216 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA 



cells in a cubic centimeter of water. Experience has shown that the 

 accuracy of the method is influenced somewhat by the number of 

 organisms in the 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 fifty and two hundred individual 

 organisms. If more than two hundred 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 ten, because the subsequent calculation is much 

 simplified. A dilution of 1 to 10 is made by adding 1 c.c. of the sample 

 to 9 c.c. of sterile water, shaking thoroughly and plating 1 c.c. If 

 the technic is all right, each colony on the plate represents one-tenth 

 the number of living bacteria in the original sample. The total number 

 of colonies multiplied by ten gives the theoretical bacterial count of 

 the sample. A dilution 1 to 100 is made by adding 1 c.c. of the sample 

 to 99 c.c. 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 streptococci, 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 con- 

 taining 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.) 



Example. A sample of milk diluted 1 to 100 shows a large number 

 of colonies after forty-eight hours' incubation. The total count of 

 nine squares (each a square centimeter) is 180 colonies, an average 

 of twenty 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 c.c. of the sample of milk would be 1260 x 100 

 or 126,000, because the number of colonies upon the plate is T^TT the 

 entire number in 1 cm. 



