248 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA 
sample. A large number of bacteria, by mutual antagonism, will fail 
to develop into a proportionate number of colonies. The most accurate 
results are obtained when the bacterial content of the sample as plated 
lies between 50 and 200 individual organisms. If more than 200 
bacteria are probably present, a dilution of the sample with sterile 
water is made before plating, to reduce this source of error. It is 
convenient in making dilutions to use a multiple of 10, because the 
subsequent calculation is much simplified. A dilution of 1 to 10 is 
made by adding 1 cc. of the sample to 9 cc. of sterile water, shaking 
thoroughly and plating 1 cc. If the technique is all right, each colony 
on the plate represents ro the number of living bacteria in the original 
sample. The total number of colonies multiplied by 10 gives the 
theoretical bacterial count of the sample. A dilution 1 to 100 is made 
by adding 1 cc. of the sample to 99 cc. of sterile water. The plating 
method is inexact, partly because an unknown proportion of organisms 
in the original sample will fail to develop for various reasons in the 
cultural medium; furthermore, certain types of organisms, as strepto- 
cocci, may remain adherent in chains of greater or lesser length and 
develop as a single colony. Anaerobic bacteria do not develop under 
aerobic conditions. 
A template of paper or glass ruled in square centimeters is used 
to facilitate the enumeration of colonies; for densely colonized plates, 
each centimeter square of the template is subdivided into smaller 
units, usually one-ninth of a square centimeter. The Petri dish 
containing colonies is placed upon the template in such a manner 
that the colonies appear superimposed upon the rulings. It is a 
simple matter, with the lines as a guide, to count either the entire 
number of colonies in the Petri dish, or a few representative areas, 
which can be multiplied by a factor. (The average Petri dish contains 
about 63 square centimeters.) 
Ea-ample.—A sample of milk diluted 1 to 100 shows a large number 
of colonies after forty-eight hours' incubation. The total count of 
9 squares (each a square centimeter) is 180 colonies, an average of 20 
colonies per square centimeter. The colonies upon the entire plate 
(63 square centimeters) is 63 x 20, or 1260. The number of living 
bacteria in 1 cc. of this sample of milk would be 1260 x 100 or 126,000, 
because the number of colonies counted upon the plate is y^^ the entire 
number in 1 cm. 
The value of the method as a convenient means of comparison of 
the bacterial content of various samples of milk, water, sewage and the 
like depends largely upon the supposition that the same types of bac- 
teria present in different samples will grow quantitatively under like 
conditions. The comparison of bacterial counts is therefore a com- 
parison of a section of the total bacterial flora, not an absolute measure 
of the number of living organisms. The method of counting bacterial 
colonies has been highly developed for the regulation of water and 
milk supplies of cities. (See section Water and Milk.) 
