ELEMENTARY BACTERIOLOGY LABORATORY EXERCISES 51 
SECTION 18: BACTERIOLOGY OF MILK 
EXERCISE 75 
THE PLATE CULTURE METHOD FOR THE QUANTITATIVE 
BACTERIOLOGICAL ANALYSIS OF MILK 
Plate out a sample of high grade milk in dilutions of 1/100 and 1/1,000. 
Plate a sample of low grade milk in dilutions of 1/10,000 and 1/100,000. Use 
lactose agar as the plating medium. Incubate the plates at 37° C. for two days. 
After incubation, count the colonies and estimate the number of bacteria 
per c.c. of milk in each case. 
EXERCISE 76 
DIRECT MICROSCOPIC DETERMINATION OF BACTERIA IN MILK 
(1) By means of a stage micrometer, using the oil immersion objective, 
adjust the tube length of your microscope so that the microscopic field has a 
diameter of 0.16 m.m. 
(2) Spread 0.01 c.c. of milk over exactly 1 sq. cm. area on a microscopic 
slide. Make duplicate smears on the same slide. 
(3) Dry the films of milk very slowly on a perfectly level surface. Extreme 
caution must be taken if heat is applied in drying. 
(4) Immerse the slide in xylol for about 1 minute to remove fat. Dry. 
(5) When dry, immerse in 95% alcohol to fix to slide. 
(6) Stain by immersion in methylene blue (1.0% methylene blue in 50% 
alcohol) until the film has a definite blue color. Avoid over-staining. 
(7) Wash the slide with water. 
(8) In case the preparation is stained too deeply, it may be decolorized to 
a light blue by immersion in 95% alcohol. When this step is taken, care must 
be used not to decolorize the bacteria. 
(9) By use of the oil immersion objective, determine the number of bac- 
teria per field. A number of fields must be counted to obtain accurate results. 
The greater the number of bacteria in the milk, the smaller the number of 
fields necessary to be counted in order to obtain accurate results. A good rule 
is to count 100 fields in the case of a high grade milk, and in the case of a low 
grade milk, 10 fields. 
(10) From the counts obtained estimate the number of bacteria per c.c. 
of milk. 
With 0.01 c.c. of milk spread over an area of 1 sq. cm., and with a micro- 
scopic field of 0.16 m.m. diameter, the number of bacteria per field multiplied by 
500,000 gives the number of bacteria per c.c. of the original milk. 
