MOTHER DAIRY FRUIT AND VEGETABLE PRIVATE LIMITED

UNIT: MOTHER DAIRY , VASHI, NAVIMUMBAI

MDV-TM-03 TESTING MANUAL

MOTHER DAIRY FRUIT AND VEGETABLE

PRIVATE LIMITED

UNIT: MOTHER DAIRY, VASHI, NAVI MUMBAI

TESTING MANUAL

Copy No.

Copyholder

Prepared By Approved By

I/C QC Unit Head

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Sr.no. Contents Page no.

i. General safety instructions for laboratory 4
ii. General care of lab equipment & instruments 5
1. Procedure for sampling of milk 6
2. Procedure for organoleptic test of milk 8
3. Procedure for clot-on-boiling (COB) test of milk 10
4. Procedure for acidity test of milk 11
5. Method for analysis of fat content in milk 13
6. Procedure for determination of SNF content in milk. 16
7. Procedure for determination of Total solid content in milk 18
8. Phosphatase test for pasteurization in liquid milk 21
9. Alcohol test of milk. 22
10. Sediment test 23
11. Hansa test 24
12. Method for detection of neutralizer in milk. 25
13. Method for detection of urea in milk. 26
14. Detection of ammonium Compounds in milk. 27
15. Test for quaternary ammonium compounds in milk (detergents). 29
16. Detection of Nitrate Fertilizers (Pond water test) in milk. 30
17. Method for detection of cane sugar in milk 31
18. Method for detection of glucose in milk. 32
19. Detection of Maltodextrin in milk. 34
20. Method for detection of starch in milk. 36
21. Procedure for detection of salt in milk 37
22. Detection of cellulose in milk 38
23. Detection of hypochlorite and chloramines. 39

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MDV-TM-03 TESTING MANUAL

Page
Sr.no. Contents
no.
24. Procedure for detection of formalin in milk 40
25. Procedure for detection of hydrogen peroxide in milk 41
26. Test for presence of salicylic acid 42
27. Test for presence of boric acid and borates. 43
28. Test for presence of dulcin. 45
29. Test for presence of saccharin. 46
30. Mineral Oil Test 47
31. Method for detection of butyro-refractometer reading for ghee 48
32. Determination of RM value: 49
33. Method of detection of keeping quality of milk at 37 o c. 50
34. Method of detection of homogenization efficiency (quick tests ) & (48 hrs test) 51
35. Test for checking of Ash and alkalinity of ash 53
36. Determination of Protein in milk. 54
37. Determination of Sodium content in milk. 58
38. Microbiological analysis liquid milk (MBRT, SPC and Coliform count) 62
39. Assessing sterility of milk equipment 68
40. Microbiological analysis water 69
41. Method of detection of purity of concentrated HNO3. 72
42. Method of detection of purity of concentrated Sulphuric acid. 74
43. Method of detection of purity of caustic flakes. 75
44. Method of detection of strength of cleaning solutions. 77
45. Method of detection of water. 82
46. Method of detection of packing material (LDPE film). 84
47. HDPE crates for milk pouch 86
48. Culture product chemical analysis Annx.1
49. Culture product Microbial analysis Annx.2
50. Culture product Packing material analysis Annx.3

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(i) GENERAL SAFETY INSTRUCTIONS FOR LABORATORY

DO’S

LABEL ALL THE CHEMICALS PROPERLY

USE GLOVES/GOGLES WHILE HANDLING CONC CHEMICALS

USE RUBBER TEAT FOR PIPETTING CONC CHEMICALS

KEEP CHEMICAL BOTTLES PROPERLY STOPPERED

CLEAN THE AREA IMMEDIATELY, IF ANY CHEMICAL IS SPILLED

USE COLD WATER BATH WHILE DILUTING ACIDS

REMOVE BROKEN GLASSWARE BY MEANS OF A DAMP CLOTH

POUR/SHAKE THE CHEMICALS AWAY FROM FACE / BODY, PREFERABLY NEAR
SINK

POUR SPENT ACIDS, CHEMICALS IN THE WASTE RESERVOIR

EMPTY WASTE RESERVOIR IN ETP COLLECTION TANK

DON’TS

DO NOT USE ANY CHEMICAL WITHOUT LABEL

DO NOT INHALE ANY CHEMICAL

DO NOT PIPETTE ANY CONCENTRATED CHEMICAL WITH YOUR MOUTH

DO NOT ADD WATER TO CONCENTRATED ACID FOR DILUTING

DO NOT PICK BROKEN GLASSWARE BY NAKED HANDS

DO NOT SHAKE BUTYROMETER IN FRONT OF FACE

DO NOT LEAVE BURNER, HOT PLATE UNATTENDED WHEN ON

DO NOT KEEP ALCOHOL NEAR NAKED FLAME

DO NOT CARRY SYRINGE/SCISSORS/SHARP INSTRUMENT WITHOUT PROPER
COVER

DO NOT THROW AWAY SPENT CHEMICALS

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(ii) GENERAL CARE OF LAB EQUIPMENT & INSTRUMENTS

1. Read the Operating Manual of the Laboratory Equipment / Instrument before using
any of the Equipment / Instrument.
2. Operate / use the Equipment / Instrument strictly as specified in the Operating
Manual.
3. Calibrate / adjust the Equipment / Instrument as specified in the Operating Manual.
Wherever calibration / adjustment is set by the supplier, refer to the supplier
concerned for calibration / adjustment.
4. Do not repair any Equipment / Instrument by yourself. Call the supplier for regular
periodic maintenance, if specified.
5. Keep the Equipment / Instrument clean. Do not spill any chemical on them.

GENERAL CARE OF GLASSWARE

1. Do not place graduated and measuring glassware in hot air oven for drying. Keep
such glassware inverted in the drain board to allow the water to run out. Rinse
such glassware with acetone and dry in oven at not more than 60 o C.
2. Do not measure any liquid when it is too hot or too cold
3. Use graduated or measuring glassware at the temperature specified
4. Use proper stands for keeping pipettes, test tubes etc.
5. Clean the glassware by thoroughly rinsing with tap water followed by brushing
with detergent solution. Immerse in chromic acid solution. Rinse again with tap
water and clean using appropriate nylon brush and detergent solution. Rinse with
tap water and finally with distilled water.

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1.0 PROCEDURE FOR SAMPLING OF MILK.

Scope: This procedure applies for sampling of milk from milk tankers.

Responsibility: Chemist

Procedure:
1. Check the seal of the incoming tanker.
2. Open the manhole cover of the tanker.
3. Check the physical appearance & smell of the milk at the manhole of the tanker
4. Dipper & plunger must be sanitized prior to use.
5. Before plungering we have to drawn the 5 liter from the bottom valve for sediment
test.
6. Plunger is thrust forward & backward, downward & pulled back. Mixing the milk
from both ends with plunger, until milk is mixed thoroughly i.e. plungering for not
less than 20 times from both side for not less than 5 min. Samples will be taken in
250 ml sample bottle. These bottles will be cleaned using liquid soap and water.
These will be properly cleaned and kept upside down to remove all traces of water
from inside. Dipper shall be fitted with a solid handle; inside portion of the dipper
is made in such a way that it can be easily cleaned.
7. Take milk in the sample bottle with dipper. Two sample bottles are drawn, one
sample bottle is used for conducting Chemical analysis having capacity of 250 ml
& other sample collected in sterilized sample bottle for Microbial analysis having
100 ml capacity.
8. Check the temperature of the milk in the sampling dipper by using calibrated
digital thermometer.
9. Close the bottle and place the plunger properly dipped in30 ppm Iodophor solution,
where in Iodophor is changed frequently and bring the sample to lab for testing.

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10. Labeling of sample: Each sample container before sampling is marked with label
bearing ‘Date of sampling’, ‘Tanker Number’ & ‘Sample Number’ shall be capped
air tight after filling.

Reference: IS11546:1999

Preparation of sample
0 0
Samples are received at low temperature. Warm the sample to 37 – 40 C by transferring
0 0
it to the beaker and keeping it in a water bath maintained at 40 - 45 C. Stir slowly for
proper homogenization. Mix sample thoroughly by pouring back into the bottle, mixing
to dislodge any residual fat sticking to the sides and pour it back in the beaker. For
mixing do not shake the bottle vigorously. Allow the sample to come to room
0 0
temperature (26 - 28 C) and withdraw immediately for analysis. If small clots or lumps
are observed in the sample, which cannot be dispersed, a few drops of liquor ammonia
may be used during homogenization. If even after homogenization the sample shows
lumps or clots or droplets of oil are visible suggestive of curdling /splitting of milk, the
sample should be deemed unfit for analysis and rejected.

(Ref: IS 1479 (Pt II) – 1961 / DGHS Manual / Mother Dairy Guideline

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MDV-TM-03 TESTING MANUAL

2.0 PROCEDURE FOR ORGANOLEPTIC TEST OF MILK.

Scope: This procedure applies for odor & taste of milk.

Responsibility: Chemist.

Procedure:1
1. Smell the milk in the tanker immediately after opening the lid. In case of foul or
abnormal smell, reject the milk or hold over for subjection to confirmatory test.
2. Properly mix the sample & take milk sample in a clean & dry beaker, which shall be
free from any extraneous odor/flavor.
3. Observe the color of the milk. If abnormal in color, it should be regarded with
suspicion.
4. Examine the milk for following taints:
a. Those due to developed acidity. This is the most important factor to be examined
when grading milk by organoleptic test.
b. Those due to feed, or exposure of milk to the atmosphere of the stable.
c. Extraneous matter, which might gain access to milk after milking.
d. Oxidized flavor due to exposure of milk to light or metallic contamination from
untinned containers.
5. Rinse the mouth using fresh water thoroughly before tasting the samples.
6. Boil some quantity of milk and check flavour then allow it to cool down at room temp.
Take a sip of a sample into mouth & perceive the taste of the milk by keeping it inside
the mouth for 15 – 30 sec.
7. Differentiate for odor:
(a) Pleasant as Normal
(b) Sour / stale / neutralized / objectionable as abnormal taste
8. Differentiate for taste:
(a) Clean as Normal
(b) Sour / Sweet / Salty / Bitter / objectionable as Abnormal taste

Reference: IS 1479(Part-1) 1960

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OR

Procedure:2
Organoleptic Evaluation
The organoleptic test permits rapid segregation of poor quality milk at the milk
receiving platform. The milk grader must have good sense of sight, smell and taste. The
appearance of the surface of the milk and the lid is observed and inspected instantly after
removing the lid of incoming milk can or container. Any abnormal colour of the milk,
visible dirt and particles, changes in viscosity etc. are observed. Any abnormal smell is
noticed by inhalation of air standing above the milk in the upper part of the milk can or
sample bottle.
• In case of large container, a sample of at least 500 gm should be taken.
• For the evaluation of flavour individual portions of at least 50-100 gm
should be available.
• During the evaluation the samples should have a temperature 16±2°C.
Procedure
• Open the bottle of milk.
• Immediately smell the milk.
• Observe the appearance of the milk
• If still unable to make a clear judgment, taste the milk, but do not swallow
it. Spit the milk sample into a bucket provided for that purpose or into a drain basin, flush
with water.
• Look at the lid of manhole of tankers and the milk surface to check
cleanliness of milk
Abnormal smell and taste may be caused by:
• Atmospheric taint (e.g. barny/cowy odour).
• Physiological taints (hormonal imbalance, cows in late lactation-
spontaneous rancidity).
• Bacterial taints.
• Chemical taints or discolouring.
• Advanced acidification (pH < 6.4).
Reference: Mother Dairy Guideline
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3.0 PROCEDURE FOR CLOT-ON-BOILING (COB) TEST OF MILK.

Scope: This procedure applies for COB test of milk for incoming raw milk sample.

Responsibility: Chemist.

Procedure:1
1. Thoroughly mix the sample.
2. Take 5 ml milk in test tube.
3. Place the tube in boiling water bath
4. Hold in water bath for 5 minutes.
5. Remove and observe for any flakes by rotating the test tube horizontally to
spread milk
on the inner glass surface.
Interpretation:
Presence of flakes indicates positive results.

Reference: IS 1479(Part-1)1960 / Mother Dairy Guideline

OR

Procedure:2
COB TEST:
Take 5 ml of milk in a dry test tube. Boil milk on flame of spirit lamp. Formation of ppts
in the test tube indicate COB test positive.

Reference: Mother Dairy Guideline

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MDV-TM-03 TESTING MANUAL

4.0 PROCEDURE FOR ACIDITY TEST OF MILK.

Scope: This procedure applies for determination of acidity in terms of % lactic acid of
milk.

Responsibility: Chemist.
Procedure: 1
1. Thoroughly mix the milk.
2. Measure 10 ml of milk in beaker.
3. Add equal volume of distilled water.
4. Add 1 ml of 1% phenolphthalein indicator solution.
5. Titrate against 0.1N sodium hydroxide solution.
6. End point: Faint pink color, which needs to be matched with the standard color or
titrate upto 8.3 pH.
7. Calculate the acidity using following formula:
Acidity (% L.A.) = 9 V1 x N
V2
V1 = Volume in ml of the sodium hydroxide solution
V2 = Volume in ml of the milk taken
N= Normality of the standard sodium hydroxide solution
Reference: IS 1479(Part-1)1960/ Mother dairy vashi

Procedure: 2
Measure accurately 10 ml of milk in two porcelain basins. Add an equal volume of
freshly boiled and cooled water. Add 1.0 ml of phenolphthalein indicator solution to one
of the basins and to the other basin, add 1.0 ml of bench solution of rosaniline acetate.
Titrate the contents of the basin to which solution of rosaniline acetate. Titrate the
contents of the basin to which phenolphthalein has been added, against standard sodium
hydroxide solution added drop by drop from the burette until by comparison the colour
matches the pink tint of the solution in the basin containing the rosaniline acetate
solution. Stir vigorously throughout. The time taken for complete titration shall not

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MDV-TM-03 TESTING MANUAL
exceed 20 seconds. The titration shall be made in north light or under illumination from a
daylight lamp.
Calculation:
Titratable acidity% (as lactic acid) = 9V1N/ V2

Where
V1 = Volume in ml of the standard sodium hydroxide required for
titration,
N = Normality of the standard sodium hydroxide solution, and
V2 = Volume in ml of milk taken for the test
Reference: IS 1479(Part-1)1960/ Reference: Mother Dairy Guideline

Procedure: 3
Taking 10 ml milk in 100 ml conical flask, add 10 ml distilled water. Add 1ml
phenolphthalein indicator and titrate against N/10 NaOH till a faint pink colour appears.
Calculate the acidity % as volume of NaOH used×0.09.

Reference: Mother Dairy Guideline

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5.0 METHOD FOR ANALYSIS OF FAT CONTENT IN MILK.

Scope: This applies to determination of fat content in Raw Milk.

Responsibility: Chemist.

Apparatus:
i. Gerber centrifuge
ii. Milk butyrometer
iii. 1 ml tilt measure for amyl alcohol and 10 ml tilt measure for Gerber acid.
iv. 10.75 ml pipette.
v. Butyrometer stoppers, keys and stand
vi. Milk pipette stand
vii.Water bath maintained at 65°C+2°C.
Reagents:
i. Gerber sulphuric acid ( 90 – 91% concentration)
ii. Amyl alcohol (Specific Gravity 0.810 to 0.812)
Procedure:1
a) Preparation of Sample:
For the fresh milk sample warm it to 27°C and mix thoroughly but do not shake
it so vigorously as to cause undue frothing or churning of the fat. Repeat this
process until a homogeneous mixture is obtained. Allow the sample to stand for
three or four minutes after mixing to allow air bubbles to rise; invert the sample
bottle three or four times immediately prior to taking milk for the test.
b) Transfer 10 ml Gerber acid in butyrometer taking care not to wet the neck of
butyrometer.
c) Transfer 10.75 ml of milk sample by holding tip of the pipette laterally as deep as
possible onto the wall of butyrometer and the milk is simply layered over to the
sulfuric acid.

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d) Transfer 1 ml amyl alcohol to the butyrometer add water if required.
e) Lock the butyrometer with a stopper without mixing the two liquids.
f) Shake the butyrometer without inverting it until the contents are thoroughly mixed,
the curd is dissolved and no curd flakes are seen in the liquid. Then slant the
Butyrometer & shake it few times to mix the contents thoroughly.
g) Centrifuge the Butyrometer for 5 minutes at 1100 - 1200 rpm.
h) Place the butyrometer in water bath at 65 ± 2°C for min 3 minutes.
i) Adjust the position of fat column to bring the lower end of column on to the main
graduation mark and note down the scale reading.

Reference: IS 1479(Part-1)1960/ Mother Dairy Vashi

OR

Principle
The milk is mixed with sulphuric acid and iso-amyl alcohol in a special Gerber
tube, permitting dissolution of the protein and release of fat. The tubes are centrifuged
and the fat rising into the calibrated part of the tube is measured as a percentage of the fat
content of the milk sample. The method is suitable as a routine or screening test. It is an
empirical method and reproducible results can be obtained if procedure is followed
correctly.

Procedure:2
• Transfer 10 ml of sulphuric acid into the butyrometer by means of automatic measure
taking care not to wet the neck of the butyrometer with the sulphuric acid.
O
• Warm the sample to approximately 27 C and mix thoroughly but do not shake it so
vigorously as to cause churning of the fat. Allow the sample to stand for 3-4 minutes after
mixing to allow air bubbles to escape, invert the sample bottle 3-4 times immediately
prior to taking milk for test.
• Transfer 10.75 ml of sample into the butyrometer by using 10.75 ml milk pipette by
following the below mentioned procedure.
• Dip the tip of the pipette in the well-mixed sample and suck in the sample until the
sample rises to a short distance above the graduation mark. Close the upper end of the
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pipette and withdraw it from the sample. Wipe the outside of the delivery tube of the
pipette, hold the pipette vertically and run out the milk until the top of the milk meniscus
is on the graduation mark.
• When this achieved, insert the jet of the pipette into the neck of the butyrometer,
holding the butyrometer vertically. Touch the tip of the jet to the base of the neck of the
butyrometer and slant the pipette so that the delivery tube of the pipette rests on the top
neck. Holding the pipette in this position, release the finger from the other end of the
pipette directing the flow of the milk against the wall of the body of the butyrometer.
When emptying the pipette, take care to have a gentle flow of the milk onto the surface of
the sulphuric acid preventing as far as possible the mixing of the two liquids. When the
outflow has ceased, wait for 3 seconds, raise the pipette and then gently touch the jet of
the pipette once against the neck of the butyrometer and then remove the pipette.
• Add 1 ml of amyl alcohol into the butyrometer by means of automatic measure and
close the neck of the butyrometer firmly with the stopper without disturbing the contents.
Shake the butyrometer carefully without inverting it until the contents are thoroughly
mixed, the curd is dissolved and no white particles are seen in the liquid. Then invert the
butyrometer few times to mix the contents thoroughly.
O
• Transfer the butyrometer quickly in the water bath at 65± 2 C and leave it there for not
less than 5 minutes.
• Take out the butyrometer out of the water bath and centrifuge for 4 minutes. Bring the
centrifuge to stop gradually, transfer the butyrometers (stoppers downwards) into the
O
water bath at 65± 2 C and allow the butyrometer to stand for not less than 3 minutes and
not more than 10 minutes and take down reading.

Reference: IS-1224 (Part-I): 1997) / Mother Dairy Guideline/ DGHS Manual

Procedure for Homogenized Milk

In case of homogenized milk, repeat the temperature adjustment and centrifuging before
taking the reading. If the second value does not exceed the first value by more than half a
smallest scale division of the butyrometer, the second value shall be recorded as the fat
content of the milk.
If the second value exceeds the first value by more than half a smallest scale division,
repeat the procedure and obtain a third value for the fat content. If the third value does
not exceed the second value by more than half a smallest scale division, the third value
shall be recorded as the fat content of the milk.

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If the third value exceeds the second value by more than half a smallest scale division,
repeat the procedure and obtain fourth value for the fat content. The fourth value shall be
recorded as the fat content of the milk, but if this value exceeds the third value by more
than half a smallest scale division, it should be regarded as of doubtful accuracy.

Reference: IS-1224 (Part-I): 1997) / Mother Dairy Guideline/ DGHS Manual

6.0 PROCEDURE FOR DETERMINATION OF SNF CONTENT IN MILK.

Scope: This procedure applies for determination of SNF content in milk.

(Validated Method)

Responsibility: Chemist
Apparatus:
1. Lactometer 15 to 40 / 20 to 40 ranges at 15.5 o C
2. Lactometer Jar
3. Thermometer.
Procedure: a) Preparation of Sample:
Warm the milk sample at 40-45 ºc and maintain this temp. for 5 min. during which time
the content of the bottle are adequately mixed. Care shall be taken to avoid the formation
of air bubble or froth when mixing the sample. Cool the sample to 15.5 ºC & maintain the
same temp until the specific gravity reading is taken.
The sample bottle is gently inverted two or three time .The milk is then poured down the
side of the lactometer jar so as to avoid the formation of the air bubble .Sufficient milk
shall be poured off into the jar to assure that some of it overflows when the lactometer is
inserted The lacto meter, held by the stem, is inserted in the sample and released when it
is approximately in its position of equilibrium thus avoiding wetting more than a very
short length of the stem above the milk surface . As soon as lactometer is at rest, the scale

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reading corresponding to the top of meniscus of the milk is noted and avoid touching of
the lactometer to the jars sides.

1. Take the Lactometer Reading at 15.5 o C.

2. Use the formula: SNF = (CLR / 4) + (0.2 X Fat%) + 0.29

3. Whenever Lactometer needs to be changed, contact Head-QC

SNF = Solid-Not-Fat, CLR = Corrected Lactometer Reading, F = Fat % in Milk.

Determination of SNF in Milk (Validated Method)

The SNF is calculated by gravimetric method by determining Total Solids as described
and Fat as described
Milk Solids Not Fat % (SNF) = Total Solids – Fat %
The SNF is calculated by lactometer method.
Procedure :2
O O
• Warm the milk sample to 40 C to 45 C and maintain at this temperature for 5
minutes.
• Mix the contents by rotating and inverting the bottle, taking care to avoid the
formation of air bubbles and froth.
O
• Cool the sample to 15.5 C.
• Invert the sample bottle two or three times, pour enough milk into the lactometer jar
taking care to avoid the formation of air bubbles, so that some milk overflows when the
lactometer is inserted.
• Insert the lactometer gently to wet the stem not more than a short length, about 3 mm
beyond the position of equilibrium. The lactometer should float freely and not touch
the sides of the cylinder.

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• Allow the lactometer to remain steady in the milk. Take the reading within about 30
seconds. Note the reading of the lactometer corresponding to the top of the meniscus on
the stem without the error of parallax.

Calculation of Solids Not Fat

Formula
CLR
SNF% = -------------- + 0.2F + 0.29
4
Where
CLR = Corrected lactometer reading.
F = Fat Percentage.
Note: The estimation of Solid Not Fat in Milk by lactometer method is compared with the
gravimetric method, i.e. SNF % = Total Solids-Fat % by Gerber Method.

Reference: Mother Dairy Guideline

7.0 Determination of Total Solids in Milk: Gravimetric Method

Apparatus
Shallow flat bottomed dishes of aluminum alloy, nickel, stainless steel, porcelain
or silica, 7 to 8 cm diameter, about 1.5 cm in height and provided with easily removable
but closely fitting lids.

Procedure : 1
Weigh accurately the clean, dry empty dish with the lid. Pipette into the dish about
5 ml of the prepared sample of milk and weigh quickly with the lid on the dish. Place the
dish uncovered on a boiling water-bath. Keep the base of the dish horizontal to promote
uniform drying and protect it from direct contact with the metal of the water-bath. After
at least 30 minutes, remove the dish, wipe the bottom and transfer to a well-ventilated
O
oven at 98 to 100 C, placing the lid by the dish. The bulb of the thermometer shall be
immediately above the shelf carrying the dish. The dish shall not be placed near the walls
of the oven.
After three hours, cover the dish and immediately transfer to a desiccator. Allow
cooling for about 30 minutes and weigh. Return the dish uncovered, and the lid to the
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oven and heat for one hour. Return to the desiccator, cool weigh as before. Repeat if
necessary until the loss of weight between successive weighing does not exceed 0.5 mg.
Note the lowest weight.

Calculation
w
Total Solids, percent by weight = ------- X 100
W
Where
w = weight in gram of the residue after drying, and
W = weight in gram of the prepared sample taken for test
Remarks
O
Keep the dish along with the lid for two hour if vacuum oven (15 mmHg, at 98 to 100 C)
is used instead of oven.

Ref: IS-1479 (Part-II) 1997/ Mother Dairy Guideline

Procedure : 2
Principle
Pre drying of a test portion on a boiling water bath and subsequent evaporation of the
remaining water in a drying oven at a temperature of 102 ± 2°C
Apparatus
(a) Analytical Balance
(b) Dessicator provided with an efficient dessicant ( for example freshly dried silica
gel with a hydrometric indicator
(c) Boiling water bath provided with openings of adjustable size
(d) Drying oven, ventilated capable of being maintained thermostatically at
102 ± 2°C throughout the total working space.

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(e) Flat bottomed dishes of height 20 - 25 mm, dia 50-75 mm and made of
appropriate material (stainless steel, nickel or aluminium) provided with well
fitted readily removable lids.
(f) Water bath capable of being maintained at 35°C - 40°C
Preparation of sample
Transfer sample to a beaker, warm slowly to 35° - 40°C on a water bath with careful
mixing to incorporate any cream adhering to the sample. Cool the sample quickly to
room temperature.
Procedure
Heat a dish with its lid alongside in the drying oven at least 1 hour. Place the
lid on the dish and immediately transfer to a dessicator. Allow to cool to room
temperature (at least 30 mins) and weigh to the nearest 0.1 mg. Add 5 ml of prepared
sample, place the lid on the dish and weigh again. Place the dish without the lid on the
vigorously boiling water bath in such a way that the bottom of the dish is directly heated
by the steam. Continue heating till most of the water is removed. Remove the dish from
the water bath, wipe the underside and place it in the oven alongside the lid and dry in the
oven for 2 hours. Place the lid and transfer to the dessicator. Allow the dish to cool and
weigh to the nearest 0.1 mg. Again

Heat the dish with its lid alongside in the oven for 1 hour. Place the lid on the
dish and immediately transfer to the dessicator. Allow to cool and weigh again. Repeat
the operation again until the difference in the two consecutive weighings does not exceed
1 mg. Record the lowest mass.

Calculation

Total Solid Content = m2 - m0 × 100

m1 - m0
Where m0 = mass in gm of dish + lid
m1 = mass in gm of dish + lid and test portion
m2 = mass in gm of dish + lid and dried test portion
Round the value obtained to nearest 0.01 % (m/m)

Ref :- IS 12333 - 1997/IS 6731 : 1989 Milk, Cream and Evaporated milk. -
Determination of total Solids Content - reference method)/DGHS Manual

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8.0 Phosphatase Test for Pasteurization in Liquid Milk

Scope: This procedure applies to Phosphatase Test for Pasteurization in Liquid Milk.

Responsibility: Chemist.

Reagent
1- Buffer solution – 3.5 gm of sodium carbonate and 1.5 gm of sodium bicarbonate
dissolved in one liter of water.
2- Buffer Substrate – Transfer 0.15 g of the substrate (disodium p- nitrophenylphosphate)
in to 100 ml measuring cylinder or stoppered graduated flask and make up to the mark
with buffer solution

Procedure:-
Into a test tube pipette 5 ml of buffer substrate solution, stopper and bring the
temperature to 37°C. Add 1 ml of test milk to it shake and replace stopper. One blank
prepared from boiled milk of the same type as that undergoing the test with each series of
sample. Incubate both tubes at 37°C. Read the yellow colour after 30 minutes, return to
the bath and take second reading after incubation for further 90 minutes (total 2 hrs)
Remove the tubes after 2 hrs. and the content should be well mixed. Place the boiled milk
blank on left hand side of the comparator stand and test sample on the right. Take reading
in reflected light by revolving the disc until the test sample is matched. Record readings
falling between two standards by affixing a plus or minus sign to the figure for the
nearest standard.

Interpretation: -
Disc reading after 30 minutes incubation
0 or trace – Properly pasteurized
6 – doubtful
10 or over – Underpasteurised
Disc reading after 2 hrs incubation
0 to 10 – Properly pasteurized
Over 10- under pasteurized

Reference: IS: 1479 Part II – 1961, DGHS Manual./ Mother Dairy Guideline
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9.0 ALCOHOL TEST OF MILK.

Scope: This procedure applies for alcohol test of milk.

Responsibility: Chemist.
Reagents:
1. Ethyl Alcohol solution
(65%, 68%, 70%. V/v).

Procedure:1
Place 5ml milk in test tube and add an equal quantity of alcohol (65%), mix the content
of the test tube by inverting several times. Note any flakes or clot. The presence of flake
or clot denotes a positive test.
For better observation pour on petri dish.
If, the test is negative then go for higher strengths and proceed for step 3 onwards.

Interpretation:
Presence of flakes indicates positive results, at the tested alcohol strength.
Reference: IS 1479(Part-1)1960/ Mother Dairy Guideline

Procedure:2
• Take 5.0 ml milk in a clean Petri dish
• Add slowly 60% (v/v, sp. gr. 0.91 g/ml) ethyl alcohol from a pipette and tilt the Petri
dish slightly on a round motion
• Notice for any presence of flakes on the Petri dish
• If the milk consume more than 5.0 ml alcohol, then the stability of the milk will be
satisfactory

Reference: Mother Dairy Guideline

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10.0 SEDIMENT TEST.

Scope: This procedure applies for estimation of sediments in milk.

Responsibility:- Chemist.

Procedure:-1

Place 10 ml of well mixed sample in a graduated tube & centrifuge for 5 minutes.
Pour of the supernatant liquid, read the volume of the deposit.
Interpretation:- Normal with clean milk the amount of deposits should not exceed
0.002 %.

Reference:IS :1479(PART 2)-1961

Test for Sediment Tanker milk

Procedure:-2

• Take out 10 to 15 liters of milk in a bucket through a small hosepipe or directly from
the tanker valve.
• Filter this milk through muslin cloth or plastic sieve provided for the purpose, check
for presence of foreign matter in the milk.
• If milk contains some undesirable matter, drain out some another 10-15 lts milk and
check again. If foreign matter is found, reject the tanker.
• If no foreign matter is found, climb on the tanker, check top seals and check the
hygienic condition.

Reference: Mother Dairy Guideline

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11.0 HANSA TEST.

Scope: This procedure applies for detection of adulteration of cow milk with buffalo
milk.

Responsibility: Chemist

PROCEDURE:-
Take 2 to 3 ml of the milk sample to be tested in the test-tube label it according to a
convenient code. Add nineteen times the quantity of water to the milk so that the sample
is diluted1/20 with water. Take a pipette put a drop of mixture on the centre of the glass
slide with another pipette take out a drop of serum on the drop of the milk (without
touching milk with the pipette). Mix the two drops together thoroughly with the clean
tooth pick or glass rod. Pickup the slide & gently rock it in your hand so as to give whole
material a swirling motion. Curd particle develop in the milk Containing buffalo milk
with in half minute. If such curd particles do not develop in half minute then the test is
negative.

Reference: IS: 1479(Part 1)-1960

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12.0 METHOD FOR DETECTION OF NEUTRALIZER IN MILK.

Scope: This method applies to detection of neutralizers as type of adulterant in

milk

Responsibility: Chemist
Reagents: 1.Rosalic acid (1 % w/v alcoholic)
2. Ethyl alcohol.
Procedure:1
i. Transfer 5 ml milk in a test tube.
ii. Add 5 ml of alcohol (Ethyl alcohol).
iii. Few drops rosalic acid and mix.
Rose red/pink colour- Neutralizers have been added to milk.

Reference: IS 1479(Part-1)1960/ NDDB KIT

Procedure: 2
Take 2 ml rosolic acid (0.05% in 60% alcohol), add 2 ml of milk. Rose red colour indicate
neutralizer test positive.

Reference: Mother Dairy Guideline

Procedure: 3
Transfer 10 ml milk in a test tube add equal volume of 95% alcohol in test tube. Add few
drops of 1% alcoholic solution (W/V) rosalic acid. If alkali is present a rose red colour
appears where as pure milk shows only a brownish colour.

Reference: DGHS Manual

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13.0 METHOD FOR DETECTION OF UREA IN MILK.

Scope: This method applies to detection of Urea as type of adulterant in Milk.

Responsibility: Chemist.

Procedure: 1
i. Take 2 ml of milk in a test tube.
ii. Add 2 ml of urea reagent & mix well.
Interpretation:
Distinct yellow color indicates urea present.

Reference: NDDB KIT.

Procedure: 2
Take 2 ml milk in test tube, add 2 ml DMAB solution (1.6%) and mix the contents.
Appearance of yellow colour indicates the urea test positive.

Reference: Mother Dairy Guideline

Take 5 ml milk in test tube, add 5 ml DMAB solution (1.6%) and mix the contents.
Appearance of yellow colour indicates the urea test positive.

Reference: DGHS Manual/ IS 1479(Part-1)1960

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14.0 DETECTION OF AMMONIUM COMPOUNDS.

Scope: Test for detection of Ammonium Compounds in Milk

Responsibility: Chemist.

Apparatus:
1. Glass pipette.
2. Test Tube.
Procedure-
Take 1 ml of homogenous milk sample in test tube and add 2 ml of Nesler’s reagent.
Shake the contents. Appearances of orange colour with brownish tinge or darker intensity
(Fig I) indicates presence of ammonium compound.
Preparation of Nesler’s Reagent
Dissolve 8 g of mercuric chloride in 150 ml of distilled water, 60 g of sodium hydroxide in
150 ml of distilled water and 16 g of potassium iodide in 150 ml of distilled water to make
three solutions. Add mercuric chloride solution and sodium hydroxide solution and mix well.
Then add potassium iodide solution and make the volume up to 500 ml. Allow the solution
to stand till it becomes clear and decant to a well stoppred bottle.

Reference: Mother Dairy Guideline

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14.1 DETECTION OF AMMONIUM FERTILIZERS

i. Take 1 ml milk
ii. Add 2 ml Ammonia reagent and mix well.

Brownish colour- Ammonia Fertilizers have been to milk.

Reference: NDDB KIT.

14.2 Test for detection of Ammonium Sulphate

Take 1.0 ml of milk add 0.5 ml of 2% sodium hydroxide, 0.5 ml of 2% sodium
hypochlorite and 0.5 ml of 5% phenol solution. Heat for 20 seconds in boiling water bath,
bluish colour turns deep blue in presence of ammonium sulphate. The development of
pink colour shows that the sample is free from Ammonium sulphate.

(Ref: Milk and Milk products Vol 5 Published by N.C.E.R.T.)/ DGHS Manual)

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15.0 METHOD FOR DETECTION OF QUATERNARY AMMONIUM

COMPOUNDS IN MILK (DETERGENTS).

Responsibility: Chemist.

PROCEDURE-1
To a centrifuge tube add 1 ml milk, 5 ml water, 1 ml indicator solution and 0.2 ml buffer
and shake hard for 10 seconds. Centrifuge for 5 minutes at 3200 rpm. If QAC is present
the bottom layer assumes a red or pink colour. Samples containing about 1 mg/kg of
QAC show a faint pink colour. If the colour is deep pink or red, the amount of QAC can
be approximately determined by titration with a standard anionic detergent solution.

Reference: DGHS manual.

PROCEDURE-2

Detection of detergents in milk

Take 5 ml of milk in a test tube and add 0.1 ml of bromocresol purple solution.
Appearance of violet colour indicates the presence of detergent in milk. Unadulterated
milk samples show a faint violet colour.

Reference - Dairy for all

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16.0 PROCEDURE FOR DETECTION OF NITRATE FERTILIZERS

Responsibility: Chemist

Apparatus: Test tubes, Pipettes

Procedure:1

Pond Water Test:

Take 5 ml of milk in a Test Tube and drain it from the tube. Add 2ml (5%) Diphenyl
amine solution in the test tube. Appearance of Blue Colour indicates Pond Water Test
positive.

Reference: Mother Dairy Guideline

Procedure: 2

i. Take 1ml milk in test tube.

ii. Add 1 ml Nitrate Reagent along the side of the tube.

Appearance of purple colour ring at junction shows pond water test +ve.

Reference: NDDB Kit.

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17.0 METHOD FOR DETECTION OF CANE SUGAR IN MILK.

Scope: This method applies to detection of cane sugar as a type of adulterant content in
milk.
Responsibility: Chemist.
Procedure: 1
i. Transfer 15 ml of well-mixed milk in a test tube.
ii. Add 1.0 ml concentrated hydrochloric acid and approx. 0.1 g of resorcinol.
iii. Place the tube in the boiling water bath for five minutes.
iv. Observe for the development of red color.
Interpretation: The development of red color indicates presence of cane sugar.
Reference: IS 1479(Part-1)1960
Procedure: 2
Take 3 ml milk in a test tube; add 5 ml dilute HCL (1:2) containing 0.1% resorcinol.
Mix well and keep test tube in boiling water for 5 minutes. Brick red colour formation
indicates sugar test positive.
Reference : Mother Dairy Guideline

Procedure: 3
Reagent- Dissolve 1.0 g of resorcinol in 100 ml HCL (1:1.5), 1 volume of concentrated
HCL of sp. Gr.1.18 mixed with 1.5 volume of water.
Procedure: Crudle an aliquot of the milk by adding a little Concentrated HCL (for 25 ml
of milk usually one ml of concentrated HCL is required). Let stand for about 10 minutes
and filter. To 5ml of the modified resorcinol – HCL reagent taken in a test tube, add 1 ml
of the filtered milk serum and mix. Place the tube in boiling water bath for exactly 1 min.
Withdraw the tube and observe the colour. Appearance of deep red colour indicates
presence of sucrose or a ketose sugar.

Reference: DGHS Manual

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18.0 METHOD FOR DETECTION OF GLUCOSE IN MILK.

Scope: This method applies for detection of Glucose as type of adulterant in milk

Responsibility: Chemist.

Procedure: 1
i. Transfer 1 ml milk in a test tube.
ii. Add 1 ml Glucose reagent 1.
iii. Place in boiling waterbath for. 3 minutes & cool.
iv. Add 1 ml Glucose reagent 2.
Interpretation: Presence of deep blue color shows glucose positive sample.
Reference: NDDB kit

Procedure: 2
The sample is checked for glucose using diastix (Bayer Diagnostics India Ltd.) strip.
Open the bottle, remove one test strip and immediately replace the cap. Hold the plastic
end of the strip and do not touch the reagent green colored reagent area. Dip the reagent
area of strip in to the test solution/sample and remove immediately. Remove excess of
liquid on strip by single jerk. Compare the colour of the reagent area with the color chart
exactly after 30 seconds of wetting. If the sample shows presence of glucose (trace and
above) the sample may be rejected and maltodextrin test need not to be performed. If it
shows negative, then proceed further for maltodextrin test.

Reference: Mother Dairy Guideline

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Procedure: 3
Reagent:
1- Modified Barfords reagent: Dissolve 24 gm of copper acetate in 450ml of boiling
distilled water. Add 25 ml of 8.5% acetic acid, shake, cool to room temp. and make
upto 500ml. After sedimentation filter the reagent and stored in dark coloured bottle.
2- Phosphomolybdic acid: To 150 gm of pure molybdic acid in an erlenmeyer flask
add 75 gm of anhydrous sodium carbonate. Add 500 ml water in small portions
with shaking, heat to boiling or until all the molybdic acid has been dissolved.
Filter and add 300 ml of 85% phosphoric acid to filtrate, cool and dilute to 1 litre.
3- Acetate buffer: 1 N Sodium acetate and 1N acetic acid in equal volume having
4.75 pH.

Procedure
To 1 ml of milk sample or 1 ml of reconstituted milk powder in a test tube add equal vol
of acetate buffer and filter. To 0.2 ml of filterate add 2.8 ml water and 2 ml of reagent.
(1) Heat the tube in boiling water for 4 minutes. After cooling for 2 minutes add 3 ml of
reagent 2 and mix the contents. Development of deep blue colour indicates the presence
of glucose.

Reference: DGHS Manual

(Ref: Manual Methods of Analysis for Adulterants and Contaminants in Foods, I.C.M.R.
1990, page 28)

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19.0 DETECTION OF MALTODEXTRIN IN MILK (ENZYMATIC METHOD)

Scope: This applies to qualitative detection of maltodextrin, an adulterant in milk and
milk products.
Responsibility: Chemist.

Introduction
This is a new highly specific and sensitive method for qualitative detection of
maltodextrin, an adulterant in milk and milk products.

Maltodextrin ((C6H10O5)n, is a type of dextrin, non-sweet nutritive saccharide

polymer that consists of D-glucose units linked primarily by α -1-4 bonds and that has a
dextrose equivalent (D.E.) of less than 20.The higher the DE, the greater the extent of
starch hydrolysis.

Requirements

Stop watch

Beaker: 100ml capacity

Graduated Glass Pipettes: Capacity 20 ml, 2ml.

Water bath: Thermostatically controlled water bath maintained accurately at 62±2°C.

pH paper: pH paper strips (Merk Ltd., India) suitable for pH measurement in a range of
3.5 to 6.

Testing strip: Reagent strips, Diastix (Bayer Diagnostics India Ltd.) is used for
qualitative test for glucose incorporating a reagent area that tests specifically for glucose
and changes colour (from green to brown) if glucose is present in test solution. Results
are obtained directly from comparison with the colour chart. Each colour block
represents a range of values of the glucose content in test solution. Diastix strips should
be stored below 30°C and out of direct sunlight. It should not be stored under
refrigeration. Desiccant pouch provided in the bottle should not be removed. Strips
should not be used after six months of opening the bottle.
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Enzyme Solution (0.2g/100ml): 200 mg of enzyme is dissolved in 100 ml water and

stored under refrigeration (2-8°C). This solution should not be used for more than 15
days as loss of enzyme activity may result in misleading results.

Lactic acid solution: 10 ml of concentrated lactic acid (88 – 92%; SD Fine Chem, India)
is taken in 100 ml volumetric flask and volume is made up (100 ml) with distilled water.

Procedure

Sample preparation: 20 ml homogenous milk sample is taken in 100 ml beaker. In case

of concentrated milk, appropriate dilution was made so as to adjust total solids in a range
of 10 – 15 %. For milk powder, 10g of skim milk or 13 g of whole milk powder is
dissolved in 100 ml water (40°C) with the help of stirrer for proper reconstitution.

Test procedure: The sample is checked for glucose using diastix strip. Open the bottle,
remove one test strip and immediately replace the cap. Hold the plastic end of the strip
and do not touch the reagent green colored reagent area. Dip the reagent area of strip in to
the test solution/sample and remove immediately. Remove excess of liquid on strip by
single jerk. Compare the colour of the reagent area with the color chart exactly after 30
seconds of wetting. If the sample shows presence of glucose (trace and above) the sample
may be rejected and maltodextrin test need not to be performed. If it shows negative, then
proceed further for maltodextrin test. Thereafter pH of sample is adjusted to 4 - 4.5 with
the help of dilute lactic acid solution. Usually 0.8 to 1.2 ml of acid solution is used to
maintain pH in this range. It is then dosed with 1 ml of enzyme solution and incubated
the contents at 62±2°C for 5 minutes. Test should be repeated if the incubation time
exceeds 5 minutes. Contents are cooled at room temperature, again checked for glucose
with Diastix strip and change in color of strip (S2) is recorded.

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Inferences

• Change in color of glucose strip indicating +ve (+, ++, +++ and ++++ as depicted on
glucose testing strip bottle) after enzyme treatment (S2), indicates presence of
maltodextrin and rejected.

Reference: Mother dairy guide lines.

20.0 METHOD FOR DETECTION OF STARCH IN MILK.

Scope: This applies to determination of starch as type of adulterant in Raw Milk.

Responsibility: Chemist.

Procedure: 1
i. Transfer 3 ml of well-mixed sample of milk in a test tube.
ii. Bring it to boil by holding test tube in boiling water bath .
iii. Cool to room temperature.
iv. Add a drop of 1 % iodine solution.
Interpretation:
Observe for the appearance of blue color for the presence of starch, which
disappears on re-boiling and reappears on re-cooling.
Reference: IS 1479(Part-1)1960/ Mother dairy guide lines

Procedure: 2
i – Boil 5ml of well mixed milk
ii– Cool it and Add few drops of Starch reagent.
Blue colour – Starch and cereal flours have been added to milk.
Reference: NDDB kit/ DGHS Manual

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21.0 PROCEDURE FOR DETECTION OF SALT IN MILK.

Scope: This procedure applies for detection of salt as type of adulterant in milk.

Responsibility: Chemist
Procedure: 1
i. Take 5 ml salt reagent solution-1.
ii. Add few drops of salt reagent solution-2.
iii. Add 1 ml of milk into it, mix well.
Interpretation:
Formation of yellow color indicates presence of salt.

Reference: NDDB Kit.

Procedure: 2
Take 5 ml silver nitrate solution (0.1341%) in a test tube, add 4 – 5 drops of Potassium
Chromate (10%) in the tube and mix well. Brick Red colour will appear in the test tube.
Add 1 ml of milk. Change of colour from red to yellow indicates salt test positive.

Reference: Mother Dairy Guideline

Procedure: 3
Take 2.0 ml of milk and add 1.0 ml of 5% potassium chromate, 2.0 ml of 0.1 N silver
nitrate. Appearance of Red precipitate indicates the absence of dissolved chloride in milk
and appearance of yellow colour indicates presence of dissolved chloride.

Reference:DGHS Manual/ Mother Dairy Guideline

(Ref: Pearsons Composition and Analysis of Foods 9th edn, 1991 - Modified Mohr
method, page 14)

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22.0 DETECTION OF CELLULOSE IN MILK

Scope:-Procedure for Detection of Cellulose in Milk.

Responsibility: Chemist

Cellulose in milk gives blue colour with Iodine - Zinc Chloride reagent.

Reagent
Dissolve 20g ZnCl2 in 8.5 ml water and when cool, introduce the Iodine Solution (3 g
potassium iodide and 1.5 g iodine in 60 ml water) drop by drop until iodine begins to
precipitate.

Procedure
Take about 10 g of milk in a 100 ml beaker. Add 50 ml of hot water and stir thoroughly
for about 2 min. Pour the mixture on a nylon cloth, wash the residue with 50 ml of hot
water twice. Scrape the residue with a spatula and place it in a spotting plate. Stain a part
of residue with Iodine-Zinc Chloride Reagent and another part with Iodine Solution.
Development of blue colour in Iodine-Zinc Chloride Reagent and absence of blue colour
in Iodine Solution confirms presence of cellulose. The method is also applicable to milk
products like curd, rabri and evaporated milk.

Reference: DGHS Manual.

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23.0 DETECTION OF HYPOCHLORITES AND CHLORAMINES.

Scope: This procedure applies for Detection of Hypochlorite and Chloramines.

Responsibility: Chemist.

Reagents:-
(1) Potassium Iodide solution - Prepare fresh by dissolving 7 gm of Potassium
Iodide in 100 ml of water
(2) Dilute HCl:– To 100 ml of Concentrated Hydrochloric acid (sp gr 1.16) add
200 ml of water.
(3) Starch solution:– Boil 1 gm starch in 100 ml water. Cool before using.

Procedure-1
To 5 ml of sample in a test tube add 1.5 ml of Pot. Iodide solution, mix thoroughly and
observe colour. A yellowish brown to deep yellow colour may be formed. If unaltered,
add 4 ml of dil HCl, mix thoroughly with a glass rod flattened at one end and note colour
of curd. A yellowish brown to deep yellow colour may be formed. Next place tube in a
large water bath previously heated to 85°C and allow it to remain for 10 minutes. The
curd will rise to the surface. The liquid and the curd will have yellowish brown to deep
blue colour. Next add 0.5 to 1.0 ml of starch solution to the liquid below curd. A blue
purple colour shall be formed.

Reference: DGHS manual.

Procedure-2

1. HYPOCHLORITE TEST :

Take 5ml milk in test tube; add 1.5ml of 7% Potassium Iodide. Appearance of
yellowish brown colour indicate hypochlorite test positive. If solution is clear add 4 ml dilute
HCL (1:2) and heat the test tube at 85°C for 1 minute. Cool it and add 0.5 ml starch solution.
Appearance of blue or red purple colour indicates presence of bleaching powder.

Reference: Mother Dairy Guideline

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24.0 PROCEDURE FOR DETECTION OF FORMALIN IN MILK

Scope: This procedure applies to the detection of presence of formalin in milk.

Responsibility: Chemist
Procedure: 1
i) Take 10 ml milk in a test tube.
ii) Add 0. 5 ml, 1% Ferric chloride solution.
iii) Add 5 ml of conc. Sulfuric acid carefully down the side of the test tube so
that it
forms a layer at the bottom without mixing.
iv) Observe the colour of the ring at the junction of the two liquids.

Interpretation: Blue or violet colour at the junction of the two liquids confirms the
presence of formalin in milk.

Reference: IS 1479(Part-1)1960
Procedure: 2
1. Take 10 ml milk in test tube
2. Add 5 ml Formalin Reagent along the side of the tube

Interpretation: Blue or violet colour at the junction of the two liquids confirms the
presence of formalin in milk.
Reference: NDDB Kit.

Procedure: 3
Test for presence of Formalin in Milk (Hehner’s Test)
Take milk sample (2 ml) in a test tube and add 2 ml of 90 percent H2SO4 containing
traces of ferric chloride from the side of the test tube slowly. Formation of purple ring at
the junction indicates formaldehyde is present in milk. If sucrose is present, distil the
milk sample (25 ml) and then carry out the test on the distillate by taking 2-3 ml of
distillate and adding 2 ml of formaldehyde free milk. The violet coloration does not
appear usually when relatively large quantities of formaldehyde are present. The milk
supplies the tryptophane that must be present for the test to operate.
(Ref :- DGHS/Pearson’s Composition and Analysis of foods, 9th edition, 1991 page 90)

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MDV-TM-03 TESTING MANUAL

25.0 PROCEDURE FOR DETECTION OF HYDROGEN PEROXIDE IN MILK

Scope: This procedure applies to the detection of presence of hydrogen peroxide in

milk.

Responsibility: Chemist

Procedure:1

1. Put 1ml of milk in a tube.

2. Add few drops Hydrogen peroxide reagent & mix well.
Pink/red colour indicates hydrogen peroxide has been added to milk.

Reference : NDDB Kit.

Procedure: 2

Take 5 ml milk in a test tube and add 2 drops of paraphenyldiamine hydrochloride (1%).
Formation of blue colour indicates hydrogen peroxide test positive.

Reference: Mother Dairy Guideline

Procedure: 3

Dissolve 1 gm V2O5 in 100 ml of 6% H2SO4 (6+94). To 10 ml of sample add 10-20

drops of reagent and mix. The development of pink or red colour indicates presence of
H2O2

(Ref : DGHS/A.O.A.C 17th edn, 2000 Official Method 957.08 Hydrogen Peroxide in
milk)

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26.0 TEST FOR PRESENCE OF SALICYLIC ACID.

Scope: This procedure applies for detection of Salicylic acid.

Responsibility: Chemist.

Procedure:
Place 50 ml (100 ml –By IS) of sample in a separating funnel. Add 5 ml of dilute HCl
(1+3) and extract with 50 ml ether, If mixture emulsifies add 10 - 15 ml Pet. Ether and
shake. If this treatment fails to break emulsion centrifuge or let stand until considerable
portion of aqueous layer separates, drain latter, shake vigorously and again let separate.
Wash ether layer with two 5 ml portions of water, evaporate ether in a porcelain dish and
add 1 drop of 0.5 % (v/v) neutral Ferric Chloride solution. A violet colour indicates
presence of Salicylic acid.

Reference: DGHS manual./IS1479(Part I)

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27.0 TEST FOR PRESENCE OF BORIC ACID AND BORATES.

Scope: This procedure applies for detection of Boric acid and Borates.

Responsibility: Chemist.

Reagents:
1. Turmeric paper dried
2. Conc. Hydrochloric acid - sp. Gr. 1.16
3. Ammonium Hydroxide
4. Lime water or caustic soda

Procedure:1 - Either of the methods given at (a) or (b) below shall be followed.
a) Immerse a strip of turmeric paper in a sample of milk previously acidified with Conc.
Hydrochloric acid to each 100ml of milk. Allow the paper to dry spontaneously. If boric
acid or borax is present, the paper will acquire a characteristic red colour. The addition of
the ammonium hydroxide will change the colour of paper to a dark green, but the red
colour may be restored by hydrochloric acid.

b) Make about 25 ml sample strongly alkaline with lime water or Sod. Hydroxide and
evaporate to dryness on a water bath. Ignite the residue on a low red heat to destroy
organic matter. Cool, digest with about 25 ml water, add conc. HCL drop by drop until
the ignited residue is dissolved. Add approx 1 ml in excess. Saturate a piece of turmeric
paper with this solution and allow it to dry. The paper would show red colour in the
presence of boric acid or borate.

Reference: IS 1479(Part-II) 1961/DGHS Manual

Procedure: 2
Test Protocol (Glycerol Method)
• Take 10 ml milk in a 50 ml beaker
• Add 1 ml 1% phenolphthalein indicator
• Titrate against N/9 NaOH soln until pink colour persist for 0-15 seconds
• Add 3-4 drops of N/9 NaOH. The milk will be pinkish in colour
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• Divide the sample in two parts in test tubes
• In one test tube, add some volume (~2.0 ml) of distilled water and in
another test tube,
add 50% Glycerol (~2.0 ml)
• Compare the colour of Glycerol added test tube with the water added test
tube

Inference
The test tube added with Glycerol will be faded or pink colour will disappear indicate the
presence of Boric acid= Boric acid Positive
Detection Level: 0.02% (200 ppm)

Reference: Mother Dairy Guideline

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28.0 TEST FOR PRESENCE OF DULCIN.

Scope: This procedure applies for detection of presence of Dulcin.

Responsibility: Chemist

Procedure:-
Extract 25 ml of milk (made alkaline with 10% NaOH) with 90 ml of diethyl ether in 3
portions. Evaporate ether on a boiling water bath. Moisten dry residue with nitric acid
and add one drop of water. Presence of dulcin is indicated by orange red colour
precipitate.
1. Alternatively expose dry residue to HCl gas for 5 minutes and add 1 drop of
anisaldehyde. Presence of Dulcin is indicated by orange red to blood red colour.

Reference: DGHS manual.

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29.0 TEST FOR PRESENCE OF SACCHARIN.

Scope: This procedure applies for presence of Saccharin.

Responsibility: Chemist.

PROCEDURE:-
Curdle an aliquot of the diluted milk sample (about 25 ml) with dilute acetic acid. Shake
well and filter. Acidify the clear filtrate with 2.0 ml of concentrated hydrochloric acid,
and extract with two 25ml portions of diethyl ether. Draw off the aqueous layers and
wash the combined ether extract with three successive portions of 5 ml of water,
evaporate the ether extract on a water bath, add a drop or two of water, mix well with
glass rod and taste a little. A characteristic sweet taste indicates the presence of saccharin.
Confirm by heating with NaOH and detecting Salicylic acid from thereby.

Reference: DGHS manual.

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30.0 MINERAL OIL TEST (HOLDE’S TEST) FOR GHEE

Scope: This procedure applies for detection of presence of Mineral oil.

Responsibility: Chemist

Procedure:-
Weigh 1g ghee in a clean dry 250 ml conical flask, add 22 ml of alcoholic KOH solution,
and heat it on a water bath or hot plate until entire ghee is saponified. Add about 25 ml
of boiling distilled water and swirl the flask. Observe for turbidity.

Clear solution means ghee is free from any mineral oil, whereas turbidity indicates ghee
is adulterated with mineral oil.

(Prepare alcoholic KOH solution by dissolving 7g KOH pellets in about 10 ml distilled

water and make up the volume to 250 ml with absolute alcohol)

Reference: DGHS manual / Mother Dairy Guideline

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MDV-TM-03 TESTING MANUAL

31.0 METHOD FOR DETECTION OF BUTYRO-REFRACTOMETER READING

FOR GHEE

Scope: This applies to determination B.R reading of ghee.

Responsibility: Chemist.

Keep the Butyrorefractometer ready with the circulating water bath maintained at 40 o C.
Clean the cell of the Butyrorefractometer with alcohol and tissue paper, followed by
distilled water.

Prepare ghee from Full Cream Milk.

For homogenized milk, do the fat test in open-ended butyrometer and collect the fat from
the fat column by means of a syringe.

Place a drop of clear ghee in the cell of the Butyrorefractometer. Adjust the focus and
read the oil scale at the junction of light and shadow.

a) For un-homogenized milk: BR reading = BR reading @ 40 o C.

b) For homogenized milk: BR reading = (BR reading @ 40 o C) X 1.08

Apply following temperature correction, if the temperature of observation is other than

40 o C:

BR reading 40 = BR reading T – [(40-T) X 0.55]

(Where BR reading T is, the BR reading observed at temperature T o C)

(BR Reading shall be 41.5 45.0 in cotton tract areas and from 40.0 to 43.0 in other areas
of Maharashtra State)

Reference: IS 1479(Part-1)1960/ Mother Dairy Guideline

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32.0 DETERMINATION OF RM VALUE OF GHEE:

Scope: This procedure applies for detection of R M value of Ghee.

Responsibility: Chemist.

PROCEDURE:-
Weight exactly 5± 0.01 g of ghee extracted from milk into a polenske flask at 20 g of
glycerol and 2 ml of 50% NaoH solution. Heat the Flask over a flame with mixing until
complete saponification takes place. Cover the flask with a watch glass.

Cool the flask, add 93 ml boiling distilled water, some pieces of beads and 50 ml of dilute
sulphuric acid ( 1N ). Connect the flask to condenser. Heat the flask without boiling its
contents until the insoluble acids are completely melted, then collect 110 ml distillate
between 19 to 21 minutes. Close flask with stopper and place it in water at 15OC for 10
minutes. Mix the distillate and filter using filter paper No.4. Reject the first few
droppings and collect 100 ml in a volumetric flask. To this add 0.1 ml of phenolphthalein
and titrate with 0.1 N sodium hydroxide to its end point. Carry out the blank also.

RM Value = 1.10 X (T1 – T2).

T1 = Volume of sodium hydroxide used (0.1 N)
T2 = Volume of sodium hydroxide used (0.1 N in blank)

Reference: Manual in Dairy Chemistry (ICAR)

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33.0 METHOD OF DETECTION OF KEEPING QUALITY OF MILK AT 37 O C.

Scope: This procedure applies for determination of keeping quality at 37ºC.

Responsibility: Chemist.

KEEPING QUALITY AT 37 O C

Test the milk sample for acidity; record it in the register with date, time, and sample
description.

Incubator temperature shall be maintained at 37 O C before incubating the sample.

Carefully transfer a portion of the same milk sample (about 50 ml) in to a clean, sterile
bottle and label it and Incubate at 37 O C. Test a small portion of the sample from this
bottle every one hour, after a lapse of first five hours, for COB until the sample turns
COB positive. Test the sample for acidity when it turns COB positive. Record the
Keeping Quality (KQ) as the number of hours until it was COB negative.

A minimum KQ of seven (7) hours is desired for pasteurized milk samples.

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34.0 METHOD OF DETECTION OF HOMOGENIZATION EFFICIENCY (QUICK

TEST ) & (48 HRS TEST).

Scope: This applies to determination H. E. (Quick test) & (48 HRS Test).

Responsibility: Chemist.

Procedure:
(This test is to be carried out for Homogenized Milk Variant of incoming tanker milk
samples only)

a) Cream Separation by Centrifugation: -

Take about 15 ml of milk sample in each test tube, and carefully balance them in the
centrifuge. Centrifuge for 3 minutes and remove the tubes carefully without shaking or
disturbing the contents. Invert the tubes gently over the sink and observe for “cream
plug”, if any.

Formation of cream plug or sufficient cream layer indicates improper homogenization.

b) Microscopic Examination: -
Dilute the milk sample about 5 times with distilled water. Place a drop of diluted milk
sample on a clean slide and put a cover slip without formation of air bubble. Observe the
slide under oil immersion nosepiece of microscope, with dim light so that the fat globules
appear shiny. Presence of large and uneven sized fat globules indicates improper
homogenization.

HOMOGENIZATION EFFICIENCY (48 HOUR HE)

(This test is to be carried out for Homogenized Milk Variant of packed milk samples
only)

Carefully pour 500 ml of homogenized milk variant from one pouch into a clean dry
plastic bottle, close it firmly with the cap, and label it with pouch code, and the date &
shift in which it shall be tested (48 hours from date & shift of placing in the refrigerator).
Keep the bottle in the refrigerator in such a manner that it shall not disturbed until it is
removed for testing.
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After lapse of 48 hours, carefully remove the bottle without shaking. Open the bottle and
place it on an elevated platform. By means of a thin plastic tube, siphon out 450 ml of
milk from the bottom most part of the bottle in to a clean dry beaker. Mark the beaker as
B.

Warm the bottle with the remaining portion of milk in a water bath, and do the Fat test
for the 450 ml portion collected in the beaker (B) and the remaining portion in the bottle
(A).

Calculate the Homogenization Efficiency (HE) as follows:

HE = (Fat% of Portion A – Fat% of Portion B) X 3

HE up to a maximum of five (5) indicates proper homogenization of milk.

Reference: IS 1479(Part-1)1960

Pateela Test for Milk

Take about 500 ml in a pateela and boil it as done in household use on a gas stove
observe for the oiling off after little cooling and afterwards transfer the milk into
another vessel and notice for the sediment sticking at bottom. Normal milk should
have marginal sediment sticking at bottom and no oiling off.

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35.0 TEST FOR CHECKING THE ASH & ALKALINITY OF ASH.

Scope: This procedure applies for detection of ash & alkalinity of ash.

Responsibility:- Chemist.

Procedure:-For Ash

Neutralization with lime water/sodium bicarbonate/caustic soda increases ash content and
alkalinity of ash.

Take 20 ml of milk in a silica dish, evaporate on a water bath and keep in muffle furnace
at 550°C to get grey ash formation. Switch off the muffle furnace and let the temperature
fall. Transfer the crucible to a desiccator, cool completely and weight. Record the lowest
mass.
Calculate the ash percentage as fallows
Where
M2 = Weight of crucible with ash
M = Weight of empty crucible.
M1 = Weight of empty crucible with milk.
B- Ash% on dry matter basis = Ash % in milk x 95.84
% SNF in milk
Reference: Mother Dairy Guideline

Procedure:- Alkalinity of ash

Take 20 ml of milk in a silica dish, evaporate on a water bath and keep in muffle furnace
at 550°C to get white ash. Dissolve the ash obtained in 10 ml of water and titrate with 0.1
N HCl. The titre of more than 1.2 ml indicates the presence of neutralizers in milk.

Reference: DGHS Manual.

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36.DETERMINATION OF PROTEIN IN MILK

1- Kjeldahl Method
Apparatus
1. Kjeldahl digestion flask - 500 or 800 ml
2. Kjeldahl distillation apparatus, - same digestion flask fitted with rubber stopper
through which passes lower end of efficient rubber bulb or trap to prevent
mechanical
carry over of NaOH during distillation.
3. Conical flask, 250 ml
4. Burette 50 ml.

Reagents
1. Concentrated Sulphuric acid – sp. gr. 1.84
2. Sodium Hydroxide solution - 50%. Dissolve 500 gm of Sodium Hydroxide in 1000 ml
water
3. Standard Sulphuric acid solution – 0.1 N
4. Standard Sodium Hydroxide solution – 0.1 N
5. Methyl Red Indicator solution - Dissolve 0.5 gm methyl red in 100 ml of alcohol

Procedure
Weigh quickly about 10 g of the prepared milk sample and transfer to a 500 or 800 ml
Kjeldahl flask taking care to see that no portion of the sample clings to the neck of the
flask. Add around 10 g digestion mixtures containing copper sulphate and potassium
sulphate in 1:50 ratio and 25 ml of concentrated sulphuric acid. Add two to three glass
beads. Place the flask in an inclined position on the stand in the digestion chamber and
digest. Heat the flask gently at low flame until the initial frothing ceases and the mixture
boils steadily at a moderate rate. During heating rotate the flask several times. Continue
heating for about an hour or more until the colour of the digest is pale blue. Cool the
digest and add slowly 200 ml of water. Cool, add a piece of granulated Zinc or anti bump
granules and carefully pour down the side of the flask sufficient Sodium Hydroxide
solution (50 ml of 50%) to make the contents strongly alkaline before mixing the acid
and alkaline layer. Connect the flask to a distillation apparatus incorporating an efficient
flash head and condenser. To the condenser fit a delivery tube, which dips just below the
surface of the pipetted volume of standard acid (0.1 N Sulphuric acid soln) contained in a
conical flask receiver. Mix the contents of the digestion flask and boil until 150 ml have
distilled into the receiver. Add 5 drops of methyl red indicator and titrate with 0.1 N

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Sodium Hydroxide solution. Carry out a blank titration using the same quantity of AR
Grade sucrose.
Per cent Protein in the sample on SNF basis=89.36466 (V-B)/ (W× 95.50*)
Where
V= vol in ml of 0.1 N sodium hydroxide
B= vol in ml of 0.1 N sodium hydroxide for blank
W= Weight in gram of sample taken
* % SNF in SMP has been considered is 95.50.

Ref: Pearson’s Composition and Analysis of Foods, 9th edn, 1991 page 17)

2- Using Pelicon/Biokjel Apparatus- Validated Method

The apparatus has 3 units namely:
1. Digestion unit
2. Distillation unit
3. Titration
Digestion
• Flow of water should be checked before starting the digestion unit.
Exhaust fan of the
hood should be switched on
• The digestion unit must be switched on by pressing the power button so that the
temperature reaches 420°C
• Weigh around 2g milk samples directly into the digestion tube
• Now place these tubes in Kjeldahl stand
• Add 5g of catalyst mix which is cupric sulphate and potassium sulphate (10g+50g)
• Add 10 ml of 98% conc H2SO4 in each of the Kjeldahl tubes with auto pipette and shake
the tubes
0
• When the temperature is reached to 420 C, then put the stand in the digestion unit and
cover the top and leave it for 1.5 hours
• Water supply should be checked continuously, it should be properly connected to the
digestion system. The lids of the Kjeldahl tubes should be properly placed
• Leave the tubes in the block for 1.5 hours, after 1.5 hours ensure the colour of samples
turns into bluish green, if not then replace the tubes in block for sometime more

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• Once the bluish green colour appears, remove the tubes and place them in cooling stand
• Keep them in the cooling stand until the fumes disappear and the colour becomes
crystal clear.

Distillation
• Now switch on the distillation system by first pressing the main switch and green
remote button, which is present at the back, then power button
• Check the water supply in the distillation unit
• Remove the tubes from distilled water; dip it in 40% NaOH (w/v)
• Two times: set alkali for 25 sec and run for flushing
• Run the process once without sample to make the apparatus ready
• Set alkali for 5 seconds 4 times
• Set distillation time 9 minutes
• Remove previous distillation tube and conical flask
• Place distillation tube containing digested sample in tube chamber and a 250 ml
conical flask containing 25 ml boric acid and add six drops of mixed indicator
(methyl red + bromocresol green in ratio 2:1) another side
• Press dilution 2-3 times for approximately 5 seconds
• Press alkali for 5 seconds 4 times till black colour appears by addition of alkali
(NaOH)
• Run distillation for 9 minutes
Titration
• Titrate distilled solution against 0.1 N HCl (standardised)
• Once the colour changes from bluish green to permanent Violet, note the burette
reading that is titer value
• Now do the calculation for finding % protein by multiplying % nitrogen with
conversion factor (6.38)
• Titer value of blank titration is subtracted from the titer value of the samples
• After distillation make the alkali addition time and distillation time display zero.
• Put the pipeline from NAOH into distilled water
• Then for 40 seconds approx that is 20 seconds for two times flushing is done.
• Press the process button and set the time for 5 minutes for cleaning
• Lastly switch off the power button then green remote button then main switch.
• Close the water supply.

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Calculation
% protein in milk on SNF basis =89.36466*(V-Blank)/ (Weight of the sample×% SNF in
milk)
Where
V= vol in ml of 0.1 N HCl for blank

3- Pynes Method
Reagents
• Phenolphthalein indicator solution: Dissolve 0.5 gm of phenolphthalein in 100 ml
of ethyl alcohol (50% of volume in distilled water)
• Potassium Oxalate solution: Saturated solution
• Formaldehyde: 40% concentrated and neutral to phenolphthalein
• Standard NaOH solution: N/10

Procedure
• Take 10 ml of milk in a conical flask
• Add 1 ml of phenolphthalein indicator solution to it followed by 0.4 ml saturated
potassium oxalate solution and set it aside for 2 minutes
• Neutralize the contents of the flask with standard N/10 NaOH solution to a pink
coloured end point
• Add 2 ml neutralized formaldehyde and again titrate with standard NaOH solution
to the same pink shade. Note the volume of NaOH solution used multiply this
value by 1.7 to obtain value of protein per cent

Reference: Mother Dairy Guideline

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37. DETERMINATION OF SODIUM IN MILK

1- Using Flame Photometer (Systronics)
Preparation of sample: Prepare a dilution of the milk to be tested in such a way that the
output comes within the range of the machine.

Preparation of working solution and milk sample

Note: All solutions to be prepared in volumetric flask.
Stock solution of 1000 ppm for sodium: Dissolve 2.5416 gm NaCl in 1 litre glass distilled
water
Stock solution of 1000 ppm for potassium: Dissolve 1.9070 gm KCl in 1 litre of glass
distilled water
Working solution: Take 1 ml. of stock solution separately for sodium and potassium in
100 ml. volumetric flasks and make the volume to 100 ml by adding glass-distilled water.
Operational Procedure
Flame Photometer is having three units:
♦ Compressor Unit
♦ Flame photometer Burner
♦ Digital F.P.M.

Connect all the three units as per instructions given by the supplier.
Attach the cooking gas cylinder to the flame photometer carefully.
2
Switch on the compressor and adjust the pressure to 0.5 kg / cm by rotating air knob,
which is fixed on the photometer.
Open the lid of the burner and release the cooking gas simultaneously and ignite the
burner and adjust the flame by rotating the knob "Gas" till the clear blue zone flame
comes and close the lid of burner.
+ +
• Select the desired filter Na or K that is fixed on the left side of the flame
photometer.
• Switch on the Digital F.P.M. Unit. It will display some digits on screen.
• Adjust the zero by using the knob "Set Zero" of the F.P.M. unit.
• Put a beaker of glass-distilled water below the siphon tube of the flame photometer
and again adjust zero. After zero adjustment, remove the distilled water beaker.
+ +
• Put the siphon tube in desired Na or K working solution and adjust 100%
transmittance by selecting LO or MD or HI Sensitivity Knobs whichever is near to 100.
Adjust 100% by rotating set zero"coarse knob".

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• Instrument is ready to use. Put the prepared sample for analysis. Before testing the
+ +
sample confirm that the required filter of Na or K is selected. Reading on Digital FPM
+ +
corresponds to mg/1000 ml (ppm) of Na or K in milk.
Caution: Sample should be free from churned fat, dirt, dust and other foreign matter.
• After completion of testing first close the valve of cooking gas, open the lid of the
burner and let the gas inside the tube ignite till it is completely burnt.
• Switch off the compressor.
• Switch off of the digital F.P.M.

2- Using Flame Photometer(ALICO INDUSTRIAL EQUIPMENTS/ Electronic India)

Use Flame Photometer to determine the Na content (ppm) in milk. Calculate the result on
8.5% SNF Basis. Follow the procedure as stated for raw milk below.

Procedure
• Determine the SNF of milk, say it is X%
• To a clean, dry 50 ml beaker, add 25 ml of double distilled water and add
accurately 0.50 ml milk sample and mix well
• Calibrate the instrument using standard solutions of Na
• Do the analysis as per the test protocol mentioned in the manual provided by the
supplier
• When the reading has stabilized, record the reading of Na content

Calculation
Reading×Dilution factor
ppm Na in milk sample= -----------------------------×8.50
% SNF in milk

3- Using Ion Meter

• To a clean, dry 50 ml beaker, add 25 ml of sample and add accurately 0.50 ml ISA
• Sure that the meter is in the concentration mode
• Place the beaker on the magnetic stirrer and begin stirring
• After rinsing the electrode tips with electrode rinse solution, blot dry and place the
electrode tips in the solution
• When the reading has stabilized, record the ppm of Na/K

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Standardization of the Equipments

• The calibration should be checked every two hours
• Adjust the meter to the concentration of the sodium/potassium standard and fix the
value in the memory according to the meter manufacturer’s instructions after
stabilization of reading
• Then, rinse the electrode with electrode rinse solution and blot dry
• Place the more concentrated solution on the magnetic stirrer and begin stirring at a
constant rate
• Lower the electrode tips into the solution
• Adjust the meter to the concentration of the sodium/potassium standard and fix the
value in the memory according to the meter manufacturer’s instructions after
stabilization of reading
• Then, rinse the electrode with electrode rinse solution and blot dry
• For low-level measurements, place the rinsed, dried electrodes into a solution
containing 25 ml of distilled water and 0.50 ml of ISA. After stabilization, fix the blank
value in the meter according to the meter manufacturer’s instructions
• Place 25 ml sample and 0.50 ml ISA in a 50 ml beaker. Place the beaker on the
magnetic stirrer, and begin stirring
• After rinsing the electrodes and blotting dry, place the electrode tips in the solution
and wait for the reading to stabilize. Read the concentration directly from the meter
display

Reference: Mother Dairy Guideline

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MICROBIAL ANALYSIS

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38.0 MICROBIOLOGICAL ANALYSIS LIQUID MILK

I- MBR TEST:

Scope: This procedure applies for detection of MBRT time of milk.

Responsibility: Chemist.

PROCEDURE:-
Take 10 ml milk in a sterilized tube, add 1 ml MBR dye solution, plug with a
sterilized cork and invert the tube to mix contents and incubate in a water bath at
37°C. Check the tube for depolarization first after 10 minutes, next after 30 minutes
and subsequently after every hour. MBRT is counted after deleting initial 30
minutes of incubation.

Recording of Results- During incubation observe colour changes as fallows.

a) If any sample is decolourized on incubation for 30 minutes, record the reduction
time as MBRT-30 minutes.
b) Record such readings as reduction times in whole hours. For example, if the colour
disappears between 0.5 to 1.5 hour readings, record the result as MBRT-1 hour;
similarly, if between 1.5 and 2.5 hours as MBRT-2 hour and so on.
c) Immediately after each reading, remove and record all the decolourized samples and
then gently invert the remaining tubes if the decolourization has not yet begun.

NOTE- Because of uneven disappearance of blue colour, that may be noted at the final
reading time, record the sample as decolourized when only a faint blue ring (about
5mm) persists at the top.

Reference: Reference: IS 1479(Part-1)1960/ Mother Dairy Guideline

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II) ANALYSIS OF STANDARD PLATE COUNT & COLIFORM COUNT

Purpose:
To test the liquid milk and report the findings for
1. Standard plate count
2. Coliform count

A. Equipments and materials

1. Water bath.
2. Autoclave
3. Hot Air Oven
4. Sampling bottles
5. Auto pipette with tips
6. Petri plate
7. Scissor
8. Laminar Air Flow
9. Test tubes
10. Durhams tube
11. Thermometer
12. Incubator, 37 +/- 1°C
13. Weigh Balance
14. Sterile graduated pipettes, 1.0 and 10.0 ml
15. Sterile utensils for sample handling
16. Refrigerator
17. Colony counter
18. pH meter/pH strip
19. Test tube rack
20. Permanent Marker pen

Composition & preparation of Media:

A) Plate Count Agar for S.P.C. (Ref. : IS: 5402 : 2002 ) :
Ingredients:
1) Tryptone 5.0 g
2) Dehydrated yeast extract 2.5g
3) Anhydrous glucose 1.0g
4) Agar 12g to 18g
5) Water 1000ml
6) Final pH 7.0 ± 0.1

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Preparation :

Suspend 23.5 grams Plate Count Agar ( Hi-Media : M091) in 1000ml distilled water.
Heat to boiling to dissolve the medium completely and fill it in flasks or bottles. Sterilize
by autoclaving at 15 psi pressure ( 121°C ) for 15 minutes.

At the time of use completely melt the medium , then cool it in water bath set at 45°C.

B) Violet Red Bile Agar ( VRBA ) for Colifom ( Ref. : IS: 5401
( Part 1 ) : 2002

Ingredients:

1) Peptone 7.0g
2) Yeast Extract 3.0g
3) Lactose 10.0g
4) Bile salts 1.5g
5) Sodium chloride 5.0g
6) Neutral red 0.03g
7) Crystal violet 0.002g
8) Agar 15g
9) Final pH 7.4 ± 0.1

Preparation :

Suspend 41.53 grams of Violet Red Bile Agar (Hi-Media: M049) in 1000ml distilled
water. Sterilize the media as per instructions given by the manufacturer.
Cool it in water bath set at 45°C and pour into sterile Petri plates.
C) Brilliant Green Bile Broth 2% for PCT
Ingredients:
1) Peptone 10.0g
2) Ox bile 20.0g
3) Lactose 10.0g
4) Brilliant green 0.0133g
5) Final pH 7.2 ± 0.1

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Preparation :

Suspend 40.00 grams of Brilliant Green Bile Broth 2% (Hi-Media : M121S ) in 1000ml
distilled water. Heat to boiling to dissolve the medium completely and distribute in test
tubes containing inverted Durham’s tubes. Cap the tubes. Sterilize by autoclaving at 15
psi pressure ( 121°C ) for 15 minutes.

Preparation of Diluent:

Phosphate Buffer:

Preparation of Stock Phosphate Buffer:

Dissolve 34 gm of Potassium Dihydrogen Phosphate (KH2PO4) in 500 ml distilled water.

Adjust pH 7.2 with 1N Sodium Hydroxide.
Make-up to one liter volume with distilled water.

Preparation of batch dilution blanks:

Take 1.25 ml of Stock Phosphate Buffer solution & make-up its volume to one liter with
distilled water.
Fill it in dilution tubes so that after sterilization each tube will contain 9 ml.
Sterilize by autoclaving at 15 psi pressure (121°C ) for 15-20 minutes.

Selection of Dilutions:

For routine testing select dilutions of samples so that total colonies on at least one plate
will be between 30 & 300. For unknown samples use two or more dilution.

STANDARD PLATE COUNT:

Materials Required :
1. Petri plates
2. Plate count agar
3. Auto pipette 1.0 ml
4. Dilution tubes containing 9 ml of phosphate buffer solution.

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Procedure :
1. Transfer 1 ml of well mixed sample to 9 ml diluent. Mix well. Transfer 1 ml of
this suspension to second tube to make second dilution. Similarly make third and/
or fourth dilution as per requirement.
2. Arrange 2 petri plates for each dilution to be tested, mark them with sample No.
and date.
3. Transfer 1 ml in each plate from respective dilution of sample to be tested.
4. Pour 10 to 15 ml of 45ºC plate count agar in each petriplate and mix well.
5. Allow the agar to set, invert and incubate the plates at 37 ± 1ºC for 48 hours.
6. At the end of 48 hours remove the plate from the incubator and count the colonies.
7. Also prepare control plate to check the sterility of the media.

Counting & Expression of results:

Count the colonies grown on Petri plates. Count only those plates, which have 30-300
colonies.
Count colonies developed on each plate for respective dilution. Multiply colonies per
plate by dilution used & report the arithmetic average as plate count per ml.

Record & report the result as Standard Plate Count/ ml. ----------- Or SPC/ml. -------------

COLI FORMS: -

a) Plating Method: -

Materials Required:
1. Petri plates
2. Violet red bile agar
3. Auto pipette 1.0 ml
4. Dilution tubes containing 9 ml of phosphate buffer solution.

Procedure:
1. Transfer 1 ml of well mixed sample to 9 ml diluent & mix well. If required,
transfer 1 ml of this suspension to second tube to make second dilution.
2. Arrange 2 petri plates for each dilution to be tested, mark them with sample No.
and date.

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3. Transfer 1 ml in each plate from respective dilution of sample to be tested.
4. Pour 10 to 15 ml of 45ºC Violet red bile count agar in each petriplate and mix well.
5. Allow the agar to solidify and pour approximately 5 ml of the media overlay the
surface of the solidified medium and allow to solidify again.
6. Invert plates and incubate the plates at 37 ± 1ºC for 18 to 48 hours.

Counting & Expression of results:

Count dark red color colonies measuring at least 0.5mm in diameter.

Count dark red with red precipitate colonies measuring at least 0.5 mm in diameter in
each plate for each dilution plated. Multiply colonies per plate by dilution used & report
the arithmetic average as coliform count per ml. of product.

Record & report the result as Coliform Count/ ml. -----------

b) Presumptive Coliform Test (PCT)

Materials Required:

Auto pipette with tips / Pipettes of 2.2 ml

Brilliant green bile broth 2% in tubes.

Procedure:

1. Transfer 1 ml from first dilution of sample into Brilliant green bile broth 2% tube
in triplicate. Incubate for 48 hours at 37 ± 10C and observe for acid & gas
production.
2. Production of gas & opacity in at least two tubes out of three constitutes the
positive presumptive test.

If PCT is positive and simultaneously typical colonies observed on solid media. This
confirms the presence of Coliforms in the sample. If required further confirmation may
be done using streak techniques on EMB agar or Microscopically.

Ref.IS: 5402:2002

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39. ASSESING STERILITY OF MILK EQUIPMENT

Materials Required:
1. Sterile cotton swab
2. Phosphate buffer/Saline

Swabbing & Testing swab:

1. Press the sterilized tube by rolling motion against the side of glass tube removing
excess liquid and take it out of the tube.
2. Rub the swab with pressure back and forth over the area to be examined and return
the swab to the buffer tube. For plain surface 900 cm2 area to be covered.
3. Let the swab in buffer solution for 5 mins. Then mix well by vigorous shaking.
4. Using 9 ml blank prepare 1st dilution and proceed as per standard plate count.

Recording Results:
Count the number of colonies and multiply dilution factor & area factor by using
following formula

= Number of colonies X Dilution factor X Area factor X 900 cm2

900 cm2

Interpretation of Results:

Standard plate count/ 900 cm2

Coliform count / 900 cm2

Ref. IS: 1479 (Part V): 1962

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40.0 MICROBIOLOGICAL ANALYSIS OF WATER

STANDARD PLATE COUNT: -

Materials Required:
Refer to SPC of milk sample.

Sampling:
Samples shall be collected in clean, sterilize & narrow mouthed neutral glass bottles of
250, 500 or 1000ml capacity.

Dechlorination:
If the water to be sampled contains or is likely to contain chlorine, Sodium thiosulphate
shall be added to the clean, dry sampling bottles before sterilization in an amount to
provide an approximate concentration of 100mg / lit. in the sample. This can be done by
adding 0.5 ml of 5% thiosulphate solution to a 250 ml bottle. Sterilize by autoclaving at
15 psi pressure (121°C) for 15-20 minutes.

Sampling from taps:

The tap should be kept open fully & water is allowed to run for 2-3 min. or for a
sufficient time to permit clearing of the service line. The flow from the tap shall then be
restricted to permit filling the bottle without splashing.
The volume of the sample shall be sufficient for carrying out all the tests required. The
bottles should be filled in such a way that 2 to 3 cm space should be left for effective
shaking of the bottle.
The initial time limit for starting analysis should be one hour but not more than six hours
after collection of water samples.

Procedure:

Refer to SPC procedure of milk sample.

Counting & Expression of results:

Count the colonies grown on Petri plates. Count only those plates, which have 30-300
colonies.Count colonies developed on each plate for respective dilution. Multiply
colonies per plate by dilution used & report the arithmetic average as plate count per ml.

Record & report the result as Standard Plate Count/ ml. --------- Or SPC/ml. -------------

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COLI FORMS (Raw Water, Treated Water, CIP Final Rince Water ): -

Principle-
Membrane filters are thin porous sheets of inert material, containing pores of varying
size, small enough to prevent passage of microorganisms. During filtration,
microorganisms present are retained on the filter. Incubation with proper media, for
proper time and at correct temperature allows microorganisms to grow into colonies.

Equipment:

Autoclave, Refrigerator, Hot Air Oven, Laminar Air Flow, Incubator, Colony counter,
Membrane Filtration Assembly & Filters

Glassware:

Sterile Petri plates, Forceps, Sterile sampling Bottles

Media, Chemicals & other requirements:

VRB Agar ( Hi –media Code : M049), Phosphate Buffer, 70% Alcohol, Absorbent
cotton,

Media Preparation:

Sterilize required glassware with dry heat in a hot air oven at 170°C for 2 hours.
Sterilize the media as per instructions given by the manufacturer. After sterilization, store
the media in cold condition.

Phosphate Buffer:

Preparation of Stock Phosphate Buffer:

Dissolve 34 gm of Potassium Dihydrogen Phosphate (KH2PO4) in 500 ml distilled water.

Adjust pH 7.2 with 1N Sodium Hydroxide.
Make-up to one liter volume with distilled water.

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Preparation of batch dilution blanks:

Take 1.25 ml of Stock Phosphate Buffer solution & make-up its volume to one liter with
distilled water.
Fill it in dilution tubes so that after sterilization each tube will contain 9 ml.
Sterilize by autoclaving at 15 psi pressure (121°C) for 15-20 minutes.

Selection of Dilutions:

For routine testing select dilutions of samples so that total colonies on at least one plate
will be between 30 & 300. For unknown samples use two or more dilution.
.

Membrane Filtration by Solid Media in Petri dish:

Preparation of Solid Media Petri plates-

Set out required sterile Petri plates and label properly. Keep control plate for each
media.Taking aseptic precautions pour sterile molten medium into sterile Petri dishes.
Allow to cool and solidify.

Procedure-

1. Prepare and arrange sterile media plates. Make membrane filtration assembly ready.
2. Sterilization of Forcep: Immerse approx. 2.5 cm of the forceps into 70% alcohol,take it
out and flame it. Let it cool for 4 or 5 seconds and use immediately.
3. Using the sterile forcep, remove the filter from sterile package.place the girded side of
the filter up onto the centre of the stainless steel support.
4. Flame the pouring lip of the sample container and pour the sample into the funnel.turn
on the vacuum and allow the sample to draw completely through the filter. If required,
rinse funnel with sterile phosphate buffered.
5. Turn on vacuum and allow the liquid to draw completely through the filter.
6. Flame the forceps and remove the membrane filter from the funnel.
7. Place the membrane filter in sterile solid media plate in upward position. Incubate the
petri plates with the lid right side up (do not invert) for proper time and temperature.
8. Count the colonies and record the results.

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Calculation:
For Coliform Count Test, observe the plates after 24 hours of incubation and
confirm the results after 48 hours.
Count the colonies developed on membrane filter disc with the help of colony
counter

Recording / Reporting Of Results:

Coliform in cfu / 100 ml
SPC in cfu/ml 1ml

Sample size and water samples :

Sr.No. Sample Attribute Sample

Raw Water( Each borewell &Storage Total Coumt 1ml

1
tank Coliforms 100ml
Total Coumt 1ml
2 Treated Water (RO/Nano/Soft Water)
Coliforms 100ml
Total Coumt 1ml
3 CIP Final Rinse Water
Coliforms 100ml

Disposal of used media:

Sterilize all used Petri plates & PCT tubes with media after observation / counting by
autoclaving at 15 psi pressure (121°C) for 15-20 minutes. Cool plates & tubes. Collect all
solid media in polythene bags & dispose it.

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ANALYSIS OF CHEMICALS & WATER

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41.0 METHOD OF DETECTION OF PURITY OF CONCENTRETED HNO3 .

Scope: This procedure applies for determination of purity of concentrated HNO3.

Responsibility: Chemist.
PROCEDURE- 1

Reagents:

1. Standard Sodium hydroxide solution - 0.1N

2. Methyl orange indicator solution - Dissolve 0.05 g of methyl orange in 100

ml of water

Procedure:

Weigh accurately 2 g of the material in a cervical flask containing 100 ml of water. Keep
cooling and shaking until the vapours are completely absorbed. Add two drops of methyl
orange indicator to the solution in the cervical flask, and Titrate with standard sodium
hydroxide solution.

Calculation:

Acidity (as HNo3) = V x 6.3 x N

Percent by mass ------------
M

V = Volume in ml of standard sodium hydroxide used in the titration

N = Normality of standard sodium hydroxide

M = Mass in g of the test portion.

Ref.: IS: 264-1976

OR

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PROCEDURE- 2

To check the purity of concentrated HNO3: -

Weigh accurately 1 g of concentrated HNO3 in a clean dry beaker and transfer this in to a
volumetric flask of 100 ml capacity already containing about 50 ml of cold distilled
water. Rinse the beaker with small portions of distilled water three to four times in
succession and transfer the rinse in to the volumetric flask. Make up the volume to 100
ml with distilled water. Stopper and mix by inverting the volumetric flask several times.

Pipette out 10 ml of the dilute acid in to a clean dry conical flask and add a few drops of
methyl orange indicator. Titrate against 0.1 N NaOH until the orange color turns yellow.

Calculate the purity as below:

Titer value (ml) Purity Percentage

12.0 75.0
11.5 72.0
11.0 69.0
10.5 66.0
10.0 63.0
9.5 60.0
9.0 57.0

(A minimum strength of 55% or as specified in the Purchase Order terms & conditions is
required)

Reference: Mother Dairy Guideline

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42.0 METHOD OF DETECTION OF PURITY OF CONCENTRETED SULPHURIC

ACID.

Scope: This procedure applies for determination of purity of concentrated H2SO4

Responsibility: Chemist.

To check the purity of concentrated H2SO4: -

Weigh accurately 0.5 g of concentrated H2SO4 in a clean dry beaker and transfer this in to
a volumetric flask of 100 ml capacity already containing about 50 ml of cold distilled
water. Rinse the beaker with small portions of distilled water three to four times in
succession and transfer the rinse in to the volumetric flask. Make up the volume to 100
ml with distilled water. Stopper and mix by inverting the volumetric flask several times.

Pipette out 10 ml of the dilute acid in to a clean dry conical flask and add a few drops of
Phenolphthalein indicator. Titrate against 0.1 N NaOH until a pale pink color appears.

Calculate the purity as below:

Titer value (ml) Purity Percentage

10.0 98.0
9.8 96.0
9.6 94.0
9.4 92.0
9.2 90.0

(A minimum strength of 96% or as specified in the Purchase Order terms & conditions is
required)

Reference: Mother Dairy Guideline

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43.0 METHOD OF DETECTION OF PURITY OF CAUSTIC FLAKES.

Scope: This procedure applies for determination of caustic flakes..

Responsibility: Chemist.

Reagents:

1. Standard Hydrochloric Acid - 1N

2. Methyl orange indicator solution - Dissolve 0.1 g of methyl orange in 100

ml of water

Procedure-1

Weigh nearest 0.01 g of the material equivalent to a little less than 50g of
caustic soda. Dissolve the material in approximately 200 ml of water and cool it to room
temperature, transfer the solution quantitatively to a 500 ml mark volumetric flask and
dilute, cool and then dilute to the mark. Transfer exactly 20ml of the sample to a 500 ml
cervical flask. Add 5 drops of methyl orange indication solution and Titrate it against
standard hydrochloric acid until the colour of the indicator changes from yellow to
orange.

Calculation:

Alkalinity (as NaOH) = V x 4.0 x N

Percent by mass ------------
M

V = Volume ml of standard hydrochloric acid solution used in the titration.

N = Normality of standard hydrochloric acid

M = Mass in gm of the sample taken fourth test

Ref.: IS: 252-1973

OR

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Procedure-2

Weigh 10 g of Caustic Flakes in a 100 ml volumetric flask and add distilled water to
dissolve the flakes and make up the volume to 100 ml with distilled water. Stopper and
mix by inverting the volumetric flask several times.

(Proceed as per Manual in Dairy Chemistry (ICAR) Page No. 16 Ex-18 Direct Reading
Titration Method)

Pipette out 10 ml of caustic solution from the volumetric flask, add a few drops of
Phenolphthalein indicator, and titrate against 2.5 N H2SO4 using 10 ml pipette until the
pink color disappears. Repeat the titration to get concordant titer value.

Calculate the Purity percentage of Caustic Flakes from the following table:

Titer value (ml) Purity Percentage

10.0 100
9.9 99
9.8 98
9.7 97
9.6 96
9.5 95
9.4 94
9.3 93

(A minimum strength of 94% or as specified in the Purchase Order terms & conditions is
required)

Reference: Mother Dairy Guideline

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44.0 METHOD OF DETECTION OF STRENGTH OF CLEANING SOLUTIONS.

Scope: This procedure applies for detection of strength of cleaning solutions.

Responsibility: Chemist.

Determination of Strength of Washing Solution (Caustic)

The strength of the detergent/washing solution is expressed in terms of sodium
hydroxide.

Reagents:

1. Standard sulphuric acid - 0.1 N

2. Phenolphthalein solution - Dissolve on gram of phenolphthalein in 100 ml of 95
percent ethyl alcohol by volume. Add 0.1 N sodium hydroxide solution until one
drop gives a faint pink colourisation. Dilute with distilled water in 200 ml.

Procedure-1
Transfer 5 ml of the test solution to a 250 ml graduated flask and dilute to
the mask with distilled water. Mix well and transfer 50 ml of the solution to an
Erlenmeyer flask. Add a few drops of phenolphthalein indicator solution and Titrate with
the standard sulphuric acid to a colour less solution.
Calculation:
Percent by mass of caustic alkali due to NaOH = A x 0.4
A = Volume in ml of standard sulphuric acid.

Ref.: IS: 1479 (Part V) – 1962

OR

Procedure-2
Draw a sample of caustic solution from the CIP tank or pasteurizer balance tank and
pipette out 10 ml of the caustic solution in to a clean conical flask, add a few drops of
Phenolphthalein indicator, and titrate against 2.5 N H2SO4 using 1 ml (0.01 divisions)
pipette until the pink color disappears. Repeat the titration to get concordant titer value.

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The number of ml of 2.5 N H2SO4 solution used in the titration = Caustic alkalinity
percentage
Required Strength of Caustic Solution is 0.9 to 1.4% for CIP system and
Pasteurizer & after acid CIP 0.3 to 0.4 %.
Reference: Mother Dairy Guideline

Determination of strength of Acid Washing solution

The strength of the Acid Washing solution may be expressed in % Nitric Acid. When
Nitric acid is used as a cleaning detergent.

Reagents:

1. Standard Sodium Hydroxide 0.1 N

2. Phenolphthalein Indicator

Sodium hydroxide N/10 solution is prepared by weighing 4 gm NaOH and dissolving it

in 1 lit of the distilled water. It is further standardised against N/100 x acetic acid by
titrating in with using phenolphthalein indicator.

Procedure-1

Transfer 5 ml of the test solution to a 250 ml graduated flask and dilute it to

the mark with distilled water. Mix well and transfer 50 ml of the solution to an
Erlenmeyer flask. Add a few drops of phenolphthalein indicator and Titrate with standard
alkali 0.1N sodium hydroxide solution till the pink red end point observed.

Calculation:

% by mass of Nitric Acid - A x 0.63

A = Volume in ml of standard Sodium Hydroxide

Ref.: IS: 264-1976

OR

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Procedure-2

Strength of Nitric Acid Solution (CIP): -

Draw a sample of Nitric Acid Solution from the CIP tank or pasteurizer balance tank and
pipette out 10 ml of the Nitric Acid Solution in to a clean conical flask; add a few drops
of methyl orange indicator. Titrate against 0.1 N NaOH until the orange color turns
yellow.

Strength of Nitric Acid Solution (%) = V X 0.063, where V is the titer value

Required Strength of Nitric Acid Solution is 0.6 to 1.0% for both CIP system and
Pasteurizer.

Titer value (ml) Strength Percentage

11.9 0.75
11.1 0.70
10.3 0.65
9.5 0.60
8.7 0.55
7.9 0.50

Reference: Mother Dairy Guideline

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STRENGTH OF SANITIZER SOLUTIONS

A- Analysis of Chlorine in Sterilizing Solution

Chlorine in sterilizing solution is expressed as PPM (Parts per million)

Reagents:

1. Standard sodium thiosulphate solution - 0.1N

2. Acetic Acid - glacial
3. Potassium iodide - Powdered
4. Starch indicator solution - Make a paste of 5 g of soluble starch in cold water, pour
into 100 ml of boiling water, boil for 5 min. Cool and boil. The solution should be
prepared a fresh every two or three days.

Procedure-1
Place 50 ml or a suitable aliquot of solution to be tested in a 250 ml in a
flask add 1 to 2 g of potassium iodide and allow dissolve. Then add 10 ml of glacial
acetic acid and mix. Titrate the solution with 0.1 N sodium thiosulphate until the yellow
colour almost disappears. Add 2 ml of starch indicator solution which gives blue colour
with free iodine continue the titration till the blue colour is discharged.

Calculation:
When 50 ml of solution is used, the available chlorine in parts per million is
obtained by multiplying the number of milliliters of 0.1N sodium thiosulphate used by
71.

Ref.: IS: 1479 (Part V) – 1962

OR

Procedure-2

Pipette 10 ml of the sodium hypochlorite sample in to a 250 ml volumetric flask. Make

up the volume to 250 ml with distilled water. Mix well.

Pipette 25 ml of this dilute solution in to a clean conical flask. Add 2 g Potassium Iodide
crystals; dissolve them followed by 10 ml glacial Acetic acid. Immediately titrate the
mixture against 0.1 N Sodium thiosulphate until the brown color turns straw yellow.

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MDV-TM-03 TESTING MANUAL
Immediately add 1 ml freshly prepared starch solution and continue the titration until the
blue color disappears. Note the total volume of 0.1 N Sodium thiosulphate as V ml.

Make a blank determination using all other reagents. Deduct the volume of 0.1 N
Sodium thiosulphate from V ml.

(One ml 0.1 N Sodium thiosulphate = 0.003546 g Available Chlorine)

Available Chlorine Percentage = V X 0.3546

Reference: Mother Dairy Guideline

B- Determination of Available Iodine in Iodophor Sanitizer: -

Weigh 10 g of the Iodophor sample in to a 250 ml volumetric flask. Make up the volume
to 250 ml with distilled water. Mix well.

Pipette 25 ml of this dilute solution in to a clean conical flask & add 10 ml glacial Acetic
acid. Immediately titrate the mixture against 0.1 N Sodium thiosulphate until the brown
color turns straw yellow.

Immediately add 1 ml freshly prepared starch solution and continue the titration until the
blue color disappears. Note the volume of 0.1 N Sodium thiosulphate as V ml.

(One ml 0.1 N Sodium thiosulphate = 0.01269 g Available Iodine)

Available Iodine Percentage = V X 1.269

Reference: Mother Dairy Guideline

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MDV-TM-03 TESTING MANUAL

45.0 METHOD OF DETECTION OF WATER.

Scope: This procedure applies for water analysis..

Responsibility: Chemist.

WATER ANALYSIS

1) Analysis of Water for Total Hardness: -

Measure 50 ml of water sample in a measuring cylinder and transfer it in to a 250 ml

conical flask. Add 1 Total Hardness Tablet and dissolve. Add 2 ml of Ammonia Buffer
Solution and titrate against N/50 EDTA until violet color turns blue. Note the titer value
as V ml.

Total Hardness of Water = V X 20 ppm

For Boiler Feed the Total Hardness shall not exceed 5 ppm

(ppm = parts per million)

a) Analysis of Water for Residual Chlorine: -

Take 50 ml of water sample in a measuring cylinder and add 5 ml of Chlorotex reagent.

Mix the contents by inverting the measuring cylinder. Observe and compare the color
with the standard color chart for Residual Chlorine.

A minimum of 0.1 ppm residual chlorine is desired in the water supply.

b) Analysis of Water for Total dissolved solids: -

Take 50 ml water sample in a glass beaker & immerse the TDS meter inwards kept for
few seconds till the reading comes on the screen of the meter. Note down the reading at
the end.

A Maximum of 1000 ppm TDS is desired in the water supply.

Reference: Mother Dairy Guideline

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PACKING MATERIAL TESTING

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46.0 METHOD OF DETECTION OF PACKING MATERIAL (LDPE FILM).

Scope: This procedure applies for detection of packing material.

Responsibility: Chemist.

TESTING PACKING MATERIAL (LDPE FILM)

 From every consignment of LDPE film received in Stores, draw a sample of film
from any roll at random, after discarding the first turn of film from the roll.
 Wipe the surface of film with a clean, dry cloth to remove dust/dirt. Measure the
thickness of film across the length and width of film on unprinted areas. Measure
the width of film.
 Stick a piece of transparent adhesive tape on the print area and remove the tape. The
print shall not fade to the extent that the print matter is unreadable.
 Test for Ink Adhesion of Milk Pouches

LEAK TEST:
The filled pouches after filling with milk at about 8°C and sealing shall not show any
leakage when subjected to a uniformly distributed load of 50 N (5.0 kgf) for 500 ml
pouch and 100 N (10 kgf) for 1 litre pouch; pouches kept in flat position for 10 minutes
with the side seal on top.

DROP TEST:
Sixteen samples of filled pouches shall be taken at random from the filling line. The
temperature of the filled pouches shall be maintained within (±)2°C of the filling
temperature of the milk.

Sample pouches drawn from the machine as above shall be dropped after 15 minfrom a
height of 1.2 meter on a flat, smooth surface. Each pouch shall be dropped four times in
the following sequences:
a) On flat side, (b) On opposite side,
c) On flat longer edge, and (d) On opposite longer edge.
The samples shall be deemed to have passed if not more than 2 pouches burst.

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PRINTING INK TEST:
Immerse a printed piece of film in milk and leave it for 12 hours in it at a temperature of
about 8°C. After removing the film from the milk wipe it with a cloth to dry it. Rub the
printed surface with tissue paper gently by hand. The ink removed from the print shall not
be to the extent so as to reduce the printed matter not readable after the test.
Reference: Mother Dairy Guideline and IS11805:2007
TABLE FOR SCALE OF SAMPLING-
No.of packages in the lot Sample size
Up to 15 2
16 to 50 3
51 to 100 4
101 to 300 5
301 to 500 6
501 to 1000 8
1001 and above 10
Reference: IS10146:1982 edition 1.1 (2002-03)
Odour and taste:
Check the odour of the polyfilm.Take the piece of the polyfilm equivalent to
require for pouch and dip it in to the same variant milk for 48 hrs under refrigeration.
Then check the odour and taste of the Milk.

Odour Test: Cut approx. 20cm x20 cm and keep it in sterilized conical flask and covered
it with aluminum foil. Keep it at @ 50 deg C in Oven for 0.5 Hour & 1 Hour. After
specified time take out the sample, wait for 2 minutes and open the sample to smell in
area free from any objectionable odour.

Acceptance criteria:

If it is normal then it should be accepted and if some smell persists then again it
should be check with the same procedure but with product and with larger panel
members. If still smell persists lot to be rejected.
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MDV-TM-03 TESTING MANUAL

47.0 HDPE CRATES FOR MILK POUCH.

Scope: This procedure applies for testing of HDPE Crates for milk pouch.

Responsibility: Chemist.

SHAPE AND DIMENSION:

The shape of crates shall be as per IS:11584-1986. The dimensions of crates are given
below:

Capacity: 10 litres. Each crate to hold 10 sachets of one litre or 20 sachets of half a litre
or 50 sachets of 200 ml or 2 sachets of 5 liter.

Dimensions and shape: the plastic crate dimensions and shape should conform to sketch
No.14.28.14.08.A4 attached for prepack type and sketch no. 14.28.15.03 A4 for ISI
type.

weight of crate: The weight of the crate should be minimum 1200 gms.

Inter Stackability and Collapsibility: inter stakcability should be smooth, even, non-
slippery and stack should stand firmly and vertically. They should be comfortably
collapsible (i.e. they should go into one another when they are empty)

Finish: The crate should free from all moulding blemishes and should have smooth and
glossy finish.

RESISTANCE TO STRESS:

A crate, when tested in accordance with method described in IS:11584-1986 (by

submerging a crate in 1% solution of suitable surface active agent at 80(±)1°C for six
hours) shall show no surface cracking.

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RESISTANCE TO APPLIED LOAD:

The compression in the height of a crate, when tested in accordance with the method
described in IS: 11584-1986, shall not exceed one percent of its original height. Twenty-
four hours after removal of the test loads the compression shall have recovered by at least
50 percent.

STRENGTH:

• Resistance to drop when a fully filled crate is tested in accordance with the method
described in IS: 11584- 1986, no crack of the crates shall occur.
• Resistance to low temperature -when a fully filled crate is tested in accordance with
the method described in IS: 11584-1986.
• Load the crates with filled poly-pouches and hold at a temperature of 5 (±1) °C in
cold storage for 6 hours. Filled crates are then dropped in a horizontal position on a
concrete surface or floor from a height of not less than 3 meters. Crate passes the drop
test if no damage other than superficial abrasion shall occur.

DIMENSIONAL STABILITY:

After testing in accordance with the method in IS: 11584-1986, the crate shall comply
with the prescribed dimension. Subject to variation of 1.5 % of the original dimension,
however inter-stackability shall not be affected in any case.

Reference IS: 11584-1986

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