FROST'S MICROSCOPIC PLATE METHOD 403 
sufficient. For pasteurized milk from ten to twelve hours may be 
necessary. 
Drying the Plates. When the colonies are sufficiently large the 
plates are dried. This should be done carefully. Too rapid drying 
will crack the films. They will dry well in the incubator or over a 
steam radiator. 
Staining. This is accomplished in the following manner: 
1. Fix in the flame. 
2. Put in a 10 per cent solution of acetic acid in 95 per cent alcohol. 
3. Stain with Loeffler’s methylene blue (1 : 4) for three minutes 
4. Decolorize in 95 per cent alcohol for a few seconds or until the 
background is a pale bluc and the colonies stand out prominently. 
5. Dry without washing. 
Counting of Colonies. For the purpose of counting the colonies, 
the slides may be examined with the low-power dry lens. If the struc- 
ture of the colonies is to be studied it is always best to use a mounting 
medium and the cover glass. Cedar oil serves quite well and in that 
case the cover glass may be removed for use with the oil immersion 
but Canada balsam may be used in the usual way. A microscopic 
examination of these plates reveals colonies of considerable size stained 
a dark blue in a light blue field. The staining process should be so 
regulated that the colonies will be stained a dark blue in a light blue 
field. At least twenty fields should be counted on each plate and the 
plate should be gone over carefully in the selection of these twenty 
fields. 
Calculation. The number of bacteria per cubic centimeter may be 
determined by multiplying the number of colonies in a microscopic 
field by the number of times the area of this field is contained in 400 
sq. mm. or the area of the little plates times the dilution used. The 
area of the microscopic field for any definite combination of lenses and 
tube length is a constant and may be determined by the formula: 
Area=R. The radius may be determined by a stage micrometer. 
The same tube length must be used that was used in the standardization 
of the microscope. When the microscope factor is known this formula 
may be used: 
Number of colonies counted 
“Number of felds counted > Reciprocal of dilution of milk. 
Microscope factor=number of bacteria per cubic centimeter in the 
milk, Example: 
