58 



PRINCIPLES OF BACTERIOLOGY 



Dilution Methods. As it is impossible to know the number of bac- 

 teria in any suspected fluid, it is usual to make a set of four different 

 plates, to each of which a different amount of material is added, so that 

 some one of the four will have the required number of colonies. The 

 dilutions are made in sterile distilled water or bouillon. In the first tube 

 we place an amount which we believe will surely contain sufficient 

 and probably too many bacteria. To the second tube we add 10 per 

 cent, of the amount added to the first, and to the third 10 per cent, 

 of the second, and to the fourth 10 per cent, of the third. Thus, if the 

 first contained 60,000 colonies the second would have 6000 (Fig. 27), 

 the third 600, and the fourth 60. If, on the other hand, the first con- 

 tained but 60, the second would have about 6, and the remaining two 

 would probably contain none at all. When there are many colonies 

 present the dishes are covered by a glass plate (Fig. 29), ruled in larger 

 and smaller squares, Wolffhiigers apparatus. With the eye or aided by 

 a hand lens the colonies in a certain number of squares are counted 

 and then the number for the whole contents estimated. 



FIG. 28 



FIG. 29 



Well-distributed colonies on agar in 

 Petri dish. 



Wolff hiigel's apparatus for counting colonies. 



When the material to be tested is crowded with bacteria it is often 

 best to make an emulsion of a portion of it, and use this rather than 

 the original substance for making the dilutions to be used. Measured 

 quantities of the diluted material can be transferred most accurately 

 through a sterilized, long, glass pipette graduated in one one-hundredth 

 cubic centimetres, or, more roughly, by a platinum loop of known size. 



The nutrient agar-agar is frequently used in a different manner. 

 About 8 c.c. are poured into the Petri dish and allowed to harden. 

 The substance to be tested bacteriologically, or a dilution of it, is then 

 drawn across the medium in a series of parallel streaks by means of a 

 platinum loop lightly over its surface. Each successive streak is made 

 with the same needle or loop without replenishing the material to be 

 tested. Each streak will therefore leave less deposit of bacteria and 

 fewer colonies will develop. While in the former method most of the 

 bacteria developed under the surface, here all develop upon it. This 



