64 SEWAGE AND ITS PURIFICATION 



the purest effluents containing the nitrifying bacteria, which 

 do not appear on ordinary media. When a minute fraction 

 (0*05 ex.) of the sample is dried, fixed, and stained on a cover- 

 shp of known area, and the counts made with a micrometer on 

 representative fields, Winslow and Willcomb, who have investi- 

 gated this method, working with pure cultures of organisms 

 thriving on ordinary nutrient media, found that the micro- 

 scopic counts closely correspond to those obtained by plate 

 cultivation, even when the number of bacteria is rapidly 

 decreasing, and conclude that the presence of lifeless forms 

 introduces no serious error, owing to the inability of the dead 

 cells to take up the stain. 



The number of spores of bacteria is determined by heating 

 the nutrient material containing the sewage to 80^-' C. for ten 

 minutes before incubating. 



The organisms which multiply at blood heat are examined 

 by means of agar plate cultures prepared similarly to the 

 gelatine plates, and incubated at ^y° to 38° C. for one or two 

 days. 



2. Anaerobic Cultures. — As I have already stated, there are 

 a number of organisms in sewage which do not thrive in the 

 presence of oxygen, and in order to develop these anaerobes 

 they must be incubated in an atmosphere of some indifferent 

 gas, such as hydrogen, or preferably nitrogen. The cultures 

 may be enclosed in a jar filled with gas, or containing a solution 

 of alkaline pyrogallate to absorb the oxygen in the air. 



I find that a certain amount of confusion has arisen from the 

 application of the words '^ aerobic " and " anaerobic " in two 

 slightly different meanings — one with reference to the chemical 

 changes that occur, the other with regard to the organisms 

 that produce them. As the words simply mean " living with 

 air" and "living without air," the chemist has applied the 

 term " anaerobic " to changes occurring by life in which free 

 oxygen takes no part. Many of these are due to hydrolysis 

 or the addition of water, like that of urea into ammonium 

 carbonate, or cellulose into starch, dextrin, and sugar. In this 

 sense the word " anaerobe " implies an organism that effects 

 its changes in surrounding matter without oxidation. But a 

 bacteriologist often uses the term ** anaerobe " in the sense of 

 " obhgate anaerobe " — i.e., one that not only does not require 

 oxygen, but is actually inhibited, or even killed, by its presence. 

 The obligate anaerobes, as is shown by our table of bacteria in 



