194 BIOLOGY AND TECHNIQUE 



differentiation, of course, can not usually be made, but much cor- 

 roborative evidence can be obtained which may guide us in the 

 methods to be adopted for further identification and for a final 

 summing up of species characteristic as a whole. 



The Counting of Bacteria. It is often necessary to determine the 

 number of bacteria per c.c. contained in water, milk, or other sub- 

 stances. For this purpose definite quantities of the material to be 

 analyzed are mixed with gelatin or agar and poured into Petri 

 plates. The exact dilutions of the suspected material must largely 

 depend upon the number of germs which one expects to find in it. 

 The plates, if prepared with gelatin, are allowed to develop at room 

 temperature for twenty-four to forty-eight hours. If agar has been 

 used, they are usually placed in the incubator at 37.5 C. At the 

 end of this time, the colonies which have developed are enumerated. 

 For this purpose, a Petri dish is placed upon a Wolffhiigel plate. 

 This plate consists of a disk or square of glass which is divided 

 into small squares of one square centimeter each. Diagonal lines 

 of these squares running at right angles to each other are subdivided 

 into nine divisions each in order to facilitate counting when the 

 colonies are unusually abundant. The Petri dish is placed upon the 

 plate in such a way that the center of the dish corresponds to the 

 center of the plate. The colonies in a definite number of squares 

 are then counted. The greater the number of squares that are 

 counted the more accurate the estimation will be. When the growth 

 is so abundant that only a limited number of squares can be counted, 

 these should be chosen as much as possible from different parts of 

 the plate, and in practice one counts usually six squares in one 

 direction and six at right angles to these, so as to preclude errors 

 arising from unequal distribution. The final calculation is then 

 made by ascertaining the average number of colonies contained in 

 each square centimeter. If standard Petri dishes have been used, 

 this is multiplied by 63.6, the number of squares in the area of the 

 dish, and then by the dilution originally used. 



Thus if twelve squares have been counted with a total number 

 of one hundred and forty-four colonies the average for each square 

 is twelve. Twelve times 63.6 is 763.2, which represents the total 

 number of colonies in the plate. Now if 0.1 c.c. of the original 

 material (water or milk) has been plated, this material may be 

 assumed to have contained 10 X 763.2, or 7,632 bacteria to each 

 cubic centimeter. 



