ALLEN’S MICROSCOPIC METHOD 405 
slide and spread it evenly over an area of 2 sq. cin. using a loopful of 
sterile water if necessary for uniform spreading. 
5. Place in the incubator until nearly dry, then finish drying at room 
temperature. 
6. Heat slightly above the Bunsen flame. 
7. Place in a tray of toluene and agitate the tray several minutes 
to remove butter fat, then take the slides out of the tray and allow the 
toluene to evaporate from them. 
8. Place the slides in 95 per cent alcohol to remove all traces of 
toluene. 
9. Stain in 25 per cent saturated aqueous solution of methylene 
blue until the film reaches a proper depth. 
10. Dry in the warm air above a Bunsen flame and examine under 
the microscope, using an oil immersion lens of which the number of 
fields per square centimeter has been determined. 
11. Count 20 representative fields on the square centimeter and 
obtain the average number of bacteria per field. 
12. The number of bacteria per cubic centimeter of milk is obtained 
by multiplying the average number of bacteria per field by the number 
of microscopic fields in a square centimeter. Usually there are a little 
over 3000 fields per square centimeter when the ordinary 1.8 oil immer- 
sion lens is used but using 3000 as an even number to multiply by is 
satisfactory in counting. 
Allen also gives the following precautions: Care must be exercised 
not to use a suspension of aluminum hydroxide too strong to give too 
thick a smear. The thickness of the plug of aluminum hydroxide after 
centrifugalization should be about 1 mm. No fat or toluene should 
appear in the field under the microscope. Some samples of pasteurized 
milk show fine particles of curd due to heating. These may be removed 
by filtering through a small amount of cotton. The film should be 
uniformly stained. Rubber stoppers should be used with the centrifuge 
tubes as cork stoppers will not hold the liquid in the centrifuge. If toe 
deep a stained film is secured, it may be decolorized sufficiently by dip- 
ping in alcohol. 
Interpretation of Results. “ The interpretation of the bacteriologi- 
cal analysis of market milk must depend upon the history of the milk. 
It is, therefore, difficult to give any general interpretation. The fol- 
lowing are a few significant conclusions: 
1. ‘‘ Where the analysis can be made immediately after the milking 
the number of bacteria enables conclusions to be drawn as to the cleanli- 
ness and care in the dairy and the thoroughness in the cleaning and 
