A402 MILK AND MILIX PRODUCTS 
instead of smears; otherwise the method is somewhat the same. In 
general, some of the same objections which are applied to Breed’s method 
may be applied to the Frost method. The method is outlined by Frost 
in the following words: 
Preparation of Glass Slides. The little plates are made on the 
ordinary microscopic glass slides (2.5 by 7.5 cm.). These are carefully 
cleaned and are then ruled with a grease pencil so that a square surface, 
over the center of the slide, is surrounded by a grease line. The included 
area is 4sq.em. The ruling is best done by having a wooden or card- 
board frame for the slide and some arrangement, as brads, to hold the 
straight-edge in place. These ruled slides are then sterilized by passing 
them through a gas flame, and then they are placed on a warm box 
(45° C., 118° F.) provided with an overhanging top to protect them from 
dust. 
Preparation of the Plates. A few tubes of ordinary nutrient agar 
are then melted and placed in a water bath at 45° C. to cool. Some 
sterile plugged test tubes are also put in this water bath to warm up (one 
for each sample of milk to be tested). The milk sample to be tested is 
then thoroughly shaken and I ¢.c. is transferred to one of the warm 
sterile test tubes. To this is added an equal amount (1 e.c.) of the 
melted agar. The milk and agar are thoroughly shaken, care being 
taken to prevent the agar from cooling below 40° C. It is best to warm 
it up frequently by putting it into the 45° C. bath. 
One-tenth (0.1 c.c.) of the milk and agar mixture is then transferred 
to a warm glass slide and spread as quickly and evenly as possible over 
the ruled surface. This may be readily done with the help of a pipctte 
while the slide is on the warm plate. The agar is allowed to set firmly 
by placing it on a level surface under cover for a few minutes. This 
makes a little plate culture containing 0.05 c.c. of milk. 
When the milk being examined is supposed to contain more than a 
million bacteria per cubie centimeter, it should be diluted with sterile 
water or sterile milk before it is mixed with the agar. The dilution 
may be 1:10. This would make the final dilution as much as 
1: 200. . 
Incubation. This is accomplished by placing the little plates in 
an incubator in a specially prepared cabinet, or moist chamber with a 
layer of water in the bottom to maintain a saturated atmosphere. A 
Petri dish may be used for a few slides. The incubation temperature 
is 37° C. for a varying length of time. Five or six hours should be 
allowed. For samples which contain many bacteria such as market 
milk which has been kept in a warm place three or four hours will be 
