BACTERIA OCCURRING IN SEWAGE 63 



ragged or circular, branchings from the centre or concentric 

 circles may appear, they may remain colourless, or develop 

 peculiar pigments. 



If there is no guidance as to the strength of the sample 

 under examination, a large number of these gelatine plates has 

 to be prepared from varying quantities of the sew^age in order 

 to hit off the right dilution. In sewage and effluents a great 

 many putrefactive species are capable of very quickly liquefying 

 the nutrient gelatine, and therefore, if the plate be too crowded 

 — containing, say, more than 200 colonies — the whole may 

 become fluid, owing to the junction of the liquefying areas, 

 before many colonies which do not develop so rapidly are 

 visible to the naked eye. In order to obtain comparable results 

 from plate cultures, it is important that the nutrient material 

 employed should conform to a definite degree of alkalinity. 

 The American Public Health Association adopted a standard 

 of +1*5 per cent, as yielding the best results; that is to say, 

 the medium, which is alkaline to litmus, would require the 

 addition of 1*5 per cent, of normal soda solution to produce a 

 pink tinge with phenolphthalein indicator. Nahrstoff agar, 

 which contains about i per cent, of Heyden's Nahrstoff, 

 incubated at room temperature, will yield nearly twice as many 

 colonies as nutrient gelatine. 



The colonies are counted with the aid of a magnifying-glass, 

 the Petri dish being placed on a glass plate ruled in centimetre 

 squares (Wolffhiigel's apparatus), and as each colony originates 

 from one individual, a factor is obtained from which the number 

 of organisms present in the original sewage can be calculated. 

 This is returned as " organisms per c.c," stating the temperature 

 of incubation and the nutrient medium employed. 



The results so obtained, however, from such a material as 

 sewage are below the total number of organisms, (i) because of 

 the difficulty of insuring that each colony has arisen from a 

 single organism, and (2) because many which are incapable of 

 sufficient development on the nutrient medium will be omitted 

 in the enumeration, such as some of the nitrifying, thermophilic, 

 and anaerobic species. In my own experience, plates that have 

 shown no colonies after three days' incubation have a little 

 later developed numerous growths of cladothrix. The direct 

 determination of the number of organisms in sewage by micro- 

 scopic counting invariably yields results many times greater 

 than those obtained by plate culture ; this ratio increases with 



