604 BACTERIOLOGY OF MILK 



spread a definite volume of milk upon a definite area on a glass slide, 

 evaporate the fluid, fix the sediment (which contains all the bacteria 

 in the sample), and stain it in such a manner that the microorganisms 

 are distinctly colored. The organisms of a definite area are counted 

 under the microscope. The number in the original sample are readily 

 computed, knowing the volume of milk examined, the area over which 

 it is spread and the size of the microscopic field. 



In practice 0.01 c.c. of a well-mixed sample of milk is spread 

 uniformly over an area of 1 square centimeter on a glass slide. (This 

 area is readily outlined with a wax pencil, using a pattern previously 

 ruled on a piece of paper as a guide and following the outline on the 

 glass slide; the wax pencil mark tends to limit the spread of the milk 

 beyond the limits of the square.) The film of milk is then air-dried 

 or dried at 40 C., immersed in absolute methyl alcohol for a few min- 

 utes to fix the sediment to the slide and to remove some of the milk 

 lipoids and fats which interfere somewhat with the staining, and 

 stained (after drying), with aqueous methylene blue. Alkaline 

 methylene blue should not be used because the alkali tends to loosen 

 the film of casein. 



The bacteria are counted with an oil immersion lens. It is necessary 

 to adjust the optical combination of lens and eye-piece so that the 

 diameter of the microscopic field is exactly 0.0016 cm., corresponding 

 to an area of 0.005 sq. cm. This can be readily accomplished with a 

 stage micrometer. 



Each organism in a microscopic field corresponds to one-five-hun- 

 dred-thousandth the number in a cubic centimeter of the original 

 sample of milk (.005 X 0.01 = 0.00005), because 0.01 c.c. of milk was 

 spread on an area of 1 sq. cm. and ^ of the volume is viewed in the 

 microscopic field. In other words, the microscopic field contains the 

 bacteria of 500000 c.c. of the original sample of milk and it is potentially 

 equivalent to an agar plate culture of the milk in a dilution of 500000 . 



If the bacteria were uniformly distributed, the number of bacteria 

 observed in one field multiplied by 500,000 would give directly the 

 number of bacteria per cubic centimeter in the milk; usually, however, 

 the organisms are somewhat irregularly distributed and in practice 

 several fields are counted and the average number of organisms per 

 field is multiplied by 500,000. Duplicate determinations should always 

 be made. The results obtained are fairly uniform when the exact 

 details of the method are closely followed. 



The advantage of the direct microscopical count are: a very material 



