Module 4. Microbiological methods of milk testing
DIRECT MICROSCOPIC COUNT
Direct Microscopic Count (DMC) is a quantitative test and is helpful in assessing the actual number of bacteria present in milk. DMC is used as a platform test to assess the microbiological quality of milk received at the Raw Milk Receiving Dock. The method is useful for rapid estimation of the total bacterial population of a sample of milk and also in giving useful information for tracing the sources of contamination of milk.
17.2 Method of DMC
It is based on the examination of stained thin film of a measured volume of milk spread over a specified area on a glass slide. The method is useful for rapid estimation of the total bacterial population (including live and dead cells) of a sample of milk.
In this test, milk smear is prepared on one square centimetre area. The smear is stained with a special stain called Newman's stain and examined under microscope. Each microscopic field examined represents a quantitative aliquot of the milk sample. The number of microscopic fields occurring in one square centimetre area of the milk smear will vary as the diameter of the microscopic field varies with different microscope. This necessitates the calculation of microscopic factor for each microscope separately.
17.2.2 Microscopic factor
Microscopic field is an area of the field observed through the microscope. It is measured by πr 2 where ‘r’ is the radius of the field. The diameter of the microscopic field is measured with the help of a stage micrometer. Each division on the stage micrometer is equivalent to 0.01 mm. The diameter of the microscopic field varies with the length of draw tube, objective lens and ocular tube. As the diameter of the field varies, the number of microscopic fields occurring in one square centimetre area (100 sq mm) will also vary and hence there is a need to calculate the microscopic factor. The microscopic factor is calculated as follows:
17.2.3 Preparation of slide
Take 0.01 ml milk with the help of a sterile Breed’s pipette and spread it evenly on a grease free slide of 1 cm2 marked area on a Breed’s slide. Dry the smear on a warm surface at 40 - 45°C. Do not heat-fix the slide on direct flame. Rapid drying results in cracked surfaces on the film or peels off during further processing. Immerse the slide in Newman's stain for ½ to 1 minute. Newman's stain removes the milk fat, fixes the smear and stains the bacteria in a single operation. The tetrachloroethane of the stain helps to dissolve the milk fat globules, ethyl alcohol fixes the smear and methylene blue stains the smear.
17.2.4 Microscopic examination
Examine under the oil immersion objective and count the number of micro-organisms (individual or clumps of cells) in a number of fields of the film. The fields for counting the bacterial cells are selected at random. The number of microscopic fields occurring in one square centimetre area of the smear is very high. Thus, a representative number of fields depending on the concentration of bacterial cells in a microscopic field are chosen for counting the bacterial cells as follows
Table 17.1 Number of microscopic fields to be counted in DMC
Calculate the average number of clumps per field and multiply by the microscopic factor to get the DMC per millilitre of milk. Interpret the results by comparing with the standards and assess the quality of milk samples as per Table 17.2.
17.2.5 Grading of milk
The quality of raw milk is adjusted using the following details
Table 17.2 Grading of milk based on DMC (BIS standards)
17.2.6 Sources of contamination of milk
This test is very helpful in obtaining useful information about the sources of contamination of milk during production.
Table 17.3 Detection of source of contamination of milk using DMC
17.2.7 Advantages of DMC
· DMC is widely used to screen incoming raw milk supplies as a platform test to determine whether the milk has an acceptable or legal bacterial load, as per BIS standards.
· Easy to perform.
· Less time is required to perform the test.
· Large number of samples can be screened in a given period of time
· Useful in providing the estimated counts, types of bacteria and somatic cells in milk.
· DMC can be used as a guide in identifying the types of bacteria present in a milk sample.
· Possible to find out the source of contamination of milk.
· A record of test, in the form of a slide for legal verification can be maintained.
· Equipment and manpower needed to assess the microbiological quality are less.
· Possible to find out the mastitis infection due to presence of somatic cells.
17.2.8 Disadvantages of DMC
· Not considered as a fool-proof/ legal method.
· Strain on the eyes of the operator is too much.
· Test is not reliable as both viable and non-viable cells are counted.
· Method is not suitable for pasteurized milk.
· Experienced person can carry out the test.
· Results are not reproducible because microbes are unevenly distributed in the smear.