 ml sample bottle). Refrigerated milk samples
are warmed up to 20ºC in water bath at 40 ºC, homogenized and a sample is then
collected monitoring its fat. Sodium azide can be used at rate of 0.1 percent.

5
Milk samples can’t be kept waiting at room temperature in the laboratory. If it can not be
processed immediately, it can be stored at freezing temperature till can be processed for
analysis. In field condition, ice box can be used to store samples.
2.4. Labeling and records keeping
Milk samples are labeled with name of the farmer, address and date such 1-23112013.
This date is also used as a code for the sample. This information is also recorded on
computerized data sheet and dead record register. The arrangements may be made so as
the farmer may be able to see the processing of his milk sample if interested but through
window. The record may be provided to the farmer if he is interested to have.

6
3. COMMON TESTS FOR NORMAL MILK
3.1. Milkoscan
Introduction
Milk of every mammalian species is a natural balanced diet for their young one. It’s
components are determined manually by colorimeteric methods but now-a-days,
Lactoscan or milkoscan machine quick analyses the milk and determine its fat (FAT),
non-fat solids (SNF), proteins, lactose and water content percentages, temperature (o
С),
pH, freezing point, salts, total solids, conductivity as well as density. The samples can be
collected as fresh milk, tank milk, processed or packed milk.
S# Parameter Measuring range Accuracy(
From To
1 Fat 0.01% 25% 0.06%
2 SNF 3% 40% 0.15%
3 Density** one kg/m3
160kg/m3
0.3kg/m3
4 Proteins 2% 15% 0.15%
5 Lactose 0.01% 20% 0.20%
6 Water content 0% 70% 3.0%
7 Temperature of
milk
15o
C 40o
C One o
C
8 salts 0.4% 4% 0.05%
9 pH 0 14 0.05
10 Conductivity 3 14ms/cm 0.05
11 Total solids 0% 50% 0.17%
Note: Density of the milk determined by adding 1000 in displayed value. For example
1000+32.30=1032.30
Materials
1. Milkoscan with pH probe and sample suction tube.
2. Milk sample
3. Washing bottle with distilled water
4. Beaker 50 ml capacity
5. Glass 250 ml capacity
Procedure
1. Bring the temperature of the milk to ambient temperature

7
2. Add 20 ml milk in a beaker and dip pH probe in it
3. Add 20 ml milk in the glass container and feed the sample through suction pipe
4. Switch on the machine
5. Within 5 minutes, it will suck the sample and display the result on the printing
role.
Results:
The results sheet can be seen as given below
POSSIBLE MALFUNCTIONS AND ERROR MESSAGES,
TROUBLESHOOTING
In the table below are described the possible malfunctions during the milk analyser’s
exploitation and ways for their repair/remedy. If the problem persists after all
recommended measures are taken, please, connect the nearest service center.
Error
message
Possible problem
/cause
Repair/remedy
2.MA
overheated
accompanied
by a continuous
sound signal
Overheated milk
analyzer
Immediately switch off the analyzer.
Pay attention the analyzer to be situated
away from direct sunlight or heating devices.
Wait 5-10 minutes the device to cool down or
to be normalized the ambient temperature
and switch it on again.
3.Empty
Camera
Insufficient
quantity of the
milk
sample sucked
in
the system or air
in
the sample
The analyzer is ready to measure the next
sample. In order to avoid the future
appearance of the same error message,
please, check the following:
- The sample is prepared according the
instructions and there aren’t air bubbles
in it.
- There is a real suction of the sample
after starting measurement, i.e. it is
obvious that the level of the milk sample
in the sample holder decreases. In other
case – there is damage in the suction
system.
- Avoid the end of the suction pipe to be
above the surface of the liquid (not

8
Table 1. Chemical Composition of Camel, Goat,Cow and Sheep Milk.
Component Camel Goat Sheep Cow
Fat % 3.24 3.21 7.52 3.8
Protein (Nx6.38)% 3.35 2.87 5.85 3.3
Lactose % 4.52 4.10 4.86 4.8
Ash % 0.80 0.79 0.92 0.71
Total Solids % 11.91 11.05 18.63 12.8
Energy (kcal/liter) 670 622 1080 785
dipped enough).
- Avoid curdling of the milk sample. Clean
immediately if there is a sample curdled
in the system.
- In mode Measurement, after starting the
measurement, remove the sample
holder and see if there is no milk poured back
in the sample holder.
-The sample is prepared according the
instructions and its temperature does not exceed
the maximum permissible sample’s
temperature.
-Complete the procedure for checking the
analyzer in case of error message Empty
Camera.

9
Species wise milk Composition
SPECIES
FAT
%
PROTEIN
%
LACTOSE
%
ASH
%
TOTAL SOLIDS
%
Antelope 1.3 6.9 4 1.3 25.2
Ass (donkey) 1.2 1.7 6.9 0.45 10.2
Bear, polar 31 10.2 0.5 1.2 42.9
Bison 1.7 4.8 5.7 0.96 13.2
Buffalo, Philippine 10.4 5.9 4.3 0.8 21.5
Camel 4.9 3.7 5.1 0.7 14.4
Cat 10.9 11.1 3.4 --- 25.4
Cow:
Ayrshire
Brown Swiss
Guernsey
Holstein
Jersey
Zebu
4.1
4.0
5.0
3.5
5.5
4.9
3.6
3.6
3.8
3.1
3.9
3.9
4.7
5.0
4.9
4.9
4.9
5.1
0.7
0.7
0.7
0.7
0.7
0.8
13.1
13.3
14.4
12.2
15.0
14.7
Deer 19.7 10.4 2.6 1.4 34.1
Dog 8.3 9.5 3.7 1.2 20.7
Dolphin 14.1 10.4 5.9 --- 30.4
Elephant 15.1 4.9 3.4 0.76 26.9
Goat 3.5 3.1 4.6 0.79 12
Guinea Pig 3.9 8.1 3 0.82 15.8
Horse 1.6 2.7 6.1 0.51 11
Human 4.5 1.1 6.8 0.2 12.6
Kangaroo 2.1 6.2 Trace 1.2 9.5
Mink 8 7 6.9 0.7 22.6
Monkey 3.9 2.1 5.9 2.6 14.5
Opossum 6.1 9.2 3.2 1.6 24.5
Pig 8.2 5.8 4.8 0.63 19.9
Rabbit 12.2 10.4 1.8 2 26.4
Rat 14.8 11.3 2.9 1.5 31.7
Reindeer 22.5 10.3 2.5 1.4 36.7
Seal, gray 53.2 11.2 2.6 0.7 67.7
Sheep 5.3 5.5 4.6 0.9 16.3
Whale 34.8 13.6 1.8 1.6 51.2

10
Questions
1. Can milkoscan determine the milk adulterants?
2. What are factors affecting amount of fat in milk?
3. Can you differentiate the natural cattle milk and fabricated milk using milkoscan?
4. Name different companies preparing milkoscan?
5. Can milkoscan check the vegetable fat in milk?
6. What is composition of milk of a healthy buffalo?
7. How can you differentiate buffalo and cow milk?

11
3.2 ORGANOLEPTIC TESTS
Introduction
Organo-leptic test rapidly detects poor quality milk at the milk collection point. Milk
grader is required to have good sense of sight, smell and taste.
Materials
There is no need of any equipment and testing reagent.
Procedure
 Open can of the milk.
 Smell the milk immediately.
 Observe the appearance of the milk.
 In case the grader is unable to make a clear decision for the quality of milk, then
taste the milk.
 Look at the lid and the container to check its cleanliness.
Comments
Abnormal smell and taste may be caused by:
1) Atmospheric smell (methane, or cowy odor).
2) Physiological smell in animal body that might be due to hormonal
imbalance, cows in late lactation or spontaneous rancidity of milk.
3) Bacterial products
4) Chemical or bacterial colors in milk.
5) Advanced acidification (pH < 6.4).
Precaution
Do not swallow but spit the milk sample into the specified container. Milk supplier may
add some desensitizing chemical for grader.

12
3.3. CLOT ON BOILING TEST
Introduction
Clot on boiling (C.O.B) is quick and simple. It is one of the old tests for too acid milk
(pH<5.8) or abnormal milk (e.g. colostral or mastitis milk). If a milk sample fails in the
test, the milk might be containing acids or acid producing bacteria or abnormally high
level of proteins such as in colostrums. Such milk turned into curd or clot on heat
treatment during milk processing so such milk must be rejected.
Materials
1. Test tube one
2. Test tube holder
3. Milk sample
4. Bunsen burner or spirit lamp
5. Matches
Procedure
1- Take 5 ml milk sample in a test tube
2- Hold the tube with test tube holder
3- Boil the milk sample in the test tube on the burner for 5 minutes
Result
Clotting or coagulation of the sample in the tube indicate the failure of the sample to
C.O.B test.
Comments: This test may fail to detect the quality if :
a. Freshly drawn milk is contaminated with bacteria.
b. Acidity of the sample is below 0.20-0.26% lactic acid.
c. Milk is contaminated non acid producing bacteria.

13
3.4. ALCOHOL COAGULATION TEST
Introduction
Alcohol coagulation test is simple and rapid. It is based on poor stability of milk proteins
in presence of alcohol when the level of lactic acid or any other acid is high or amount of
rennet is increased. Increased level of milk albumin such as in colostrums or increased
salt concentration as in mastitic milk may fail to qualify the test.
Materials
 Test tube one
 Milk sample
 Reagent in glass stoppered bottle (86% ethanol solution)
Preparation of alcohol solution
Ethanol solution is prepared by mixing 68 cc of 96%(absolute) alcohol and 28 cc of
distilled water.
Procedure
Take 2 ml milk sample in a test tube
Add 2 ml of 86% ethanol solution
Incubate the mixture at 25-37C for 5 minutes.
Result
Coagulation, clotting or precipitation of the milk sample even up to small lumps in the
test tube indicate failure of the sample to qualify the test.,
Comments
Clotting can first be seen if milk contains more than 0.21-0.23% lactic acid.

14
3.5. ALCOHOL-ALIZARIN TEST
Introduction
This test is just like alcohol test. Alizarin is a pH indicator. Alcohol–Alizarin (A-A test)
test is more informative. It will indicate the coagulation as well as intensity of acidity.
Materials
1. Alcohol–Alizarin solution
2. Test tube one
3. Milk sample
Preparation of Alcohol–Alizarin solution
Alizarin powder 0.2 grams
Ethanol solution (61%) 500 ml
Alcohol /ethanol solution is prepared by mixing 61 ml of 96% (absolute) alcohol and 39
ml distilled water.
Procedure
 Take 2 ml milk sample in a test tube
 Add 2 ml of Alcohol–Alizarin solution
 Incubate the mixture at 25 0
C for 5 minutes.
Result
Coagulation, clotting or precipitation of the milk along with its color as given below:
Parameter Normal milk Slightly acid Milk Acid milk Alkaline Milk
PH 6.6 – 6.7 6.4 – 6.6 6.3 or lower 6.8 or higher
Color Red brown Yellowish-brown Yellowish Lilac
Condition of
milk
No coagulation
no lumps
No coagulation Coagulation * No coagulation
**
Note:* Sour milk will appear yellowish with small lumps or completely coagulation while
alkaline milk ** will look like lilac. It may be mastitis milk and may show flakes or clots.

15
3.6 ACIDITY TEST
Introduction
Bacteria that normally grow in raw milk produce ferment lactose and produce lactic acid.
In the acidity test, lactic acid is neutralized with 0.1N solution of sodium hydroxide. The
amount of sodium hydroxide is measured and from this amount, percentage of lactic acid
is calculated.
Materials
 Small conical flask (100 ml capacity) One
 Graduated pipette (10 ml) One
 Graduated pipette (1 ml) One
 Burette having 0.1 ml graduations One
 Glass rod for stirring the milk in the flask One
 Phenophtalein indicator solution ( 0.5% in 50% Alcohol)
 N Sodium hydroxide solution.
Procedure
 Take 9 ml milk in the flask.
 Phenopthalein (one ml) is transferred to the milk in the flask
 Add sodium hydroxide (0.1 N) under continuous mixing from the burret until
development of faint pink color.
 Amount of sodium hydroxide solution in ml is noted and divided by 10 expresses the
percentage of lactic acid.
Comments
Fresh milk contains "natural acidity" that is due to the natural ability to resist pH changes
.The natural acidity of milk is 0.16 - 0.18%. The acidity higher than this is due to
fermentation of lactose by lactic acid producing bacteria.

16
3.7 BUTTERFAT TEST
Introduction
Fat content of milk /cream of dairy animals is one of the important factors in determining
the price of the milk/cream. Butter/fat % of milk or amount of milk produced is
considered while calculating the ration of the lactating animal.
Testing of butterfat is important to know in order to make correct adjustments of the
butterfat percentage in standardized milk and milk products.
Materials
 Water bath
 Sulphuric acid, (1.82 g/cc)
 Amyl alcohol
 Rubber stoppers for butyrometers
 Butyrometers, 0-6% or 0-8% BF
 20 ml glass pipettes for milk
 10 ml pipettes for Gerber Acid
 1 ml pipettes for Amyl alcohol
 Stands for butyrometers
Procedure
 Fresh milk at 20-25 ºC should be mixed well. Samples kept cool for some days
should be warmed to 40ºC, mixed gently and cooled to 20ºC before the testing.
 Add 10 ml sulphuric acid to the butyrometer followed by 11 ml of well mixed milk.
Avoid wetting neck of the butyrometer.
 Add one ml of Amyl alcohol, insert stopper and shake the butyrometer carefully until
the curd dissolves and no white particles are seen. Place the butyrometer in the water
bath at 65ºC and keep it there until a set is ready for centrifugation.
 The butyrometer is placed in the centrifuge with the stem (scale) pointing towards its
center and spin for 5 min. at ll00 rpm.
 Remove the butyrometers from the centrifuge.

17
 Put the butyrometers in a water bath maintained at 65ºC for 3 min. before recording
results.
Precautions
1. Butyrometers are all the time held with the neck pointing upward.
2. The fat column is required to be read from the lowest point of the meniscus of the
interface of the acid-fat to the o-mark of the scale and read the percentage of the
butterfat.
3. The butyrometers are required to be emptied into a special container for the very
corrosive liquid of acid-milk
4. The butyrometers should be washed in warm water and dried before the next use.
Appearance of the test
Color of the fat column should be straw yellow.
Ends of the fat column should be clearly 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.
Problems in test results
 Too lightly colored or curdy fat column can be due to:
1. Temperature of milk or acid or both is too low.
2. Acid too weak.
3. Insufficient acid.
4. Milk and acid not mixed thoroughly.
 Darkened fat column containing black specks at the base is due to:
o Temperature of milk-acid mixture too high.
o Acid too strong.
o Milk and acid mixed too slowly.
o Too much acid used.
o Acid dropped through the milk.

18
3.8 MEASUREMENT OF MILK DENSITY
Introduction
Milk has specific gravity or density. It can be determined lactometer. Middle men add
water in milk to increase its volume so as to get high price of the same pure milk. It is
way to deceive the buyers. Every body know that middle men add water in milk. Buyers
therefore assure the addition of water in milk.
The lactometer test is designed to determine the change in density of such watered milk.
If it is tested along with butterfat test, it enables us to calculate the milk total solids (%
TS ) and solids not fat (SNF).
Materials
1-Measuring cylinder
2-Milk sample 500 ml
Procedure
 Mix the milk sample gently and pour it into a measuring cylinder (500 ml capacity).
 Drop the lactometer slowly into the milk sample.
 Read and record the last lactometer reading (ºL) just above the surface of the milk.
 If the temperature of the milk is different from the calibration temperature
Note: Calibration temperature of the lactometer may be 200
C. Calculate the correction
factor for the temperature. For each one ºC above the calibration temperature, add 0.2ºL;
for each ºC below calibration temperature subtract 0.2 ºL from the recorded lactometer
reading. EXAMPLE: Calibration temperature of lactometer 20ºC.
Sample Milk temperature Lactometer reading Correction True reading
No.1 17 ºC 30.6 ºL - 0.6 ºL 30.0 ºL
No.2 20 ºC 30.0 ºL Nil 30.0 ºL
No.3 23 ºC 29.4 ºL + 0.6 ºL 30.0 ºL
In normal milk SNF should not be below 8.5% according to Kenya Standards

19
3.9 FREEZING POINT DETERMINATION
The freezing point of milk is always a constant value as compared to all its other
measurable properties.. A small adulteration of milk with water will cause a detectable
elevation of its freezing point from its normal values of -0.54ºC.
This test is accurate and sensitive to added water in milk. It is therefore used to monitor
the adulteration of milk with water.

20
3.10 INHIBITOR TEST
Introduction
Milk is some times adulterated with antibiotics or disinfectants during its transportation
over long distance to cities. These antibacterials agents inhibit growth of lactic acid
producing bacteria. Moreover, it is health hazards for end consumers.
Principle
The suspected milk sample is subjected to a fermentation test with starter culture and the
acidity checked after three hours. The values of the titratable acidity obtained are
compared with titratable acidity of a similarly treated sample which is free from any
inhibitory substances.
Materials
 Test tubes
 Starter culture
 One ml pipette
 Water bath
 Material for determination of titratable acidity (Fig.9)
Procedure
 Three test tubes are filled with l0 ml of the sample to be tested and three test tubes
filled with normal milk.
 All tubes are heated to 90 0C by boiling them in water bath for 5 minutes.
 After cooling to 30 ºC temperature of the starter culture, add one ml of starter culture
to each test tube, mixed and incubated for 3 hours.
 After each hour, one test tube from each of the “test” and the “control” tubes is
removed and processed for estimation of its acid content.
Results
If acid production in suspected sample is the same as the normal sample, then the suspect
sample does not contain any inhibitory substances;
If acid production as suspect sample is less than in the normal milk sample, then, the
suspect sample contains any antibacterial or other inhibitory substances.

21
3.11. PHOSPHATASE TEST
Introduction
Normal milk is sterile secretion and is free from pathogenic organisms. It may contain
pathogenic bacteria if animal is suffering from mastitis. Some of the mastitic causing
bacteria are zoonotic in nature (Mycobacterium tuberculosis, Brucella abortus, Quexilla
burnetti etc) and may cause infection in milk consumers. Milk also contains phosphate
that is normal enzyme present in somatic cells of milk. The enzyme is heat labile and is
inactivated at pasteurization temperature (63ºC for 30 min or 72ºC for 15 seconds). The
above mentioned pathogenic bacteria are heat labile and are destroyed at the at
pasteurization temperature. Pateurized milk is therefore considered as safe for human
consumers.
Inactivation of the enzyme in the pasteurized milk is indication that milk is properly
pasteurized and all the pathogenic bacteria are destroyed. If the milk is positive for the
enzyme, it means the pasteurization process was inadequate and the milk may not be safe
for human consumption and will have a short shelf life.
Materials
 Test tubes
 Pipettes (one and 10 ml capacity)
 Volumetric flask (100 ml and 500 ml capacity)
 Water bath at 37ºC
 Buffer solution is prepared by mixing 0.75g anhydrous sodium carbonate and
l.75g Sodium bicarbonate in 500 ml distilled water
 Buffer-substrate solution is prepared by placing 0.l5 g of di-sodium para-
nitrophenylphosphate (the substrate) into a clean 100 ml measuring cylinder.
 Note: All glassware must be rinsed, cleaned, demineralized in dichromate solution
and boiled in water for 30 min. Store this buffer and substrate solution in a
refrigerator and protected against light. It should not be used after one week. Prepare
a fresh stock.

22
Procedure
 Pipette 5 ml buffer-substrate solution into a test tube, cover with suitable stopper and
warm in the water bath at 37ºC.
 Add to the test tube 1ml of the milk to be tested, stopper and mix well and place in
water bath at 37ºC.
 Prepare a blank sample from boiled milk of the same type as that undergoing the test.
 Incubate both the test samples and the blank sample at 37ºC for 2hrs. After
incubation, remove the tubes and mix them thoroughly.
 Place one sample against the blank in a Lovibond comparator" All purposes using
“A.P.T.W”. disc and rotate the disc until the color of the test sample is matched and
read the disc number.
Interpretation:
Disc Reading after 2 hrs incubation at 37ºC Remarks
0-10 Properly pasteurized
10-18 Slightly under pasteurized
18-42 Under pasteurized
> 42 Not pasteurized
Questions
1. What is phosphatase?. What are its common source? What are its substrate
and color of the end product?
2. What type of bacteria could be present in milk?
3. What is thermoduric, thermophilic, mesophilic and psychrophilic bacteria?
4. Enlist at least 10 pathogenic bacteria likely to be present in milk?

23
4 CHEMICAL ADULTERANTS IN MILK
4.1 CANE SUGAR
Introduction
Generally sugar is mixed in the milk to increase its solids not fat contents i.e. to increase
the lactometer reading of milk, which is normally below the normal value if diluted with
water. In laboratory, cane sugar (sucrose) in milk reacts with acidified resorcinol and
gives red color.
Materials
1-Test reagent
2-Test tubes
3- Pasteur pipette
4- Spirit lamp
5-Match stick
6-Milk sample
Composition of the reagent
Resorcinol 5.0 grams
Hydrochloric acid 500 ml
Dissolve 5.0 g of resorcinol in 500 ml HCl (1:1.5). One ml concentrated HCl (sp. gr.
1.18) is mixed with 1.5 ml of distilled water. The resorcinol flakes should be white in
color.
Preparation of hydrochloric acid solution
Hydrochloric acid (concentrated) 200 ml
Distilled water ` 300 ml
NOTE:
1. Hydrochloric acid solution should be made in fume hood because fumes of the
concentrated HCl are toxic for laboratory worker.
2. Add the acid in the water along sides of the container.
Procedure
 Take two test tubes and label one as “test” and other as “control”
 Transfer one ml milk sample to each of the tubes.

24
 Add 0.5 ml of the reagent in the tube marked as “test” and 0.5 ml distilled water
in the tube marked as “control”.
 Mix the contents of each tube.
 Boil contents of each test tube
Results
Appearance of deep red color indicates presence of sucrose, or a ketose sugar.
Detection of minimum level of cane sugar in milk
Tube # Dilution of adulterated milk Amount of cane sugar (%) Result
1 Unadulterated milk 0 -
2 Adulterated undiluted milk 5.0 : 5000 mg/100 ml +
3 1:10 of the adulterated milk 0.5 : 500 mg/100 ml milk +
4 1:100 of the adulterated milk 0.05 : 50 mg/100 ml milk -
Comments: Chemical test can detect the Cane sugar up to 0.5 % in raw milk. Addition of Cane
sugar below 0.5% is hardly detectable in the laboratory.
Effect of volume of milk sample on cane sugar test
Tube # Volume of adulterated milk Result
1 5 ml +
2 2.5 ml +
3 1 ml +
Comments: Volume of milk sample did not affect the laboratory testing of the cane sugar.
Effect of boiling of adulterated milk on cane sugar testing
Tube # Nature of milk Result
1 Un-boiled and un-adulterated milk -
2 Un-boiled but adulterated milk +
3 Boiled adulterated milk +
Comments: Boiling of market milk did not affect the laboratory testing of the cane sugar.
Questions
1. How will you prepare following solutions?
a. One Normal solution of HCl acid
b. One molar solution of HCl acid
c. Five percent solution of the HCl acid
2. Why the middle man add cane sugar in milk?.
3. What is minimum volume of the milk used in the test (5 ml, 2.5 ml, 1.0 ml)
4. What is effect of boiling of milk on detection of cane sugar?
5. Is it possible to develop strip test for detection of cane sugar in milk?

25
4.2. UREA
Introduction
Urea is generally added in the preparation of synthetic milk to raise the SNF value.
Turmeric paper method is used to test the urea in milk. The test is applicable to
formaldehyde treated milk and can detect urea concentration greater than 0.1% and thus
differentiate inherent urea in milk below 0.1%.
Boil 50 grams of coarsely powdered turmeric-root in 1/2 a pint of water for 30 minutes
and strain. Dip a filter paper in the liquid and dry it. It is rendered brown by alkalies and
also by boric acid and borates.
Materials
1-Test reagent
2-Test tubes
3- Pasteur pipette
4-Milk sample
p –Dimethyl amino benzaldehyde (DMAB) 8.0 grams
Hydrochloric acid 500 ml
The reagent is prepared by dissolving 1.6 gm of p –DMAB in 100 ml of 10 % HCl.
Preparation of hydrochloric acid solution
Hydrochloric acid (concentrated) 50 ml
Distilled water ` 450 ml
NOTE:
3. Hydrochloric acid solution should be made in fume hood because fumes of the
concentrated HCl are toxic for laboratory worker.
Procedure

26
 Add 1.0 ml of the reagent in the tube marked as “test” and 1.0 ml distilled water
Results
Distinct yellow color is observed in milk containing added
urea. However, the control (normal milk) may show a slight
yellow color due to presence of natural urea.
Detectable level of urea in milk
Tube # Dilution of adulterated milk Amount of urea (%) Result
5 1:1000 of the adulterated milk 0.0005:0.5 mg/100 ml milk -
Comments: Chemical test can detect the urea in raw milk up to minimum level up to 0.05 %.
Addition of urea below 0.05 is hardly detectable in the laboratory.
Effect of volume of milk sample on urea test
1 5 ml +
2 2.5 ml +
3 1 ml +
Comments: Volume of milk sample did not affect the laboratory testing of the urea.
Effect of boiling of adulterated milk on urea testing
Comments: Boiling of market milk did not affect the laboratory testing of the urea.

27
Questions
2. Why the middle man add urea in milk?.
4. What is effect of boiling of milk on detection of urea?
5. Is it possible to develop strip test for detection of urea?

28
4.3. FORMALIN
Introduction
Milk is produced in rural and suburban areas of the country and is transported twice a day
to big cities. During summer months, milk usually gets spoiled. The milk transporters add
ice blocks to keep milk cool and formalin to preserve the milk in the containers. Formalin
is highly toxic and carcinogenic therefore formalin added milk is hazardous for end
consumers.
Materials
1-Test reagent
2-Test tubes
3- Pasteur pipette
4-Milk sample
Sulphuric acid containing traces of ferric chloride 90%
It is prepared by 10 ml distilled water along the sides of the concentrated sulphuric acid
container.
NOTE:
5. Sulphuric acid solution should be made in fume hood because fumes of the concentrated
sulphuric acid are toxic for laboratory worker.
Procedure
 Take two test tubes and label one as “test” and
other as “control”
 Add 1.0 ml of the reagent in the tube marked as
“test” from the side of the test tube slowly and
1.0 ml distilled water in the tube marked as
“control”.

29
Results
Formation of purple ring at the junction indicates formaldehyde is present in milk.
Detectable level of formalin in milk
Tube # Dilution of adulterated milk Amount of formalin (%) Result
2 1:10 of the adulterated milk 0.1 : 100 ul/100 ml milk +
4 1:1000 of the adulterated milk 0.001 : 1.0 ul/100 ml milk +
5 1:10000 of the adulterated milk 0.0001 : 0.1 ul/100 ml milk +
6 1:1000000 of the adulterated milk 0.00001 : 0.01 ul/100 ml milk -
Comments:
Chemical test can detect minimum 0.0001 % formalin in raw milk. Addition of formalin below
this level is hardly detectable in the laboratory.
Note: It is noteworthy that formalin more than 1% in milk is undetectable by this test.
Effect of volume of milk sample on formalin test
1 5 ml +
2 2.5 ml +
3 1 ml +
Comments: Volume of milk sample did not affect the laboratory testing of the formalin.
Effect of boiling of adulterated milk on formalin test
Comments: Boiling of market milk did not affect the laboratory testing of formalin.
Questions
2. Why the middle man add formalin in milk?.
4. What is effect of boiling of milk on detection of formalin?
5. Is it possible to develop strip test for detection of formalin?

30
4.4. HYDROGEN PEROXIDE
Introduction
During long transportation from villages to big cities in summer months, milk usually
gets spoiled. The middle men add ice block to keep milk cool and hydrogen peroxide to
preserve the milk in containers. The hydrogen peroxide is highly toxic, therefore
hydrogen peroxide added milk is health hazard for end consumers.
Materials
1-Test reagent
2-Test tubes
3- Pasteur pipette
4-Milk sample
Composition of the test reagent
Validium pentaoxide (V2 O5) =5 grams
Sulphuric acid (6%:H2SO4) = 500 ml
Preparation of 6 % sulphuric acid (H2SO4)
Sulphuric acid (H2SO4) =030 ml
Distilled water =470 ml
NOTE:
This solution should be made in fume hood because fumes of the concentrated sulphuric acid
are toxic for laboratory worker.
Add the acid in the water along sides of the container.
Procedure
 Take two test tubes and label one as “test” and other as
“control”
 Add 0.5 ml of the reagent in the tube marked as “test”
and 0.5 ml distilled water in the tube marked as
“control”.

31
Results
The development of pink or red color indicates presence of H2O2 in the sample while
color of the control sample will remain unchanged.
Tube # Dilution of adulterated milk Amount of sodium bi-
carbonate (%)
Result
2 Adulterated undiluted milk 1.0 : 1000 ul/100 ml +
4 1:100 of the adulterated milk 0.01 : 10 ul/100 ml milk -
5 1:1000 of the adulterated milk 0.001: 1 ul/100 ml milk -
Comments:Chemical test can detect the H2O2 up to 0.1 % in raw milk. Addition of H2O2 below
0.1% is hardly detectable in the laboratory.
1 5 ml +
2 2.5 ml +
3 1 ml +
Comments: Volume of milk sample did not affect the laboratory testing of the hydrogen
peroxide.
1 Un-heated and un-adulterated milk -
2 Un-heated but adulterated milk +
3 Heated adulterated milk +
Comments: Boiling of milk did not affect this test in detection of H2O2
Questions
How will you prepare following solutions?
a. One Normal solution of sulphuric acid
b. One molar solution of sulphuric acid
c. Five percent solution of the sulphuric acid
Why the middle man add hydrogen peroxide in milk?.
What is minimum volume of the milk used in the test (5 ml, one ml, 0.1 ml, 0.001
ml)
What is effect of boiling of milk on detection of hydrogen peroxide?
Is it possible to develop strip test for detection of hydrogen peroxide in milk?

32
4.5. SODIUM BI-CARBONATES
Introduction
Sodium bi-carbonates or other neutralizers such as sodium hydroxide, sodium carbonate
etc, are used to avoid curdling of milk during transportation. Addition of neutralizers is
generally prohibited.
Materials
1-Test reagent
2-Test tubes
3- Pasteur pipette
4-Milk sample
Preparation of the reagent
Resolic acid 5 grams
Ethanol 500 ml
NOTE:
 This solution should be made in fume hood because fumes of the alcohol are health
hazard for laboratory worker.
Procedure
 Add one ml of the reagent in the tube marked as “test” and one ml distilled water
Results
Development of pink red color indicate that the milk is adulterated with sodium
carbonate / sodium bicarbonate
MINIMUM DETECTABLE LEVEL OF SODIUM BI-CARBINATE IN MILK
Tube # Dilution of adulterated milk Amount of sodium bi-carbonate (%) Result
5 1:1000 of the adulterated milk 0.001: 1 mg/100 ml milk -
Comments:

33
Chemical test can detect the sodium bi-carbonate up-to 0.1 % in raw milk. Addition of sodium
bicarbonate below 0.1% is hardly detectable in the laboratory.
EFFECT OF VOLUME OF MILK SAMPLE ON SODIUM BI-CARBONATE
TEST
1 5 ml +
2 2.5 ml +
3 1 ml +
Comments
Volume of milk sample did not affect the laboratory testing of the sodium bi-carbonate.
EFFECT OF BOILING OF ADULTERATED MILK ON SODIUM BI-
CARBONATE TESTING
Comments
Boiling of market milk did not affect the laboratory testing of the sodium bi-carbonate.
Questions
One, 5, 10 % solution of rosalic acid in acohal
2. Why the middle man add sodium bi-carbonate in milk?.
3. What is minimum volume of the milk used in the test (5 ml, one ml, 0.1 ml, 0.001
ml)
4. What is effect of boiling of milk on detection of sodium bi-carbonate?
5. Is it possible to develop strip test for detection of sodium bi-carbonate in milk?

34
4.6. STARCH
Introduction
Starch of cereal flours such as flour of sangaraha (water caltrop) is commonly added to
increase the density of milk (Lactometer reading) that reduces if it is adulterated with
water. Uncooked starch may be health hazard for consumers but it is deceiving/ cheating
to the end consumers. The presence of starch or cereal flours is detected in the laboratory.
Materials
1-Test reagent
2-Test tubes
3- Pasteur pipette
4-Milk sample
Preparation of reagent
Iodine crystal 6.5 grams
Pot. Iodide 7.5 grams
Distilled water 500 ml
Procedure
 Add one ml of the reagent in the tube marked as “test” and one ml distilled water
Results
Presence of starch is indicated by the appearance of a blue color which disappears when
the sample is boiled and re appears on cooling.

35
DETECTION OF MINIMUM LEVEL OF STARCH IN MILK
Tube # Dilution of adulterated milk Amount of starch (%) Result
Comments: Chemical test can detect the starch up to 0.01 % in raw milk. Addition of starch
below 0.01% is hardly detectable in the laboratory.
EFFECT OF VOLUME OF MILK SAMPLE ON STARCH DETECTION
1 5 ml +
2 2.5 ml +
3 1 ml +
Comments: Volume of milk sample did not affect the laboratory testing of the starch.
EFFECT OF BOILING OF ADULTERATED MILK ON STARCH
Comments: Boiling of market milk did not affect the laboratory testing of the starch.
Questions
One, 5, 10 % solution of iodine crystal in alcohol
2. Why the middle man add starch in milk?.
3. What is minimum volume of the milk used in the test (5 ml, one ml, 0.1 ml)
4. What is effect of boiling of milk on detection of starch?
5. Is it possible to develop strip test for detection of starch in milk?

36
4.7. SOAPS/ DETERGENTS
Introduction
During transportation of milk from rural to urban areas, milkers remove fat and add water
in the skimmed milk. This result the milk watery and lack of foams so the milkers add
detergent to improve the milky foam formation.
Materials
1-Test reagent
2-Test tubes
3- Pasteur pipette
4-Milk sample
Preparation of the reagent
Phenolphthalein 5 grams
Ethanol 500 ml
Procedure
 Add 0.5 ml of the reagent in the tube marked as “test” and one ml distilled water
Results
Development of pink or red color appears

37
DETECTION OF MINIMUM LEVEL OF SODIUM CHLORIDE IN MILK
Comments: Chemical test can detect the detergent up to 0.25 % in raw milk. Addition of
detergent below 0.25% is hardly detectable in the laboratory.
EFFECT OF VOLUME OF MILK SAMPLE ON SODIUM BI-CARBONATE TEST
1 5 ml +
2 2.5 ml +
3 1 ml +
Comments: Volume of milk sample did affect the laboratory testing of the detergent.
EFFECT OF BOILING OF ADULTERATED MILK ON DETERGENT
Comments: Boiling of market milk did not affect the laboratory testing of the detergent.
Questions
One, 5, 10 % solution of phenolphthalein in alcohol
2. Why the middle man add soap/detergent in milk?.
4. What is effect of boiling of milk on detection of soap?
5. Is it possible to develop strip test for detection of soap in milk?

38
4.8 SORBITOL
Introduction
Addition of water in milk reduces its lactometer reading. Addition of sorbitol will
increase the lactometer reading. Lactometer fails to detect addition of water in the milk
Sorbitol is a major problem in production of powder milk. During evaporation through
milk nozzles, water evaporates while sorbitol does not so it make large lumps of milk. In
this way it deteriorates the quality of powder milk.
Materials
 Test tubes
 Pasteur pipette
 Ferric sulphate solution (One percent aquous solution)
 Sodium hydroxide (ten percent aquous solution)
 Milk sample
Procedure
 Add one ml solution of ferric sulphates (1% aquous solution) in the tube marked
as “test” and one ml water in the tube marked as control.
 Add one ml solution of sodium hydro-oxide solution (10 % aqueous solution) to
the tube marked as “test” and one ml distilled water to the tube marked as
“control”.
Result
There is development of brownish color in tube marked as “test”. If milk does not
contain sorbitol, the precipitate will settle down as precipitate. If the milk contains
sorbitol, then precipitate will not settle down.

39
DETECTION OF MINIMUM LEVEL OF SORBITOL IN MILK
Tube # milk dilution Amount of sorbitol Result
1 Adulterated milk One ml/100 (1%) Color precipitate does not settle
2 Adulterated milk 0.25 ml/100 (0.25%) Color precipitate does not settle
3 Normal milk 0 Color precipitate settles
Comments: Chemical test can detect 0.25 % Sorbitol in raw milk.
EFFECT OF VOLUME OF MILK SAMPLE ON SORBITOL
Tube # Volume of adulterated
milk
Result
1 5 ml Color precipitate did not settle
2 2.5 ml Color precipitate did not settle
3 1 ml Color precipitate did not settle
Comments: Volume of milk sample did not affect the lab testing of the Sorbitol.
EFFECT OF BOILING OF ADULTERATED MILK ON SORBITOL
Comments: Boiling of the milk did not affect the lab testing of the Sorbitol.
Questions
One, 5, 10 % solution of aquous solution of ferric sulphate
2. Why the middle man add soap/detergent in milk?.
4. What is effect of boiling of milk on detection of sorbitol?
5. Is it possible to develop strip test for detection of sorbitol in milk?

40
4.9. SODIUM CHLORIDE IN MILK
Introduction
Sodium chloride is normal components of milk but its normal concentration is not
detectable by laboratory test. It is added in the milk to increase its density. Addition of
table salt in milk is toxic for the consumers if suffering from blood pressure.
Materials
 Test tubes
 Pasteur pipette
 Solution A
Potassium dichromate 25 grams
 Solution B
Silver nitrate 840 grams
 Milk sample
Procedure
 Add one ml of solution A in the tube marked as “test” and one ml water in the
tube marked as control.
 Add one ml of solution B in the tube marked as “test” and one ml water in the
tube marked as control.
Result
Appearance of red precipitate indicate the absence of dissolved chloride in milk and
appearance of yellow color indicate presence of dissolved chloride

41
4 1:20 of the adulterated milk 0.25 : 250 mg/100 ml +
5 1:40 of the adulterated milk 0.125: 125 mg/100 ml -
Comments: Chemical test can detect the sodium chloride up to 0.25 % in raw milk. Addition
of sodium chloride below 0.25% is hardly detectable in the laboratory.
EFFECT OF VOLUME OF MILK SAMPLE ON SODIUM CHLORIDE TEST
1 5 ml +
2 2.5 ml +
3 1 ml +
Comments: Volume of milk sample did not affect the lab testing of sodium chloride.
Questions
1. How will you proceed for preparation of one, 5, and 10 percent solution of
sodium chloride solutions?
2. How will you proceed for preparation of one, 5, and 10 percent solution of silver
nitrate?
3. Why the middle man add sodium chloride in milk?.
5. What is effect of boiling of milk on detection of sodium chloride?
6. Is it possible to develop strip test for detection of sodium chloride in milk?

42
4.10. HYPOCHLORITES AND CHLORAMINES
Introduction
Sodium hypochlorite is an important disinfectant that can be incorporated in drinking
water. Middle man add the chlorine solution in milk to minimize bacterial growth during
transportation of milk to cities.
Materials
 Test tubes
 Pasteur pipette
 Potassium iodide solution
 Potassium iodide (7% aqueous solution) 150 ml
 Hydrochloric acid (50% aqueous solution 400 ml
 Starch solution (Boil 1 gram starch in 100 ml water, cool before use)
 Milk sample
Procedure
 Add one ml of the potassium iodide solution in the tube marked as “test” and one
ml distilled water in the tube marked as “control”.
 Mix the contents of each tube and place the tubes in water bath to heat upto 85 C
for 10 minutes
 The curd will rise to the surface the liquid and curd will have yellow brown to
deep brown color
 Add one ml of starch solution to the liquid below curd and one ml distilled water
in control tube..
Result
Formation of a blue purple color is indication of Hypochlorites

43
Tube # Dilution of adulterated milk Amount of sodium hypochlorite (%) Result
Comments: Chemical test can detect the sodium hypochlorite up to 0.01 % in raw milk.
Addition of sodium hypochlorite below 0.01% is hardly detectable in the laboratory.
EFFECT OF MILK VOLUME ON SODIUM HYPOCHLORITE TEST
1 5 ml +
2 2.5 ml +
3 1 ml +
Comments:Volume of milk sample did not affect the laboratory testing of the sodium
hypochlorite.
EFFECT OF BOILING OF ADULTERATED MILK ON SODIUM CHLORIDE
Comments: Boiling of THE milk did not affect the lab testing of the sodium hypochlorite.
Questions
1. How will you proceed for preparation of one, 5, and 10 percent aquous solution of
potassium chloride?
2. How will you proceed for preparation of one, 5, and 10 percent solution of starch?
3. Why the middle man add sodium hypochlorite in milk in milk?.
5. What is effect of boiling of milk on detection of sodium hypochlorite?
6. Is it possible to develop strip test for detection of sodium hypochlorite in milk?

44
4.11: QUATERNAY AMMONIUM COMPOUND (QAC)
Introduction
This is a disinfectant commonly used to disinfect the dairy farms. It is added in milk to
control the bacterial growth during transportation of milk from suburb areas to
metropolitan cities. It is highly toxic for human being or even for animals so milk
containing such type of health hazard materials should be declared unfit for human
consumption.
Materials
 Test tubes
 Pasteur pipette
 Indicator solution: Prepare a stock solution by dissolving 0.05 gm eosin in 100
ml acetone. Shake 10 ml of stock solution with 90 ml of tetrachloroethane and 1
gm citric acid and filter before use.
 Buffer: Dissolve 25 gm citric acid in 100 ml water and adjust to pH 3.5 with 50
% Sodium Hydroxide solution ( approx 15 ml required ).
 Milk sample
Procedure
 Take two test tubes and label one as “test” and other as
“control”
 Add one ml indicator solution and 0.2 ml buffer in the tube
marked as “test” and 2.2 ml water in the tube marked as control.
 Shake well the contents of each tube.
Result
Development of pink to red color indicate the presence of QAC. Deep pink or red if more than
5mg/litter and faint pink color if QAC is equal to one mg/litter

45
DETECTION OF MINIMUM LEVEL OF QAC IN MILK
Tube # Dilution of adulterated milk Amount of QAC (%) Result
2 Adulterated undiluted milk +
3 1:10 of the adulterated milk 10 +
4 1:100 of the adulterated milk 1 -
5 1:1000 of the adulterated milk 0.1 -
Comments: Chemical test can detect the QAC up to 1mg/litter in raw milk. Addition of QAC
less than this is hardly detectable in the laboratory.
EFFECT OF MILK VOLUME ON CHEMICAL TESTING OF QAC
1 5 ml +
2 2.5 ml +
3 1 ml +
Comments: Volume of milk sample did not affect the laboratory testing of the QAC.
EFFECT OF BOILING OF ADULTERATED MILK ON QAC
Comments: Boiling of market milk did not affect the laboratory testing of the QAC.
Questions
1. How will you proceed for preparation of one, 5, and 10 percent aquous solution of
QAC?
2. How will you proceed for preparation of one, 5, and 10 percent solution of eosin?
3. Why the middle man add QAC in milk in milk?.
5. What is effect of boiling of milk on detection of QAC?
6. Is it possible to develop strip test for detection of QAC in milk?

46
4.12 BORAX AND BORIC ACID
Introduction
Boric acid or borax is added in milk to increase its density. It is an adulteration of milk
and is against the policy of pure milk.
Materials
 Test tubes
 Pasteur pipette
 Concentrated HCL solution
 Strips of turmeric papers
 Ammonia solution
 Milk sample
Procedure
 Add one ml concentrated HCl in the tube marked as “test” and one ml distilled
water in the tube marked as control.
 Dip a tip of turmeric paper in the acidified milk and is dried over small flame. Its
color turns red.
 Add a drop of ammonia solution on the turmeric paper.
Result:
If the red color changes to green, it shows the presence of boric acid.

47
Doc Title: Lab Accreditation Data Form Doc. No: QMS/LA/Tox/001
REV: 01
Quality Management Systems.9000
Prepared By: _________ Approved By: ________________
Dated: Dated:
Please attach more pages of similar format if required.
2. SCOPE
S.#
Products
Tested
Types of Test/Properties
measured/Range of
Measurement
Standard
Specifications
/Techniques
Used
Reference of
Test Methods
Remarks
1) Milk 1.1. Normal constituents
1.1.1. Organo-leptic test
1.1.2 Clot on boiling (C.O.B.)
test
1.1.3 Alcohol test
1.1.4 Acidity test
1.1.5 Methylene blue reduction
test
1.1.6 The Gerber butterfat test
1.1.7 The lactometer test
1.1.8. Inhibitor test

48
Doc Title: Lab Accreditation Data Form Doc. No: QMS/LA/Tox/001
REV: 01
Quality Management Systems.9000
Prepared By: _________ Approved By: ________________
Dated: Dated:
2. SCOPE
S. #
Materials/P
roducts
Tested
Types of Test/Properties
measured/Range of Measurement
Standard
Specifications/Te
chniques Used
Reference of Test
Methods
Remarks
1 Milk 2.2 Milk Adulterants
2.2.1. Formaldehyde
2.2.2 H2O2
2.2.3. Urea
2.2.4. Sodium chloride
2.2.5. Detergent (surf)
2.2.6. Sorbitol
2.2.7. Sodium hypochlorite
2.2.8. Neutralizers / Carbonate
2.2.9. Starch
2.2.10. Cane sugar
2.2.11. Quaternary Ammonium
Comp
2.2.12. Gelatin

49
5.0 MONITORING OF HYGIENIC STATUS OF MARKET MILK
Milk is a sterile lacteal secretion of healthy dairy animals. After milking, it may get
contamination from contaminated environment, containers, water, etc. However, some
time the lactating animals are suffering from sub-clinical mastitis. Milk from such cases
may contaminate the bulk of milk.
Some pathogenic bacteria such as tubercle bacilli, brucella spp, streptococcus spp, etc,
are incriminated to be cause of mastitis in dairy animals. These pathogens are transferred
to raw milk consumers. Such pathogens can be eliminated by pasteurization of the milk,
but spore forming and thermoduric bacteria can survive in the pasteurized milk.
Contamination of non-spore forming and thermoduric bacteria mitigate the shelf life of
milk and some time spoils the milk. In testing laboratories, following microbiological
assays are routinely performed to determine the bacterial load in milk (Also read
Annexure III).
5.1-DIRECT MICROSCOPIC SOMATIC CELL COUNT
Introduction
Somatic Cell Count (SCC) is an indicator of milk quality. The majority of somatic cells
are leukocytes (white blood cells). These cells enter in milk secretions after an infection
in udder tissue or memory gland or immune response of the udder tissue to infectious
agent and increase in their number in milk. In addition to leukocytes, there is small
number of epithelial cells in milk. These are milk-producing cells shed from inside of the
udder when an infection occurs.
Materials
Functional fume hood.
Microscope Slides, Clean (see item 18), 2.54 x 7.62 cm with 11.28 mm diameter
delineated area
Optionally, with center marks on sides of delineated area

50
Optionally, 5.08 x 7.62 or 5.08 x 11.43 cm with 11.28 cm delineated areas
Syringe suitable for rapid and convenient transfer of 0.01 mL of milk
Micropipettor, with appropriate tips
Dissecting Needle, Bent Point suitable for spreading milk film
Drying Device, Slide Drier or Incubator
Heat source regulated at 40-45C
Forceps or slide holder required for dipping and holding slides
Staining Jars or Trays
Microscope with:
i. Binocular with 1.8 mm oil immersion objective, rack and pinion sub-stage, condenser
with iris diaphragm
ii. Oculars, 10X (12X or 12.5X), Huygenian or wide-field
iii. Optics provide a Single Strip Factor of 6070 or smaller
iv. Each analyst measures field diameter and calculates SSF
annually, round to three significant figures
2. Calculation of Single Strip Factor
a.Using a stage micrometer, measure field diameter (D) of oil immersion objective lens in
mm
D = ________ mm
Compute SSF with formula
SSF = 10,000/(11.28 x D)
Mechanical Stage Suitable for examination of slides, smooth action, does not drift, allows
proper tracking of smears.
Microscope Lamp, provides adequate illumination.
Stage Micrometer Ruled with 0.1 and 0.01 mm Divisions.
Hand Tally, accurate.
Immersion Oil.
Refractive index 1.51-1.52 at 20C.
Pyronin Y-Methyl Green Stain for Goat or Sheep Milk
a. Carnoy's fixative
l. Combine:

51
60 mL chloroform
20 mL glacial acetic acid
120 mL 100% ethyl alcohol
Pyronin Y-methyl green stain
1. Mix one gram Pyronin Y, 0.56 g methyl green in 196 ml water
2. Filter through Whatman No. 1 paper before use
3. Stain is light sensitive; store in brown bottle
Slides, Cleaning
a. Physically clean
b. New slides may be cleaned by soaking in strong cleaning solution
c. Rinse thoroughly in flowing water 10-15 sec and water
d. Used slides may be soaked in hot detergent or wetting agent until all residues are
removed, rinsed as above
e. Air or heat dry with minimal exposure to dust, insects, etc. and store dry
f. store slides in alcohol and flame just before use
PROCEDURE
Slide Identification
a. Legibly and indelibly identify each sample area on margin of slide
Sample Agitation
a. Mix samples by shaking 25 times in 7 sec with 1 ft movement.
b. Optional: Warm high fat samples to 40C for no longer than 10 minutes prior to testing
(discard after testing)
Sample Measurement and Smear Preparation
a. Before use and between successive samples, rinse syringe 2 - 3 times in clean, 25-35C
water
b. Before transferring test portion to slide, insert syringe not over 1 cm below surface
(avoiding foam) of milk and repeatedly rinse.
c. Holding tip beneath surface, rinse syringe three times with milk, then fully depress and
release plunger and withdraw test portion.
d. With clean paper tissue, remove excess milk from exterior of tip.

52
e. Holding instrument vertical, place tip near center of area for smear, touch the slide with
the tip and expel the test portion.
f. Spread milk with point of bent needle point (item 5), not hockey stick style.
g. Wipe needle dry between samples on tissue.
h. After spreading test portion, dry smears at 40-45C within 5 min on level surface.
g. To prevent smears from cracking and peeling from slide during staining, do not heat
too rapidly.
h. Protect smears and slides from damage until read
Staining Films
Stain is light sensitive and must be protected from overexposure to light.
1. Slide is run through the following staining scheme.
Carnoy's fixative 5 minutes.
50% Ethanol 1 min
30% Ethanol 1 min
H20 1 min
Stain 6 min
N-Butyl alcohol flush briefly.
Xylene flush briefly.
a. Optionally, if smears will not adhere to slides:
1. Allow slide to dry, (approx 10 min) protected from overexposure to light.
Allow slide to dry (approx 10 min) protected from overexposure to light, after stain step
but before flushing with N-Butyl alcohol.
2. Cells stain blue or blue-green; RNA and background stain pink.
Examination
a. Adjust microscope lamp to provide maximal optical resolution.
b. Locate edge of smear to be read using low power.
c. Place 1 drop immersion oil on smear.
d. Carefully lower oil immersion lens.
e. Focus and locate center of edge of area and begin counting cells.
f. Count all cells in field wide strip across diameter of a single smear, focusing up and
down as necessary.

53
Identifying and counting somatic cells
l. Cells possess a nucleus stained dark blue (bovine) or blue or blue-green (caprine)
2. Cells generally 8 microns or larger (bovine; caprine may be smaller); do not count cells
less than 4 microns; fragments counted only if more than 50% of nuclear material visible.
3. Cluster of cells counted as one unless nuclear units are clearly separated; focus up and
down to ensure that there are no bridges connecting nuclear masses.
4. Count cells touching only top or bottom half of strip.
5. If in doubt, do not count.
h. After examination of each smear record strip count
Records and Reporting
a. Maintain record of strip count for each smear examined.
b. Compute DMSCC/mL, multiply number of cells counted (strip count) by the SSF.
c. Report somatic cell counts as DMSCC/mL, record only first two left hand digits, round
as necessary.
1. If the third digit is 5 round the second number using the following rules.
a. When the second digit is odd round up (odd up, 235 to 240).
b. When the second digit is even round down (even down,
225 to 220).
Reference
1. FDA approved protocol for somatic cell count in milk “FORM FDA 2400d Direct
Microscopic Somatic Cell Count rev 2/10”.

54
5.2. METHYLENE BLUE REDUCTION TEST
Introduction
Methylene Blue is a blue colored dye. Aerobic bacteria are common contaminates of
milk. Such bacteria release respiratory burst metabolites as a result of active metabolism.
These metabolites reduce the methylene blue that ultimately turns its color white. Rate of
reduction of methylene blue is directly related with bacterial load in milk. Milk is
classified on the basis of methylene blue reduction time.
Materials
Sterilized test tubes with rubber stoppers 2
Test tube stand 1
Medium glass jars 2
Saucepan 1
Tongs 1
Thermometer 1
Calibrated (cc) medicine dropper 1
Methylene Blue Solution
Milk sample
Negative control standing milk
Procedure
1. Transfer 9 ml of the milk sample into both the tubes.
2. Add 1 ml methylene blue solution to the first test tube, noting the time,
than place the stopper on the test tube and shake it until the blue color
mixes thoroughly with the milk sample.
3. Place both the tubes at 37o
C
4. Check them on every half an hour for the first two hours and once an hour
after that.
5. If the blue color starts disappearing in streaks
6. Gently mix the sample. If milk containing the methylene blue becomes
white as that of milk without methylene blue, the test is over.
7. Note the time it takes to disappear the color.
8. Record observations.
Classification
Class 1 Excellent, not decolorized in 8 hours
Class 2 Good, decolorized in less than 8 hours but not less than 6 hours.
Class 3 Fair, decolorized in less than 6 hours but not less than 2 hours
Class 4 Poor, decolorized in less than 2 hours

55
5.3. RESAZURIN TEST.
Introduction
Resazurin test is the most widely used test for hygiene and the potential keeping quality
of raw milk. Resazurin is a dye indicator. Under specified conditions Resazurin is
dissolved in distilled boiled water. The Resazurin solution can later be used to test the
microbial activity in given milk sample.
Resazurin can be carried out as:
i. 10 min test.
ii. 1 hr test.
iii. 3 hr test.
The 10 min Resazurin test is useful and rapid, screening test used at the milk platform.
The 1 hr test and 3 hr tests provide more accurate information about the milk quality, but
after a fairy long time. They are usually carried out in the laboratory.
Apparatus and reagents:
 Resazurin tablets
 Test tubes with 10 mls mark
 1 ml pipette or dispenser for Resazurin solution.
 Water bath thermostatically controlled
 Lovibond comparator with Resazurin disc 4/9
Procedure
The solution of Resazurin as prepared by adding one tablet to 50 mIs of distilled sterile
water. Rasazurin solution must not be exposed to sunlight, and it should not be used for
more than eight hours because it losses strength.
Mix the milk and with a sanitized dipper put 10 mls milk into a sterile test tube.
Add one ml of Resazurin solution, stopper with a sterile stopper, mix gently the dye into
the milk and mark the tube before the incubation in a water bath, place the test tube in a
Lovibond comparator with Resazurin disk and compare it colourimetrically with a test
tube containing 10 ml milk of the same sample, but without the dye (Blank).

56
READINGS AND RESULTS (10 MINUTE RESAZURIN TEST)
Resazurin disc No. Colour Grade of milk Action
6 Blue Excellent Accept
5 Light blue v. good Accept
4 Purple Good Accept
3 Purple pink Fair Separate
2 Light pink Poor Separate
1 Pink Bad Reject
0 white Very bad Reject

57
5.4. COLIFORM COUNT
Introduction
Coliform is a group of bacteria including species of genera Escherichia and Aerobacter.
These are normal inhabitant of intestines of man and animals. Presence of coliform in
milk indicates fecal contamination. Fecal contamination may transfer salmonella and
other bacteria of zoonotic importance to raw milk consumers.
Coliform bacteria produce black colonies with metallic sheen on ENDO agar or EMB
agar so are easily assessed.
Procedure
1. Make a serial 10 fold dilution of milk sample (From 101
to 105
) in sterilized
bottles (Annexure III and IV).
2. Take 4 sterility tested Eosin Methylene Blue (EMB) agar plates and mark them 1
to 4.
3. Spot aseptically 5 drops (50 ul each) from each of the bottles having milk dilution
105
, 104
, 103
, 102
on the agar plate number 1, 2, 3, 4, respectively.
4. Incubate each of the plate at 37 o
C for 24 hours.
5. Count the number of countable colonies in each of the 5 spots on each agar plate
and calculate average.
6. Calculate the bacterial load using following formula.
Bacterial count/ml= Average number of colonies x dilution factor/ volume of
spot
For example
Average number of colonies per spot 2
Volume of the spot 0.050 ml or 50 ul
Dilution factor (suppose countable colonies are recorded in plate No. 2 (104
)
Bacterial count/ml = 2 x 10,000 / 0.05 ml
= 2 x 10, 000 x 20
= 4 x 100, 000 per ml

58
5.5 STANDARD PLATE COUNT
Introduction
Standard plate count is a sensitive, accurate and reliable technique to determine the
bacterial in the milk. It measures all the aerobic viable bacteria. However, it is time
consuming, bacterial clumps gives single bacterial count, and can only be performed in a
microbiological laboratory.
Procedure
 Make a serial 10 fold dilution of milk sample (From 101
to 108
) in sterilized bottles
(Annexure-1).
 Take 4 sterility tested nutrient agar plates and mark them 1 to 4.
 Spot aseptically 5 drops (50 ul each) from each of the bottles having milk dilution
108
, 107
, 106
, 105
on the agar plate number 1, 2, 3, 4, respectively.
 Incubate each of the plate at 37 o
C for 24 hours.
 Count the number of countable colonies in each of the 5 spots on each agar plate and
calculate average.
 Calculate the bacterial load using following formula.
 Bacterial count/ml= Average number of colonies x dilution factor/ volume
of spot
 Average number of colonies per spot 12
 Volume of the spot 0.050 ml or 50 ul
 Dilution factor (suppose countable colonies are recorded in plate#2 (107
)
 Bacterial count/ml= 12 x 10,000,000 or 107
/ 0.05 ml
 = 12 x 10,000,000 x 20
 = 24 x 100, 000, 000 per ml

59
Annexure-1
Aseptic dilution and transfer
Aseptic Plating

61
ANNEXURE-III
Milk Hygienic Standards
 Somatic cell count equal to or less than 200,000
 Standard plate count equal to or less than 5,000
 Preliminary incubation count equal to or less than 10,000
 Drug residue free
 Sediment count equal to or less than 1.0
 Cryoscopy reading equal to or less than -.530° H
Listed below are possible causes of high Counts.
 Failure to thoroughly clean equipment after each use or neglecting to sanitize
equipment before using (a major cause)
 Slow cooling farm bulk tank or temperature above 40° F (bulk tanks should be
less than 40° F within two hours of milking and kept below 45° F during
subsequent milkings). These points out the importance of the recording
thermometer and its use.
 Problems with debris buildup in plate coolers and chillers.
 When milking fresh cows and problem cows in bucket milkers, hoses need to be
keep clean
 Dirty animals; may need clipping
 Poor udder sanitation practices (dirty, excessive water used to wash teats &
udders); teats need to be clean, sanitized, and dry
 Contaminated water supply, especially Coliform or other spoilage-causing
bacteria
 Wash water temperature should start at 155-170°F and drain at above 120° F
 Gaskets and rubber parts need to be clean, free of cracks and deposits, and
replaced when necessary
 Improperly drained milking equipment
 Teat cup liners should be clean and free of cracks and changed on schedule
 Pulsator and main vacuum supply lines need cleaning on a regular basis and
especially whenever milk enters the line

62
REFERENCES
1-Pearson Composition and Analysis of Food 9th
Edition , 1991. Modified Mohr Method:
Page 14.
2-Pearson Composition and Analysis of Food 9th
Edition , 1991. Modified Mohr Method: Page
14.
3- BS 1741: Part II, Pearson Composition and Analysis of Food 9th
Edition, 1991. Page 548
4-Milk Processing Guide Series, Vol. 2, published by FAO/TCP/KEN/6611 Project,
Dairy Institute, Naivasha, 2004
5-ILCA Manual No.4, Rural Dairy Technology. Experiences from Ethiopia.
IDF Doc. No.9002, Handbook on Milk collection in Warm Developing Countries.
International Dairy Federation, Brussels, Belgium.
6.Marshall, R.T. 1992. Standard Methods for the determination of Dairy Products, 16th
ed. Publ. American Public Health Association
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