BIHAR ANIMAL SCIENCES UNIVERSITY

LABORATORY MANUAL
MILK AND MEAT HYGIENE, FOOD SAFETY AND PUBLIC HEALTH
VPE-311 (2+1)

Prepared by
Dr. Anjay, Ph.D.,
Dr. P. Kaushik, Ph.D.

Department of Veterinary Public Health and Epidemiology

Bihar Veterinary College, Patna– 801302

2018-19

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BIHAR ANIMAL SCIENCES UNIVERSITY

LABORATORY MANUAL
ENVIRONMENT AND ENVIRONMENTAL HYGIENE

VPE-511 (2+1)

Name:

Roll No.:

Department of Veterinary Public Health and Epidemiology

Bihar Veterinary College, Patna– 801302

2018-19

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 CERTIFICATE

Certified that this laboratory manual represents the work done by

Mr./Miss. ………………………………… I.D. No.: ……………….for the course VPE- 311
MILK AND MEAT HYGIENE, FOOD SAFETY AND PUBLIC HEALTH (2+1) in the
Department of Veterinary Public Health and Epidemiology, Bihar Veterinary College, Patna
during the academic year ………………………………

Marks awarded: Signature of the course teacher

Submitted to the Annual Board Practical Examination in Veterinary Public

Health and Epidemiology held on …………………………………………

Signature of Signature of Signature of

Internal examiner Internal examiner External examiner

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 INDEX

SI Practical Page Date Signature of

No. Instructor
1. Sanitary collection of samples for chemical
and bacteriological examination.
2. Grading of milk by MBR test
3. Test for pasteurization and plant sanitation.
4. Microbiological examination of raw and
pasteurized milk, milk products and water.
a. Standard plate counts.
b. Psychrophillc counts.
c. Mesophilic counts.
d. Thermophilic counts
e. Coliform counts.
f. Faecal streptococcal counts.
5. Detection of adulterants and preservatives in
milk and milk products.
6. Isolation and identification of organisms of
public hearth significance from milk.
7. Ante-mortem and post mortem inspection of

food animals.
8. Methods of slaughter (demonstration at the

slaughter houses).
9. Demonstration of speciation of meat.
10. Physical and bacteriological quality of meat
and aquatic foods (fish).
11. Demonstration of toxic chemical and
microbiological residues in milk and meat.
12. Visit to abattoirs, meat processing plants,
marketing centers and food service
establishments.

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 PRACTICAL No. 1

AIM: SANITARY COLLECTION OF MILK SAMPLES FOR CHEMICAL AND

BACTERIOLOGICAL EXAMINATION
Milk is ideal and perfect food for both new born and mature human beings. The high
nutritive value and moisture content of milk make it a good medium for the growth of
microorganisms. So it is very important to examine its bacteriological and chemical quality.
Objective:
To collect a portion of milk small enough in volume to be transported conveniently
and handled in laboratory while still accurately representing the material being sampled.
The sampling is often underestimated but very crucial step in process of determining
food safety. Many things can go wrong before the sample reaches the laboratory like
inappropriate sample type and locations, incorrect sampling techniques, sample
contamination, incorrect labeling, sample homogeneity and delivery time frames. The careful
and accurate sampling of milk is of utmost importance in all analysis of milk viz., for
detection and fraudulent practices. The most careful analysis is useless if the sample is faulty.
The most important thing to bear in mind is that the whole body of milk from which a sample
to be drawn should be uniform throughout in its composition and must be a true
representative of the whole body of milk.
Materials required:
Sterilized dipper, plunger, agitator and milk collection bottles.
(A) Sanitary collection of milk sample from:
(a) Milk container
Procedure:
1. Milk should be thoroughly mixed with the help of plunger and the sample should be
drawn with the help of sterile dipper from well below the surface of milk.
2. Transfer the sample in a sterile stopper or screw capped bottle.
3. Dispatch the sample immediately (within 24 h) to laboratory in ice box at 4-5°C for
bacteriological examination.
4. Examine the sample in lab as soon as possible.
(b) Individual cow/ mastitic animal:
Procedure:
1. Wipe the udder with clean cloth soaked in suitable disinfectant like KMnO4 solution
and allowed to dry.
2. Level the four-sample bottle as RF, RH, LF and LH.
3. Collect the sample directly from teat into the respective bottle & immediately dispatch
to laboratory in ice for examination.

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(c) Storage tanks and milk tankers:
The milk in storage tanks and milk tankers should require very careful mixing
because the volume of milk is very large. Mixing can be done using large plungers,
mechanical agitator or compressed air.
Sampling methods:
There are two methods of sampling
i) Manual sampling
ii) Automatic sampling
Preservation of Sample:
Samples of milk should be examined as fresh as possible. When samples are to be sent
to laboratory situated at distance place it should be preserved by addition of certain
preservatives to prevent souring. The composite samples could be kept for longer period.
a. Formalin -20 drops of formalin per liter of milk.
b. Dichromate potash (0.5 g/ liter) for metal container.
c. Mercuric chloride used at milk plants.
General precautions during sampling:
1. All precautions should be taken to prevent contamination of the sample.
2. Cleaned and sterilized bottles should be used for sampling.
3. Put a level or an-identification mark on the sample container before or at the time of
sampling.
4. Collected samples should be packed properly and marked with detail information of
animal, date, time and place of sample collection.
5. A uniform quantity of milk should be taken for each sample; it is advisable not to fill
the bottle more than three quarters so that milk may be shaken before examination.
6. The samples should be dispatched to the laboratory as soon as possible.
7. Make a record of every sample collected with sufficient information to provide
sample identification at a later date.
8. In case any preservative is added to the sample, the details should clearly be
mentioned on the label.
9. The samples should be analyzed preferably within four hours of collection.

Questions. Draw the diagram of plunger, dipper, sample collection bottle and milk can?

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7
 PRACTICAL No. 2

AIM: GRADING OF MILK BY MBR TEST

Methylene Blue in presence of oxygen remains blue, but colorless in absence of
oxygen. In the test, color imparted to milk by the addition of a dye such as methylene blue
will disappear more or less quickly. The removal of the oxygen from milk and disappearance
of color is due to bacterial metabolism which leads to the formation of reducing substances.
Though certain species of bacteria have considerably more influence on reduction than
others, yet it is generally assumed that the greater the number of bacteria in milk, the quicker
will the oxygen be consumed, and in turn the sooner will the color disappear. Thus, the time
of reduction is taken as a measure of the number of organisms in milk. Although actually it is
more truly a measure of the total metabolic reactions proceeding at the cell surface of the
bacteria.
Materials required: Test tubes with rubber stoppers, Pipette or dipper graduated to deliver
10 ml of milk; Water bath for maintaining the samples at 35o to 37oC. Methylene blue (1 mg
in 250 ml distilled water).
Procedure:
1. Sterilize all glassware and rubber stoppers either in an autoclave or in boiling water.
2. Add 10 ml of milk and add 1-2 drops of methylene blue solution.
3. Keep the tubes immediately in water bath with a incubation temperature of 37oC.
4. Check samples for decolorization after 30 minutes of incubation. Make subsequent
readings at hourly intervals thereafter.
5. Record reduction time in whole hours between last inversion and decolorization.
Reading:
1. According to American Public Health Association (APHA):
Observation Interpretation
I. Not decolorized in 8 hours. Excellent
II. Decolorized between 5-8hrs Good
III. Decolorized between 2-5hrs Fair
IV. Decolorized in less than 2 hours. Poor
V. Decolorized in less than 20 min. Very poor
2. According to Chalmers:
Reducing time Bacteria /ml Quality Class of milk
I. Above 4.5 hrs 200000 Good Class I
II. Between 2.5 -4.5 hrs 200000-2000000 Average Class II
III. <2.5 hrs 2-10 millions Poor Class III
Advantages:
1. The test is inexpensive & requires minimum equipments.
2. Do not require much skilled & experience technician.

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3. Good indicator of keeping quality of milk. Short reduction time indicates poor keeping
quality & vice-versa.
Disadvantages:
1. Lack of uniformity in distribution of bacteria, because in creamy layer, the bacteria are
swept along a large extent.
2. Difference in oxygen consumption of different bacteria.
3. Difference in amount of dissolved oxygen present sample.
4. Presence of other reducing substances in milk such as leucocytes.
5. Doesn’t furnish information about type of bacteria.

Observation:

Result:

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 PRACTICAL No. 3

AIM: TEST FOR PASTEURIZATION AND PLANT SANITATION

Test for pasteurization:
The most common method of milk pasteurization being followed in India is either the
holder process (63oC for 30 mim) or high temperature short time (72oC for l5 seconds).
Phosphatase enzyme is naturally present in raw milk which is destroyed at pasteurization
temperatures. In case the heat treatment is inadequate or less the phosphatase enzyme remains
active and in the presence of suitable substrate (Di-sodium p-nitro-phenyl phosphate) in buffer
solution liberates phenol at pH 9.5.
Milk is added with a suitably buffered solution of disodium p-nitophenyl phosphate
which is hydrolyzed by the phosphatase enzyme present in raw or inadequately pasteurized
milk to form p-nitrophenol. This free nitro-phenol gives yellow colour in alkaline solution.
Materials required:
Milk sample, Water bath, Colorimeter, Test tubes with rubber stoppers, Test tube rack, Disc
comparator.
Reagents:
i. Buffer solution: Weigh 3.59 of sodium carbonate (analytical grade) and 1.59 sodium
bicarbonate and dissolve in 1000 ml of distilled water.
ii. Substrate: Di-sodium p-nitrophenyl phosphate.
iii. Buffer substrate solution: Weigh 0.15gm of substrate disodium p-nilrophenyl phosphate and
transfer into 100-ml measuring cylinder and make up to the mark. (Do not store the solution
for longer duration but keep refrigerator and use within a week. Practically the solution is
colorless).
Method:
1.Take 10 ml buffer substrate solution in two test tubes.
2. Keep both test tubes in water bath at 37- 38oC.
3.Take 5 ml milk from the test sample and boil for few minutes in boiling water.
4.Cool the boiled milk under running tap water
5.Use the boiled & cooled milk as blank control (boiling destroys the phosphatase enzyme).
6.Add 2 ml of milk sample to one of the test tubes containing buffer substrate.
7.Add 2 ml of blank control to the second test tube containing buffer substrate.
8.Close the tube with rubber stopper and invert to mix the contents. Repeat the process 4-5
times to mix thoroughly.
9.Incubate the tubes at 37-38oC and see within 30 min for development of yellow colour.
10. Return these tubes to the water bath and take the second reading after incubation of 90
minutes.
11. Compare the colour development using colour disc comparator and interpretate result or
use any other calorimetric method to detect the intensity of yellow development.

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Interpretation:
Disc reading
(a) After 30 minutes of Incubation Interpretation
i. 0 or trace Property pasteurized
ii. 6 Doubtful
iii. 10 or over Under pasteurized
(b)After 2 hours of incubation Interpretation
i. 0-10 Properly pasteurized,
ii. 10-18 Slightly under pasteurized
iii. 18-42 Under pasteurized
iv. 42 or over Gross under heating of milk or raw milk

Note: The 30 minutes test will reveal only serious fault in pasteurization but to enable
detection of minor errors reading shall be taken after further incubation of 90 minutes.

Result:

Tests for Plant Sanitation:

In addition to testing of incoming milk for bacteriological milk and compositional

quality, the control of outgoing milk is the primary duty of the dairy control laboratory,
which speaks all about the plant sanitation.
Objective:
a. To assess the state of cleanliness of pasteurizing plant
b. To assess the cans and tanks before they go out to the milk producers or
collecting depots.
c. To assess the external hygiene in plant sanitation.
Materials required: test tubes: Sterile swabs, Test tubes, Test tube stand, Petri dishes,
Pipettes (1, 5 and 10 ml), Incubator, Normal saline solution, Glass marker, Culture medium.
Procedure: Assessment of equipment hygiene by
A) Visual Inspection:
A few simple objective tests are often useful to demonstrate an unsatisfactory
condition such as:
a. The sense of sight and smell enable serious neglect to be quickly pointed out.
b. A pocket knife or spatula can be used to scrape the surface to demonstrate the
presence of a film of residues on improperly cleaned equipment.
c. A piece of muslin cloth wiped over the inside of a can or over metal surfaces of other
equipment will be soiled if the surface is improperly cleaned.

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B) Laboratory tests:
There are two methods for the laboratory control of the hygienic condition of the
equipment i.e. bacteriological tests to be performed on the equipment and bacteriological tests
to be performed on the final products.
Swab technique: To assess equipment hygiene swab or rinse technique is applied on the
surface of equipment such as Vats, Tanks, Coolers and Pipelines.
Procedure:
1. Swab the surfaces of the equipment with sterilized swabs
2. Isolate the bacteria from the equipment using the spread plate.
3. Identification of the bacteria following routine methods of identification of bacteria
viz. study of colony characteristic, morphology, staining growth pattern on differential
and selective medium, biochemical reactions.
Rinse technique: Chiefly used for cans and bottles.
Procedure:
1. Rinse the equipment with sterilized water.
2. Collect the rinsed water in sterilized flask and process for isolation of bacteria
3. Inoculate the rinsed water by following SIC technique.
4. Isolate and identify the bacteria following standard procedure of identification

Bacteriological test on the final product:

Perform coliform, yeast, and mould count.
Interference: It gives an idea about efficiency of can washing and quality of detergent used.
It also gives idea about the concentration and efficiency of sterilizing agents and the
bacteriological quality of water used for cleaning the equipment.

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 PRACTICAL No. 4

AIM: MICROBIOLOGICAL EXAMINATION OF RAW AND PASTEURIZED

MILK, MILK PRODUCTS AND WATER
a. Standard plate counts.
b. Psychrophillc counts.
c. Mesophilic counts.
d. Thermophilic counts.
e. Coliform counts.
f. Faecal streptococcal counts.

a. Standard Plate Count (S.P.C.)

The Standard plate count (S.P.C.) is a method for estimating the total number of viable
bacteria in food products and water. This can be performed by using the pour plate method or
spread plate method.
Materials required:
Test tubes, Test tube stand, Petri dishes, Pipettes (1, 5 and 10 ml), Incubator, Colony
counter, Normal saline solution, Glass marker, Blower, Culture medium.
Procedure:
Pour plate Method:
1. Weigh and dissolve the required amount of nutrient agar medium and sterilize by
autoclaving (1210C, 15lb, 15 min) and bring down its temperature between 45-55oC.
2. Make a 10 fold serial dilution of sample after thorough mixing.
3. Take 1 ml each of the diluted samples and transfer to petridishes.
4. Pour about 15-20 ml agar medium to each of the petri-dishes under the flame of a blower.
5. Mix the content thoroughly but gently by agitating the plate in all directions (side to side,
up and down, clock wise and anti clock wise) taking care that there is no spillage of
content from the plate.
6. Make a duplicate plate for each dilution.
7. Allow the agar medium to solidity.
8. Place these petri-dishes in the incubator at 37oC.
9. Take out the plates after 24 to 48 hrs and observe for the development of the colonies.
10. Select the plates having colony range between 30-300 and count the colonies of both
plates of a particular dilution using colony counter and take the average.
Note: For spread plate technique, first prepare the agar plate and then take 0.1ml of diluted
sample on it and spread it with the help of spreader.
Disadvantages:
1. Each colony appears on the plate arises from varying number of organisms.
2. One ml or 0.1ml of milk is not true representative of the sample as the groups of organism
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 aren’t uniformly distributed.
3. Conditions of incubation favor the growth of certain type of bacteria.
4. Count doesn’t help to detect the type of organism.

Calculation:
Total no. of viable bacteria/ml = Dilution factor x Average no. of colonies of the
of sample same dilution/ volume taken on inoculation
Interpretation of Result:
The following standards have been suggested by ISI for grading sanitary quality of
milk by SPC Method.
Raw milk SPC/ml Grade
Less than 200,000 very good
Between 200,000 and 10, 00,000 Good
Between 10, 00,000 and 50, 00,000 Fair
Over 50, 00,000 Poor

Pasteurized milk
Less than 30,000 very good

Result and interpretation:

Psychrophilic, Mesophilic counts and Thermophilic counts:

The psychrophilic organisms grows optimally <15 0C (0-20 0C), the mesophilic organism
optimum growth temperature is between 20 – 450C and the thermophilic organism optimum
growth temperature is > 450 C.
Procedure:
Similar to SPC except incubation temperature which are:
Psychrophilic counts: Incubate plates at 4-70C for 7 days
Mesophilic counts: Incubate plates at 30 – 350C for 24 h.
Thermophilic counts: Incubate plates at 550C for 24 h.
Result and interpretation:

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Most probable number for Coliform:
Coliform is a group of aerobic or facultative anaerobic, gram-negative, nonspore-
foaming, rod shaped bacteria which ferment lactose with gas formation within 48 hours at
37°C. Coliform includes Escherichia coli, Citrobacter, Enterobacter, Hafnia and Klebsiella.
Most probable number technique is widely used for enumeration of coliform, the indicator of
sanitary quality of food and water. This technique is also useful in the quality assessment of
foods that may have extremely low number of organisms.
Materials required:
Test tubes, Test tube stand, Durham’s tube, Pipettes, Incubator, Glass marker, Blower,
MacConkey broth.

Procedure:
1. Take 10 ml of sample and make 10 fold serial dilutions upto 1:1000.
2. Take 1 ml inoculums from each dilutions and inoculate in triplicate tube containing
10 ml MacConkey broth with inverted Durham’s tubes.
3. Incubate the tubes at 370C for 48 h.
4. Observe for the trapped gas bubble in inverted tubes.
5. Count the number of tubes showing acid or gas production in each dilutin and
compute the MPN of coliform using MPN statistical table/MacCardy’s table.

Result and interpretation:

MPN coliform/ml of sample =

Faecal Streptococcal count

Faecal streptococci are aerobic Gram positive cocci occurring in long chains. The
important species in the group are Streptococcus faecalis, S. faecum, S. bovis and S. equines.
The faecal Streptococcal count is used as an indicator of sanitary quality of milk, food
or water. The presence of faecal Streptococci in food and water indicates recent pollution
with faeces. The number of faecal Streptococci is determined by plating the known amount of
sample in suitable medium
Materials required:
Test tubes, Test tube stand, Petri dishes, Pipettes (1, 5 and 10 ml), Incubator, Colony
counter, Normal saline solution, Glass marker, Blower, Culture medium.
Procedure:
Similar to SPC except in place of nutrient agar KF Streptococcal agar or Karl
Friedrich Streptococcal agar is used.
Result and Interpretation:

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 PRACTICAL No. 5

AIM: DETECTION OF ADULTERANTS AND PRESERVATIVES IN MILK

AND MILK PRODUCTS
Adulteration of milk is addition of inferior and cheap quality substances in milk
which doesn’t have nutritional value like pure milk for the purpose of profitability. It is a
illegal process under law. Adulteration of milk is practiced in India on a large scale and
basically aims for reduction of fat and addition of preservatives. The adulteration of fat in
milk is done by the following ways.
a. By adding water,
b. By skimming of milk
Both will make milk less viscous. To make the milk more viscous some thickening agents
are added. The thickening agents most commonly used are starch, gelatin, cane sugar, etc.
Detection of Adulteration:
Synthetic milk is the spurious form of milk, which mostly contains toxic compounds. It is
not at all milk but mimics for milk. In gradient of synthetic milk are soda,. urea, refined oil,
salt, glucose, ammonium sulphate, detergent, liquid soap etc.
1. Test for detection of starch: Take 10 ml of milk in a test tube which has been boiled &
cooled to room temperature + add 1 ml of 5% iodine solution.
* Blue color will develop in presence of starch.
Observation:

2. Detection of Cane sugar: Take 2 ml of milk in a test tube + add 1 ml of HCl and 20mg of
resorcinol + Boil for a few minutes.
*Development of brick red color in presence of cane sugar.
Observation:

3. Detection of Milk Powder: 10ml of milk is taken in a test tube + add one drop of formalin
& incubate at 600C for 10 min + add nitric acid.
*Light violet color indicates presence of skim milk powder.
Observation:

4. Detection of Annato: Take 10ml of milk in a test tube + add 10ml of ether & shake well +
Mixture is left until ether separates on the top of the milk.
*Yellow color of ether layer indicates annatto presence.
Observation:

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5. Detection of Gelatin: Take 10ml of milk is taken in a test tube+ add 10ml of Acid Mercuric
Nitrate (5gm of mercury, 10ml of nitric acid & 20mlof water) + Mixture is shaken well & then
filtered. + If gelatin present in large quantities of filterate will be opalescent (turbid) & not
clear + To this filtrate saturated aqueous solution of picric acid is added.
*Development of yellow precipitate indicates presence of gelatin. In absence of gelatin,
solution will be clear.
Observation:

6. Detection of Coal tar dyes: 10ml of milk is taken in a clean test tube + 10ml of strong HCl
is added.
* Development of pink color indicates the presence of dyes in milk.
Observation:

Detection of Preservatives:
Preservatives means a substance which when added to milk or milk products, will
retard decomposition. The objective is to prolong the period of sweetness of milk and
freshness of food and food products, to destroy pathogenic and non-pathogenic bacteria, to
delay curdling of milk/decomposition of food and food products.
The disadvantage of adding preservatives of milk may be accounted for poisoning.
Milk may become unfit for consumption and preservative may interfere in the process of
digestion.
The agents commonly used to preserve milk are boric acid, borax, formalin, salicylic
acid, benzoic acid, sodium carbonate and sodium bicarbonate.

1. Boric Acid and Borax

Requirement: Test tube, Phenolphthalein, N/10 caustic soda sol, 50% water sol. of neutral
glycerine.
Procedure: Take 5 ml of milk in a test tube + Add few drops of phenolphthalein + Add N/10
caustic soda solution drop by drop until pink color develops + Take half the quantity of this
mixture in another test tube + Add equal volume of distilled water in one and equal volume of
50% water solution of neutral glycerine, in the other tube. Observe the reaction changed to
white in the tube in which glycerine is added (in case of presence of boric acid and borax in
milk.)
Observation:

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2. Formalin
Requirement: Test tube, 1% ferric chloride sol, Conc.H2S04
Procedure: Take 1O ml of milk in a test tube + Add 0.5 ml of l% ferric chloride sol+ Dilute
the fluid by adding equal quantity of water + Pour concentrated H2SO4 by the side of the test
tube slowly and observe the reaction.
*Violet colour appears at the junction of the two liquids in case of the presence of
formalin.
Observation:

3. Salicylic acid and Benzoic acid

Requirement: Test tube, H1S04, 0.5% ferric chloride solution
Procedure: Take 10ml of milk in a test tube and add one drop of sulphuric acid a few drops
of 0.5% neutral ferric chloride solution and observe.
*A purple violet color will develop in case of presence of salicylic. A buff color will
develop in case of presence of benzoic acid.
Observation:

4. Hydrogen Peroxide
Requirement: test tube, 2% solution of paraphenylene diainine
Procedure: Take 10 ml of milk in a large test tube + Add few drops of 2% solution of
paraphenylene diamine + observe the reaction.
*Blue color will develop in case of presence of hydrogen peroxide.
Observation:

5. Test for detection of Sodium Carbonate as Sodium bicarbonate

Requirement: Test tube, Alcohol, 1% solution of resolic acid.
Procedure: Take 10ml of milk in a test tube. Add 10 ml of alcohol add few drop of 1%
solution of resolic acid and observe reaction.
* Pink Rose colour will develop in case of the presence of carbonates or bicarbonates.
Observation:

Detection of Adulteration in Ghee

1. Detection of Animal body fats by opacity test:
There is a common malpractice to admix the costly ghee with cheaper fats like
vanaspati, vegetable oils and animal body fats. These adulterants can be detected using
Baudouin test, Phytosterol acetate test and Thin Layer Chromatography (TLC).
The method used to detect animal body fat is based on the principle of their melting
points. The melting point of ghee is lower than the animal body fats. There is a significant

18
difference in solidification points of ghee and animal fats at lower temperature. The fat is
transparent above its melting points and has a tendency to solidify with a decrease in
temperature. The fat looses transparency during its solidification at the lower temperature and
consequently opacity develops which is used to differentiate the ghee from animal body fat.
Material required: Oven, Filter paper (Whatman No.1), Colorimeter, Colorimeter tubes,
Water bath.
Method:
1. Take the fat sample in a beaker and place it in an oven maintained at 60°C and allow to it
melt.
2. Filter the fat at the same temperature through Whatman filter paper (No.1).
3. Pour the fat sample (transparent) in the colorimeter tube (8cm x 1.5 cm) to a height of about
7 cm. and place the tube in water bath at 50°C.
4. Transfer the whole assembly for 30 min. in an oven maintained at 50°C.
5. Transfer the colorimeter tube containing sample in another water bath maintained at 23 ±
0.5°C.
6. Observe the opacity after every 2 minutes in a colorimeter (Spectronic-20) at 590 nm
wavelength using green filter.
Observation:
Opacity observed within 20 minutes- suspected to be admixed with body fats (possibly of
buffalo, goat or sheep)
Limitation of the test:
It is difficult to detect the pig body fat up to 10% level. This method fails to detect the
presence of cotton tract fat, which behaves like ghee adulterated with animal body fats in all
properties. To differentiate the cotton tract ghee from normal ghee, Methylene blue test could
be done. Cotton tract ghee reduces Methylene blue dye instantaneously, whereas normal ghee
and adulterated ghee do not reduce this dye.
Conclusion
1. Test Positive -Suspected to be cotton tract fat (No need to conduct opacity test).
2. Test negative- Cotton tract fat absent and opacity test could be used).
Observation:

2. Detection of vegetable oils and fat by Baudouin test:

The sesame oil is present in almost all brands of vanaspati available in the market. The
stable pink color develops with furfural solution in the presence of hydrochloric acid that
indicates the presence of sesame oil in ghee.
Materials required:
Reagents: Hydrochloric acid (Sp. Gr. 1.19), Furfural solution (2%)
Note: The furfural solution is not distilled earlier than 24 hrs prior to the test in the spirit

19
(95%).
Method:
1. Take 5.0 ml of melted ghee in a test tube.
2. Add 5.0 ml of hydrochloric acid (Sp. Gr. 1.19) and 0.4 ml of furfural solution
3. Insert a rubber stopper in the test tube and agitate vigorously for two minute.
4. Allow the mixture to settle and separate.
5. Observe the development of color.
* Development of pink/ red color in the acid layer- Vanaspati present
Confirmation of the result:
Add 5.0 ml water to the mixture and shake to confirm the result. If the pink color
persists in the acid layer, vanaspati is present and if the color disappears, vanaspati is absent.

20
 PRACTICAL No. 6
AIM: ISOLATION AND IDENTIFICATION OF ORGANISMS OF PUBLIC
HEALTH SIGNIFICANCE FROM MILK, MILK PRODUCTS, MEAT AND MEAT
PRODUCTS

Sl. Organisms Enrichment Selective plating Colony

No. characteristics
1. Escherichia coli MacConkey broth Eosin methylene Metallic sheen
Enterobactericeae broth blue
MacConkey agar Small, round pink
colour
2. Salmonella Pre enrichment: Brilliant green agar Pink colonies
Buffered peptone water surrounded by red
medium
Enrichment: Hektoen enteric agar Black colonies
Tetrathionate broth surrounded by green
Selenite cystine broth margin
Rappaport-Vassiladis MacConkey agar Colourless
broth Bismuth sulfite agar Black centered,
bright edged
colonies surrounded
by a precipitate
(rabbit or fish eye
appearance)
Salmonella- Shigella Colourless with
agar black centre
3. Staphylococcus Trypticase soy broth Baired parker agar Shiny jet black
aureus colour colonies
Mannitol salt agar Yellow colour
colonies
4. Aeromonas Alkaline peptone water Ampicillin dextrin Yellow coloured
agar small smooth honey
drop like

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5. Campylobacter Bolton broth, Preston Skirrow’s, Butzler’s, Small flat,
broth Blaser wing, Preston translucent and or
selective medium effuse
6. Listeria UVM I&II, PALCAM, DRA Greenish yellow,
monocytogenes Fraser broth I &II glistening, pointed
surrounded by black
zone

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23
 PRACTICAL No.7

AIM: ANTE-MORTEM AND POST MORTEM INSPECTION OF FOOD ANIMALS

Ante mortem examination:
Ante mortem examination is the examination of live animal conducted 24 hrs prior to
slaughter at lairage by a qualified veterinarian.
Objectives:
1. To detect evidence of cruelty to animals by overcrowding, over driving or other
methods.
2. To find out the disease symptoms which may affect general health of animals or may
depreciate the value of meat.
3. To note the presence of schedule of infectious and contagious disease or symptoms
which may suggest that the disease is in developing condition.
4. To prevent the food borne infections.
5. To segregate the disease animal from the healthy animals.
6. To diagnose some of zoonotic diseases.
7. To detect certain conditions and disorders due to diseases which may be difficult to
detect by examination of organs, carcass after the slaughter, but can be diagnosed
easily during A.M. inspection eg. Mastitis, rabies, tetanus, metritis, etc.
8. To ensure the selection of normal rested animal which will produce high quality meat
for human consumption.
Required Facilities:
1. Sufficient space is required to examine and to restrain the animal.
2. Identification of live animal either by paint, ear tags, tattooing or leg bands.
3. Lairage should be well ventilated and properly lighted to hold the animals for 24
hours.
4. Isolation pen is needed for separation of diseased animal from the healthy individuals.
5. A trained stock assistance is needed for handling the livestock during the examination.
Procedure for A.M. Examination:
1. A veterinarian is to enter into pen quickly, while animals are in rest and by clapping
24
 the animals are forced to move. The healthy animals will move faster, while the
diseased or injured animal will be left without responding to environment and these
animals are looked for general appearance (alert or dullness), their respiration,
temperature, pulse and lameness.
2. The inspector should tag to diseased animal so that such animal can be examined
carefully during P.M. by other meat inspector.
3. Record the evidence of cruelty.
4. Temperature, Pulse, Respiration rate, Gait, Posture any discharge are recorded.
5. Record for emaciation and poorness.
6. Record the condition of skin and hair.
7. Note the condition of digestive system.
8. Examine the genital organs for mastitis, metritis and orchitis.
9. Pregnant animal should not be slaughtered and send back immediately.
10. Special attention must be given to the recumbent animals.
11. The inspection is repeated immediately before slaughter in certain condition even if
they were examined 24 hrs before slaughter.
12. Animals suffering from metabolic infection or disorders should be treated first.
13. Animals which have recently suffered with accident or injuries should be subjected to
emergency slaughter.
14. Dirty animals must be cleaned prior to slaughter by washing and should be re-
inspected before slaughter.
Decisions taken under AM Inspection:
A. Animal fit for slaughter: Animals showing all physiological parameter normal are
generally passed for slaughter.
B. Animal unfit for slaughter –Condemnation of the animal and not be allowed for
slaughter e.g. rabies, tetanus, hog cholera, anthrax, B.Q., dropsy (general) emaciation
etc.
C. Suspected– Animal affected with localized conditions be segregated for detailed
examination to see localized or systemic involvement. Temperature, respiration,
colour of mucous membrane should be noted and slaughtered separately. A tag is
attached to the animals and examined thoroughly during post mortem inspection.
D. Delayed slaughter – Fatigued, excited and inadequately rested animals fall in the
category of delayed slaughter as these animals require rest and treatment.
E. Emergency and casualty slaughter: Animals suffering from acute conditions are
sent for emergency slaughter and in chronic conditions refereed for casualty slaughter

Post Mortem Examination:

25
 P.M. Examination refers to the systematic examination of dressed carcass in a
hygienic manner immediately after slaughter by a meat inspector with the object of providing
wholesome meat to consumers.
Essential requirements for P.M. examination:
1. Well constructed slaughter house.
2. Arrangement for natural light.
3. Proper equipment for hoisting and splitting.
4. Arrangement for tanks to retain the carcasses for 24 hours and one each for head, for
sides, and for viscera.
5. Sufficient arrangement should be there to record age, sex, weight of animal, so as to
help in identification of carcasses and to known the incidence of diseases at different
age and in various sexes.
6. The staff should have proper clothing.
7. There should be atleast one laboratory.
8. There should be well experienced meat inspector.
Type of examination:
1. Visual examination: To detect hemorrhages, infarction, fatty liver abscesses, cyst,
jaundice, presence of foreign body, melenosis, etc.
2. Palpation: To detect hematoma, calculi, pneumonia, pleurisy, cyst and tumor.
3. Incision of organ and tissue: Examination of lymph node, glands, liver, kidney, etc.
4. By smell: Ketosis, Uremia, decomposition.
5. Laboratory test: For suspected pathological lesions the samples should be submitted to
laboratory e.g. leptospirosis, listeriosis, Q fever, salmonellosis and some of protozoan
and fungal disease.
Inspection of carcass and organs: It should be preceded in following order:
1. Head: Examine submaxillary, retropharyngeal and parotid lymph node, inspection of
gums, lips and tongue is done for foot and mouth disease, rinderpest, Cysticercus
bovis, actinobacillosis, actinomycosis, stomatitis. The other diseases are T.B., horn
cancer and glander. Nostrils are also examined for unilateral, bilateral discharges and
masseter muscles are examined for Cysticercus bovis infection.
2. Viscera:
Lungs: Lungs are examined for pluricy, pneumonia, T.B., fasciolisis, hydatid cyst and
examine bronchial and mediastinal lymph node.
Heart: Pericardium is examined for pericarditis, hemorrhage, hydatid cyst,
Cysticercus bovis, sarcocyst, traumatic and tubercular pericarditis. Heart is incised and
examined for pin point hemorrhages on epicardium or on endocardium. In perfectly
bled carcass, the heart stops at systole, while in imperfectly bled carcass it stops at
diastole.

26
 Liver: Portal lymph node is examined for its size, shape and color. In diseased
condition it shows enlargement and changes in colour. Liver is examined for fatty
changes, actinobacillosis, fascioliosis, Cysticercus bovis, hydatid cyst and T.B. There
is deposition of excess amount of fat due to diet which is rich in fat and carbohydrate
liver becomes larger and heavier than normal, edges become rounded, yellow or
yellowish brown in color, soft in consistency, it pits on pressing with finger, are the
changes generally seen in the liver. Microscopically due to physiological fatty change
its cells shows fat globules and the nucleus of the cell is displaced to the edge of cell.
Pathological fatty changes have the presence of numerous fatty droplet and the
nucleus is absent.
Stomach and Intestine: Anterior aspect of reticulum may show evidence of
penetration due to foreign body, mesenteric lymph node examined for disease
condition of intestine. They are incised and examined microscopically.
Spleen: Surface and substance be examined for T.B. anthrax, haematoma,
haemorrhagic infarction, fungal and protozoan diseases.
Uterus: It is examined for evidence of pregnancy, for recent parturition and metritis.
Complete carcass is condemned on evidence of pyometra. In Brucella abortus
infection the uterus should not be handled as this disease is highly infectious to
human.
Udder: It is examined for presence of abscess, mastitis, cow pox, pseudocowpox,
actinomycosis, T.B. and brucellosis and for these diseases supramammary lymph node
should be examined.
Testes: These should be examined for orchitis, abscess, haematoma, haemorrhages or
injury.
3. Carcass: It is examined for bruises, state of nutrition, local or general oedema.
Examine visually to see whether smooth shining or have dull appearance, examine
pelvic and thoracic cavity and abdomen for presence of inflammation and abscess due
to T.B. Examine the cut surfaces of bone and color of bone marrow. Examine kidney
visually and also incise the renal lymph node.
If the carcass on examination reveals no abnormality then it may be passed for
food. During routine following lymph node should be examined.
1. Prepectoral 2. Lymph node of lower and upper thoracic wall
3. Prescapular 4. Lumbar
5. Precrural 6. External and internal iliac
7. Superficial inguinal 8. Popliteal.

27
 PRACTICAL No. 8

AIM: METHODS OF SLAUGHTER

Slaughter implies putting an animal to death and subsequently preparing the carcass
and organs for human food. Methods of slaughter should thus be aimed at complete bleeding
as long as possible & least unnecessary suffering & minimum struggling to animal. Good
bleeding influences the keeping quality of meat. Bleeding is most thorough when the heart
and respiratory functions remain in action as long as possible. For good bleeding more than
half blood must drain out.
Meat in a broad sense refers to all parts of the carcass that are fit for human food in its
restricted sense; it includes the skeletal muscles and accompanying body tissues (i.e.
connective tissue, fat, tendons, bone and blood vessels).
Bleeding as the keeping quality of meat depends considerably on through bleeding;
this should be as complete as possible. It depends on the following conditions:
i. Health of the animal.
ii. Sufficient rest of the animal before slaughter.
iii. Strong and long continued respiratory and cardiac action.
iv. Slaughtering and stunning methods used.
Methods of slaughter: Following are the chief.
(A) Humane methods: In these methods animal feels least possible pain and bleeding is
complete. Humane conditions during slaughter can be achieved by the use of appropriate
stunning procedure which makes the animal unconscious and insensitive to pain prior to
bleeding.
Stunning is done by various methods:
(i) Mechanical instruments: various type of mechanical instruments are used.
(a) Captive bolt pistol: This is most widely used stunning instrument in modern abattoir
which is effective in cattle, sheep, goat and calves. In this a bolt is propelled forward
on discharge of cartridge & automatically recoil into barrel. It is less effective in bull
& pig due to thick frontal bone structure. It is not a suitable method if no. of animal to

28
 be slaughtered is over 250 cattle/hr because difficulty in reloading. The instrument
has to be placed at correct site on the head of animal. The correct position in adult
cattle is at the middle of forehead where to lines drawn from medial canthus of each
eye to the base of opposite horn meat. In calves pistol must be placed slightly lower
at the forehead than that for adult cattle. In sheep & goat the muzzle is placed behind
the ridge which runs between the horns. In pigs the pistol is placed about 2.5 cm
above the level of eyes.
(b) Free bullet pistol: Free bullet pistol is frequently used to destroy horse and
sometimes cattle humanly. Bullets are fired from a small bore rifle. The point of
application in horse is immediately below the point where two lines from the medial
canthus of each eye to base of opposite ear crosses. Due to destruction of brain. It is
not utilized for edible purpose.
(c) Non penetrative percussion stunner: Non penetrative percussion stunner are used
in calves. Where brain are used for edible purpose.
(ii) Stunning By Gas: Carbon dioxide is used fairly widely throughout the world.
Carbondioxide is usually stored in cylinders or bulk tanks as a liquid under
pressure. It is also available in solid form for which a converter is necessary. Solid
CO2 is occasionally used for refrigeration purpose.
Various concentrations of gas have been tried, but it is now accepted that a
concentration of 65-70% CO2 in air is most suitable for stunning. It is important
that in addition to the correct concentration of gas, the period of exposure should
be 45 seconds and bleeding should takes place within 30 seconds suitable for pigs,
sheep.
There are 3 forms of apparatus used (i) The oval tunnel (ii) The clip lift (iii)
The ferris wheel.
The advantages claimed for CO2 anesthesia include meat and offal free
from harmful residues, relaxed carcasses allowing easier dehairing and dressing,
less noise, reduced labour requirements, bleeding is more efficient & blood
splashing is eliminated.
(iii) Electrical stunning: The method consists in passing a low voltage alternating
current through the brain of the animal; the instrument most commonly employed
being one which resembles a pair of tongs. It is widely used for the stunning of
pigs & poultry and in some abattoirs for sheep & calves.
The conditions necessary for the production of genuine anesthesia are as
follows:-
(i) The strength of the electric shock should be of sufficient magnitude; this is
dependent on the strength of the current, which should not be less than 250 mA,
and the voltage which should not less than 75 volts.
(ii) A genuine electric shock will be induced if the current is applied for a
sufficient time of 10 seconds.
29
 (iii) The electrodes should be correctly positioned so that the current will pass
through the thalamus and cortex, the chief sensory centers in the foe brain.
(iv) The animal should be bled immediately after unconciousness has been
produced, otherwise it may regain conciousness. Bleeding performance is better in
this method.
In electrical stunning a rapid rise in blood-pressure because of
vasoconstriction & an increase heart rate are noticed so immediate bleeding
should be done after stunning. If there is delay in bleeding then bleeding
performance becomes poor & muscular hemorrhage or muscular splashing or
blood splashing condition may occur.
(B) Ritual method:
(i) Halal method: This method is used by Muslim in India. Majority of animals are killed
by this method. In this method bleeding is performed by severing the carotid
arteries and Jugular vein. The halal method does not forbid the practice of stunning
however stunning is not performed prior to slaughter. In this method bleeding is
satisfactory as nerve center which control the heart and respiratory rate remain
intact & functioned continuously & bleeding is perfect. So meat produced by this
method is of good quality.
(ii) Jhatka method (Hindu/Sikh method): In this method head is decapitated by one
stroke of sword. In this method bleeding is not perfect as head is removed and there
remains no contact with nervous system so heart & lung does not work for more
times.
(iii) Jewish method: In this method stunning is forebidden and act of slaughter has to be
carried out by incising the throat without pause, pressure, stabbing, slanting &
tearing.
All the soft structure anterior to cervical spine is severed & neck is cut with help of
sharp sword including the carotid artery and jugular vein. The process of Jewish
slaughter is known as shechita. The person performing the slaughter process is
known as sochet. Meat which is fit for human consumption is known as Kosher
while meat which is unfit under Jewish method is known as Terefa. In this method
bleeding is perfect but blood cannot be used because it is contaminated by food
material from oesophagus.

30
 PRACTICAL No. 9

AIM: SPECIATION OF MEAT

Meat species speciation or specification is an area which needs specialized attention
in the food quality management system. It is a vital field to ensure the food safety to the
consumers and it conserves the laws related to meat and meat products. Fraudulent
substitution is a malpractice in meat industry, in which inferior or cheaper quality meat is
mixed into superior quality meat. The common frauds are the substitution of meat of another
species, i.e., horse for beef especially in Britain and Ireland, beef in kangaroo meat in
Australia, cat for chicken or rabbit, goat meat for mutton, mutton for venison, dog meat and
cat meat for chevon in other countries including India. As per an estimate about 25-30% of
meat sold in India is adulterated. These practices are more common in comminuted meat
products. For detection of meat species in adulterated meat have several techniques started
from simple physical tests to recent sophisticated molecular techniques.
Various methods are available right from physical, chemical, anatomical, histological
and biological to sophisticated molecular techniques. If the meat is in carcass form, it can be
easily identified with the physical, chemical, anatomical and histological methods but the
reproducibility and quantitative identification is not possible. In biological methods we use
the simple method of antigen-antibody reaction for visual identification. In electrophoresis,
the migration of the protein moiety according to their molecular weight under the influence of
electric field principle is applied. The band patterns produced in this technique is them
visualized for result interpretation. In recent molecular techniques DNA and RNA
amplification is done to produce the fingerprints as per the characteristics of an identical
genetic material for a particular meat species. Now the development of PCR technique makes
easy to identify the meat species even from the cooked and spoiled meat in which protein is
easily destroyed. Real time PCR is the revolution in this field in which we can identify and
monitor the product during its amplification. Although no single technique is sufficient for
differentiation of all types of meat species and meat products.

31
1. Physical techniques: It is a combined perception of colour, texture, odour and
presence of other body parts along with meat. It gives the primary idea about the meat
species on the basis of quality characteristics of the meat.
Physical quality characteristics of meat of different animals:

Physical quality characteristics of fat of different animal species:

2. Anatomical techniques: The primary identification method for meat species is dental
formula if teeth are attached with the carcass. Another anatomical technique for
carcass identification is on the basis of vertebrae and ribs number present on the
carcass.
3. Histological techniques: In this technique muscle fiber length, diameter, density and
pattern of the muscle fibers in different meats of animal origin measured. Frequently
encountered case of cow and buffalo meat mixing and illegal slaughtering may be
identified by the histological techniques.
4. Chemical techniques: For meat species specifications various chemical tests are of
immense value. In these tests the amount of certain chemicals presents in the meat of

32
 different animal species is estimated. On the basis of its contents present in particular
meat we can easily get an idea about the meat species.

Chemical characteristics of meat of different animal species:

5. Biological techniques: These techniques are mainly based on the principles of

antigen antibody reactions. The homologous antigen binds with the antibody which is
visualized by various methods. These techniques are simple and can be performed
anywhere with little efforts. These tests are also known as Serological or
Immunological methods. Eg. Precipitation test, complement fixation test (CFT)/
Immunodiffusion Test or Agar Gel Precipitation Test (AGPT) or Agar Gel
Immunodiffusion Test (AGID), Enzyme-linked immunosorbent assay (ELISA).
6. Electrophoresis techniques: These techniques are based on the separation of proteins
by their differential migration through a supporting medium under the influence of
electric field. The protein bands thus resolved are visualized by general,
enzymological, chemical or immunological means. Eg. Poly acrylamide gel
electrophoresis (PAGE), Sodium Dodecyl Sulphate PAGE (SDS-PAGE), Counter
immunoelectrophoresis (CIE), Isoelectric focusing (IEF), Immobiline gels and
immunoblotting.
7. Molecular techniques: DNA is molecule of choice for species specifications due to
its stability during heating and processing. DNA molecular based species specification
is possible in the foods obtained from identification rendered meat products,
genetically modified foods etc. Eg. DNA hybridization technique, Polymerase Chain
Reaction (PCR), PCR techniques using multi-locus primers, Random amplified
polymorphic DNA fingerprinting (RAPD), Amplified fragment length
polymorphism, PCR techniques using mono-locus primers.

33
 PRACTICAL No. 10

AIM: PHYSICAL AND BACTERIOLOGICAL QUALITY OF MEAT AND AQUATIC

FOODS (FISH)
1. Physical methods: Measurement of pH changes and measurement of refractive index of
muscle juices
2. Direct bacteriological methods: Determination of total aerobes & determination of total
anaerobes
3. Physicochemical methods Determination of extract-release volume & determination of
water-holding capacity
A). Determination of pH of meat sample:

Result:

Interpretation:

B). Extract release volume (ERV)

The Extract release volume is a valuable tool in determining incipient spoilage in
meat as well as predicting the refrigerator shelf-life of meat.
Extract release volume is based on the amount of aqueous extract released from a
slurry of meat when allowed to pass through filter paper for a given period of time. Fresh beef
of good organoleptic quality with a relatively low bacterial number releases large volume of
extract (high ERV), whereas beef in the process of microbial spoilage with a high bacterial
number releases less (less ERV) or none. An ERV value of 25 m1 acceptable and
unacceptable ground (minced) beef to be more meaningful, the data of ERV must be
correlated or compared with other routine tests viz. colour, odour, total bacterial count (SPC)
and ninhydrin- positive substances(amino acids) from the same meat.

Determination of ERV:
Materials: Meat sample, 0.05 M PBS (pH 5.8), weighing balance, petri dishes, homogenizer,
beaker, funnel, Whatman filter paper No.1, measuring cylinder, and pH paper.
Procedure:
A 15 gram minced meat sample is blended in a tissue homogenizer for 2 minutes with
60 m1 of 0.05 M PBS (pH 5.8). Homogenate is immediately filtered through Whatman filter
34
paper No.1. Filtration is carried out at room temperature for 15 minutes and filtrate is
collected in a measuring cylinder. The volume of extract depicts ERV. Meat with an ERV
above 25 ml is considered satisfactory for human consumption. As the meat storage time
increases, there is reduction in ERV progressively and increase in microbial count.

Results and Interpretation:

C) Water holding capacity (WHC)

Water holding capacity of meat which mainly depends on the degree of hydration or
muscle protein, plays an important role in maintaining consumer qualities of meat such as
appearance, texture, tenderness, juiciness as well as weight loss or shrinkage during cooking.
In muscles, most of water is present in myofibrils and in spaces between actin and myocin
filaments. Hydration water in muscles is found mainly in two forms. One part of hydration
water of muscle is tightly bound which has different physical properties such as freezing
point, dissolving power, vapor pressure and density than simple water. This water layer is
called as "fixed bound hydration water" or the "true hydration water". The other part of water
is physico-chemically defined as "free water" or "loose water" which can be immobilized on
applying any force. Thus, water holding capacity is the ability of meat to hold all or part of its
own and or added water during application of any force such as pressing, heating and
grinding. A beef of good organoleptic quality and low bacterial count has a low WHC (high
free water on pressing) whereas microbiologically spoiled beef has a progressively higher
WHC manifested by smaller free water.
The changes in water holding capacity of meat are influenced by number of factors
such as age and sex of animal, concentration of salts, stage of rigor and the pH of meat. At pH
5.0, the WHC of meat is at minimum but it continue to increase when bases are added to
muscle, WHC of meat decreases with age and is more in females than in males.
The amount of free water in beef, pork, veal and mutton varies from 30-50% of the total
moisture contents, depending on the kind of meat and period of aging.

Determination of WHC
The measurement of WHC of meat is carried out in different ways all over the world. Most of
the methods are based on measuring the loose water liberated by applying pressure on the
muscle tissue.
This pressure can be produced in different ways to determine the WHC of meat.
1. Method of sedimentation
2. Method of centrifugation
3. Method of filtration

35
 4. Filter paper press method
To measure the WHC in relation to microbial quality of meat, filter paper press
technique has been widely used which is very simple and rapid as described below.
Filter paper press method
A definite amount of meat sample is placed on a filter paper with defined moisture content
(Whatman No.1) and pressed between two plexi glass plates making thin film. The water
which is squeezed out is absorbed by the filter paper. The area of the ring of t1uid, which is
obtained by subtracting the area of the meat film from the total area, is proportional to the
amount of water released. Below the area of pressed meat, the pressure is so high that no
water or very insignificant amount of water is absorbed by the filter paper. This simple
technique can be carried out in a: slaughter house.
Materials: Meat sample, Whatman filter paper No. I, forceps and scissors; Plexi glass plates,
weights and electronic balance.
Procedure: An exactly 1 gram finely ground meat sample is weighed and placed between
two previously weighed Whatman filter papers. Then, these filter papers with meat sample
are placed between two plexi glass plates and pressed with at least 18 kg weight for one
minute forming a round thin film. After removing the upper plate, the meat film area and
wetted area are marked with a coloured pencil. After removing the meat film, the filter papers
are stored for surface measurement.
Note: Another sample of same meat is also processed to determine the total moisture content.
For this purpose, one gram minced meat sample is weighed by difference in dried aluminum
disk and kept in hot air oven removing the lid at 100-105° C for 16-18 hour. After cooling in
desiccator, loss in weight is calculated as moisture content of the sample.

Observations and interpretations:

The surface area of the free moisture (wetted ring) around the pressed muscle is determined
by subtraction of the surface of the meat film from the entire surface.
Then, the difference is multiplied by regression coefficient of 61.1 (water absorbance
factor per square inch) which gives the amount of free water in the meat sample being
pressed. The results are best expressed as the percent of the free water out of the total
moisture contents of the meat.

(Total wet area- meat film area) x 61.1

Percent free water = _________________________________ x 100

Total moisture (mg) in muscle sample

The per cent bound water or WHC equals 100 minus per cent free water.

Modification of pressing method:

36
 In a modification of the above technique, after pressing the meat sample between two
plexi glass plates, the amount of free water is detected by subtracting the weight of dry filter
paper from the weight of wet filter paper. Then the water holding capacity of meat is
estimated by subtracting the amount of water released on pressing from total moisture content
of meat.
Free water = weight of wet filter paper - weight of dry filter paper

Total moisture content - Free water X 100

____________________________
WHC=
Total moisture content
The amount of tree or bound water can be expressed as percent of the meat weight or as the
amount of bound or free water per unit weight of protein of the muscle.
Advantages: 1.The technique is applicable for ground (minced) and unground tissues with
without added water and heat-denaturized meat can also be used.
2. Only smaller quantity of tissue (about 0.3g to 1 g) or homogenate is required.
3. Technique requires only few minutes to obtain results.
4. Results are fixed on filter paper for future use.
5. Apparatus, technique and evaluation are very simple.

Disadvantages: The technique is not applicable for samples that contain salts and large
amount of fat such as sausages.

Results and interpretations:

37
 Practical No. 11
AIM: TOXIC CHEMICAL AND MICROBIOLOGICAL RESIDUES IN MILK AND
MEAT
Residues in the widest sense may be defined as undesirable substances present in
milk/meat. These substances are chemical or biological in nature and have always been
present in a small amount or can be introduced into the environment by various technological
practices, can arise as a result of incorrect storage of food stuff, can get in to the food chain
due to modern agricultural practices and thus introduced into the foods or they may be results
of medicines given to the animals or of processing methods.
Detection of Residues in Milk and Meat:
Pesticides Detection:
1. Conventional Methods: A number of analytical techniques such as colorimetric
method, Thin Layer Chromatography (TLC), High Performance-Thin Layer
Chromatography (HP-TLC), Gas Liquid Chromatography (GLC), Gas
Chromatography-Mass Spectrometry (GC-MS), High Performance Liquid
Chromatography (HPLC), Liquid Chromatography-Mass Spectrometry-Mass
Photometry (LC-MS-MS) etc., are established for detection and quantification of
pesticide residues in animal tissues
2. Modern Methods: The new assays like Biosensors based on enzyme inhibition or
biosensors based on immunological assays used for quantification of either an
individual or a class of pesticides. However, there are very limited applications of
biosensor detection of pesticide compounds in meat system.
Veterinary Drugs Detection:
Conventional Methods: For determination of veterinary drug residues in foods, currently, 6
types of detection methods are commonly used. These include microbial growth inhibition
assays, microbial receptor assays, enzymatic colorimetric assays, receptor binding assays,
chromatographic methods and immunoassays. But microbial growth inhibition assays and
later 2 methods are popular for monitoring of antimicrobial residues in meat and meat
products as are capable of detecting a broad range of these drugs.
Modern Methods: Most of the biosensors developed are aimed at determining them in
biological or food samples. The Surface Plasmon Resonance (SPR) technique has been
developed and demonstrated for on-line/at-line detection of veterinary drug residues in milk,
porcine bile and bovine urine, including a commercial handling robot.
Mycotoxins and bacterial toxins Detection:
Conventional and Modern Methods:

38
 The TLC and HP-TLC is regularly used for determination of mycotoxins in foods. The
specific sensors for bacterial toxins and mycotoxins using an integrated optical sensor,
potentiometric immunosensor, impedance-based immunosensor are also developed.

Detection of Antibiotic Residues in Milk

Release of antibiotics in milk (treatment of mastitis is usual source of antibiotic in

milk is significant from the point of view of economic and public health significance since it
interferes in the preparation of dairy products and also responsible for allergic reaction.
Principle: Tests depends on the growth of bacteria in the test and control milk sample in the
presence and absence of antibiotics.
Requirement: Sterile Petri plates, incubator, Hot air oven, water bath 45oC; Refrigerator,
well cuffing device, desiccators, etc.
Reagents: Nutrient Agar, NSS, Sample milk containing 0.02 IU Penicillin/ml; milk
containing 0-01 IU penicillin/ml standard bacterial culture (Sarcina lutea, Bacillus subtilis,
B.stearothermophilus).

Procedure
1. Prepare nutrient agar plates.
2. Inoculate the plate with broth culture properly diluted with NSS (usually 2 parts of
culture with 3 parts of diluents)
3. Pour the dilute culture over the agar and allow to stand for 5 minute
4. Drain off excess fluid.
5. Dry the uncovered plain for 30 min in the desiccator.
6. Punch the wells a the agar with pinching device
7. Remove the agar from the punched well
8. Seal the bottom of the holes with melted agar medium (optional)
9. Fill the holes with the milk to be examined in its present state.
10. Place the plates in the refrigerator at+4oC for 2 hours.
11. Put the filter paper in the lid and incubate at 37oC for 24 hours.
Control: In order to test whether the bacterial strain is sensitive;
- Add 0.02 and 0.01 IU of penicillin/ml to two milk samples and repeat the procedure as
mentioned above.
Result:
No growth of bacteria or : Milk contains
zone of inhibition around antibiotics
punched holes.
Growth of bacteria or no : Milk is free of
zone of inhibition antibiotics
39
 (Always compare the results with control)

Interpretations:

Practical No. 12

AIM: VISIT TO ABATTOIRS, MEAT PROCESSING PLANTS, MARKETING

CENTERS AND FOOD SERVICE ESTABLISHMENTS

40