Laboratory examination of milk

The laboratory examination of milk is one of the most valuable and necessary aids to the overall milk
quality control program. Without this service the safety and sanitary quality of milk is difficult to
determine. Although the environmental health worker may not personally conduct such tests, he or she
must be able to send samples to the laboratory and able to interpret the results of laboratory tests. In
order to make milk safe it may have to be handled with maximum care during milking, processing or
storage. Milk may also undergo pasteurization or other types of heat treatment. Regardless of this the
condition of the milk has to be checked at all stages. For example:

It has to be cheeked for its bacteriological content just after milking to see the extent of the bacterial
colony • It also has to be checked after pasteurization to see if the process has inactivated all enzymatic
activities.

Sample taking • If the milk is contained in bottles, one unopened bottle is enough. • If the milk is in
bulk containers it must be mixed with a sterile plunger before a sample is collected from well below the
surface with a sterile dipper and poured into a sterile stopper or screw cupped bottle with a capacity of
about 125ml. • Place the sample in an insulated box, label and dispatch to the laboratory.

Types of laboratory examination of Milk

Clot on Boiling Test (COB) The COB test is an old but very easy test designed to determine the overall
quality of milk. Though it should be considered a preliminary test, it can be easily done in the feld or on
the farm. In the COB test, the milk is boiled, and once the milk cools, physical characteristics are
examined. When high-quality milk is boiled and cooled, it will remain a fuid with a general milk
appearance. Milk that is acidic (an indication of bacterial growth) or milk that contains colostrum or
other adulterations often cannot withstand boiling; once it cools, it will coagulate and form lumps or
fakes. MATERIALS • Milk sample • Spoon • Flame PROCEDURE 1. Start fame away from all fammable
objects. 2. Place milk in spoon using a pipette or similar device. 3. Hold milk/spoon over fame until milk
boils. 4. Allow milk to cool. 5. Check for coagulation.

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Ethanol/Alcohol Test

The ethanol/alcohol test is a rapid way to determine overall quality of milk. It relies on how milk reacts
with ethanol. High-quality or otherwise normal milk will not react with ethanol. Milk that is high in
acidity (low pH), contains colostrum, or contains salt concentrates (indication of mastitis) often will
coagulate or form fakes when exposed to ethanol. MATERIALS • Milk sample • 68% ethanol (often
abbreviated “EtOH”) • Mixing container PROCEDURE 1. Combine equal parts 68% ethanol and milk
sample (example: 10 mL of milk sample + 10 mL of 68% ethanol). 2. Mix thoroughly. 3. Incubate at room
temperature for 10 minutes. 4. Check samples for coagulation. READING RESULTS The samples pictured
to the right are milk samples after the addition of ethanol and a 10-minute incubation at room
temperature. Sample A is fresh milk with little to no microbial growth. It does not react with alcohol and

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retains a normal milk appearance. Sample B has a low pH (high acid) due to microbial growth. This milk
reacts with alcohol, and heavy coagulation and faking can be seen on the sides of the fask

Sediment Test

This is done in the field or laboratory. The purpose is to determine the amount of insoluble visible filth
or extraneous matter in the milk. To undertake the test: From a mixed milk sample force a measured
amount of milk through a tester made of stiff cotton or pad discs measuring about 1 inch in diameter
whereupon the sediment is collected. If this is not available a simple filtration through a filter paper can
do the same job.

Interpretation: Although the amount of sediment on test discs often has little relation or no relation to
the bacterial content of the milk, results of this test provide information about the care exercised during
the handling of the milk. The sediment on the pad is graded as clean or fairly clean, compared with
ready-made photographic standards. Therefore poor/dirty sediment is indisputable evidence of
carelessness while clean sediment may merely represent efficient straining on the farm. Methylene-Blue
Reduction Test The purpose is to determine the quality of milk before pasteurization. This test is
particularly applicable to raw milk. The test involves determination of time required for the
disappearance of color when methylene blue thiocynate solution is added to raw milk. In a test tube
with 10 ml of milk add 1 ml of standard methylene blue solution. The sample is mixed and then place

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either in a hot water bath or in an incubator at 350 C -370 C. Observations are made at intervals of 15-20
minutes for an 8 hour period to determine the time required for the disappearance of the blue in the
sample. Interpretation: Milk with a high bacterial content will decolorize the dye quite rapidly whereas
milk with a low bacterial content retains the blue color for several hours. On the basis of this test milk
can be graded as follows:

Excellent: Very low bacterial count. Its decolorized time is about 8 hours i.e. not decolorized in 8 hours
Good: Low bacterial count. Decolorization time is 6-8 hrs i.e. decolorized in less than 8 hours but not less
than 6 hours Fair: High bacterial count. Decolorization time is below 2 hours i.e. decolorized in less than
2 hours Poor: Very high bacterial count. Decolorization time is below 2 hours i.e. decolorized in less than
2 hours Results of the test may be effectively used as a basis for improving the milk supplies, particularly
milk intended for pasteurization. This test, combined with field investigations, serves as an effective
means of correcting improper production methods and improving the general sanitary conditions of the
dairy farm.

Resazurin Reduction Test

The purpose is to determine the sanitary quality of raw milk. This test is quite similar to that of the
methylene blue test, except with changes in color in a very short time. This test also undergoes a series
of color changes whereas methylene blue changes from blue to colorless. In a test tube with 10ml of
milk add 1/10 (0.1ml) of resazurin solution (0.05%). After mixing properly the sample is placed in a water
bath or incubator at 350 C-370 C. Observations for color changes are made at 15 minute intervals. The
two most common applications of the resazurin test are: 35 The one-hour test: In this test the color of
the milk is compared after 1 hour at 370 C with several exactly described color standards. The three
hours (triple reading) test: In this test 3 readings are made at 1-hour interval to see changes in color.
Interpretation: Milk with a high bacterial count and other infections of the animal is detected quite
sharply with the resazurin test. A resazurin pink of 3 hours or longer represents a good quality of milk.
High-grade (acceptable) milk requires at least 3 hours.

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Standard plate count (agar plat count)

The purpose is to estimate the bacterial population of the milk and dairy products prior to heat
treatment. In this test a 1ml portion of the milk sample is diluted with distilled water to give dilutions
ranging from 1/10 to 1/1000. A 1 ml portion from the dilutions is then placed in a Petri dish/plate
containing a growing media (milk agar). This sample is incubated at 350C-370C for 48 hours. At the end
of 48 hours a count of colonies developing on the plates is done with help of a “Queven bacterial colony
counter.” Interpretation: The number of colonies counted multiplied by the dilution factor gives what is
known as the standard plate count (SPC) or the estimated number of bacteria per milliliter of sample.
For 36 example, if 30 colonies are counted in a Petri dish of 1/100 dilution, the SPC is 30x1000= 30,000.
The standard is as follows: • Raw milk: 200,000 maximum acceptance • Pasteurized milk: 30,000
maximum acceptance. . The interpretation of the results is based upon the fact that the bacterial
population in milk serves as an index of sanitary quality i.e. a low bacterial population indicates correct
handling and processing, whereas a high count many indicate the reverse.

Using a Lactometer

A lactometer can be used for several purposes. It is basically a tool to measure the specifc gravity or
mass of milk. For example, milk has a higher specifc gravity than water, so a lactometer can be used to
test for added water. It can also be used to estimate total solids of milk. The total solids are basically the
non-water portions of milk and make up about 13% of all milk (~4% fat and ~9% protein, lactate and

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other non-fat solids). The quality of milk for further processing, such as cheese or yogurt, is dependent
in many ways upon the total solids concentration of the starting milk.

MATERIALS • Cylinder

PROCEDURE 1. Heat fresh milk sample to 40C and cool to room temperature. (Heating reduces the size
of fat globules, which can impact the lactometer) 2. Mix cooled milk sample and add to cylinder. Add
enough milk so that the lactometer will not touch the bottom of the cylinder. 3. Lower the lactometer
into the milk sample and allow it to settle (foat). This can take up to 1 minute. 4. Record the lactometer
reading at the bottom of the meniscus. 5. Record the temperature

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In the example above, the lactometer reads 120 and the temperature is 65ºF. If the milk is too warm or
too cold, the lactometer reading has to be corrected. The acceptable range with the lactometer pictured
above is 60ºF +/10 degrees. The lactometer reading must be adjusted for the temperature if it is outside
of this range (+1 for every 10 degrees above 60ºF and ‐1 for every 10 degrees below 60ºF). The
lactometer pictured above is “New York” style lactometer. The “quevenne” style is also common
throughout the world. The only diference is the scale. With both lactometers, a reading of 0 translates
to a specifc gravity of 1 (water). To determine the specifc gravity with the New York scale:

Specifc gravity = (lactometer reading X 0.29)/ 1,000 +1

From the sample above:

Specifc gravity =(120 X 0.29)/1000 + 1 = 1.0348 Kg/mL

This is the normal specifc gravity for fresh, unadulterated milk. The examples below illustrate how a
lactometer can be used as a frst step in determining if milk is adulterated.

Coliform Test The purpose is to determine the presence of organisms of coli form group in milk. It is
used primarily to detect contamination after pasteurization. In this test the dilutions 10ml, 1ml, 0.1 ml
from the milk sample are transferred into appropriate media and then incubated for 48 hours at 370C.

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At the end of this period these preparations are observed for the presence of gas (refer to water analysis
for complete procedures). Interpretation: A positive test on pasteurized milk indicates contamination
after heat treatment. That is the presence of coli form organisms in pasteurized milk is a signal for the
source of contamination in the milk processing plant.

Phosphate Test

The purpose is to determine the efficiency of pasteurization or to check the adequacy of pasteurization.
The test is based on the fact that raw milk contains an enzyme, phosphatase,the presence of which is
easy to detect and which is practically destroyed by heating at pasteurization temperature and time. The
test depends upon the hydrolysis of a disodium phenyl phosphate to form phenol and phosphate. The
addition of BQC (dibromo quimone chloramines) causes a blue color reaction to occur with the phenol.
Thus the presence of phosphatase is demonstrated by a blue reaction while the destruction of
phosphates by proper pasteurization fails to produce a blue color. The intensity of color is measured by
a colorimeter. Interpretation: If the test reveals little or no phosphatase, one may assume that the milk
was adequately heated, but if the enzyme is present it means that pasteurization was incomplete or that
raw milk has been added to pasteurized milk. It is possible to detect a 1oF temperature differential or 5
minutes under heating or the addition of only 0.5 % raw milk.

Using a pH Meter

Used properly, a pH meter is a valuable tool for testing milk quality. Most pH meters require regular
calibration for accurate measurement. A proper calibration prior to starting any experiments can save a
lot of time and produce much more accurate results. Diferent pH meters have diferent protocols for
calibration; calibration should follow the manufacturer’s instructions. Below is a standard calibration
protocol.

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Soap Test

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When soap is added to milk, the milk often becomes thicker. Therefore, milk with soap as an adulterant
can appear to have more cream. Soaps are bases, so testing for the presence of soap is a basic test of
pH. This can be done using the same phenophtalein solution that is used for the Acidity test. MATERIALS
• Milk sample

• Phenophtalein solution (0.5% phenophtalein in 50% ethanol)

PROCEDURE

1. Add 10mL of milk sample to test tube.

2. Add phenophtalein solution in drops to the milk sample.

3. Check for color changes.

NOTES ON THE SOAP TEST

Starch Test

Like soap, starch can be added to milk as a thickener to give the appearance that the milk is high in
cream. Starch can also be added to adulterated milk to increase its density; sugars are part of the total
solids of milk and part of what makes milk heavier than water. In doing so it can mask the efects of
diluting milk with water by giving the appearance that the adulterated milk has the same density as
unadulterated milk. Starch is a complex carbohydrate (polysaccharide) that reacts with iodine, unlike the
primary milk sugars lactose and galactose, which are single and disaccharides. Thus, the presence of
starch can easily be detected by adding a small amount of iodine.

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 MATERIALS • Milk sample • Test tubes • Water bath (or other device that can hold boiling water) •
Iodine solution (1%) PROCEDURE 1. Add 5mL milk sample to a test tube that can be boiled. 2. Boil milk
samples for 5 minutes. 3. Allow samples to cool to room temperature. 4. Add 2 to 3 drops of iodine
solution to each sample. 5. Observe color changes.

Total Coliform Test

TPC tests measure total bacteria in a milk sample (good and bad). A coliform test is more specifc.
Coliforms are bacteria that are generally found in the intestinal systems of animals and, in turn, animal
feces. So, the presence of coliforms in a milk sample (or water sample, etc.) is an indication of fecal
contamination. The coliform test is done just like a TPC test but uses a “selective” medium. This medium
allows coliforms to grow while inhibiting other types of bacteria, thereby “selecting” coliforms. In the
protocol here, violet red bile agar (VRBA) is used; it’s regularly recommended for testing milk and dairy
products for coliforms. It is a standard medium to detect lactose fermenting coliforms. The medium
contains crystal violet and bile salts, which inhibit the growth of most gram-positive bacteria. Neutral
Red is a dye and is included as a pH indicator. Lactose fermenting colonies (likely coliforms) appear
pink/red and may be surrounded by pink/red zones, indicating precipitation of bile salts. Note that VRBA
does not get autoclaved. It is only boiled prior to pouring plates. The procedure is exactly the same as
the TPC — either the pour-plate or spread-plate method. Only the growth medium is diferent

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