NITRIFICATION 33 



salts [33]. After inoculating such media with soil, Wino- 

 gradsky readily obtained excellent growth accompanied by 

 nitrification, and the cultures were easily maintained 

 through successive transfers in a mineral medium containing 

 potassium phosphate, NH4CI, MgS04 and K2CO3 . Using 

 the enrichment culture technique, the isolation of organisms 

 oxidizing NH3 was facilitated by repeated subculture in an 

 inorganic medium containing NHt but no added NOT: 

 similarly, a medium containing N07 and no NH^ was used 

 for organisms oxidizing NOT. Winogradsky solved the 

 problem of how to obtain discrete colonies on a solid yet 

 completely inorganic medium by using silica gel as the 

 matrix for the mineral salts [34]. The colonies are minute 

 (diam. 200 [.i) and micro-manipulators are sometimes used 

 to pick out and transfer those required for inoculating sub- 

 cultures. Winogradsky was the first to obtain indisputably 

 pure cultures of organisms which specifically oxidized either 

 NH3 (Nttrosomonas europaed) or NOT {Nitrohacter wino- 

 gradsky). About the same time, Warington [31] and the 

 Franklands [8], working independently, obtained cultures 

 which oxidized NH3 to NOT and which appeared to be 

 pure as judged by microscopic examination and the absence 

 of growth on gelatin plates. The isolation of pure cultures 

 of the nitrifiers is difficult because their slow growth favours 

 the appearance of heterotrophs which grow rapidly even if 

 only traces of organic matter are present in the medium. 

 Great care is therefore required in assessing the purity of 

 any culture of nitrifying bacteria and no growth should be 

 detectable after incubating plates of nutrient agar streaked 

 with such cultures. Lees has found that adequate aeration 

 of the medium greatly facilitates the isolation and culturing 

 oi Nttrosomonas [14]. 



Some strains of the nitrifiers are actively motile whilst 

 others appear to be habitually associated with surfaces, e.g. 

 they readily adhere to granules of CaCOg [cf. 19]. Many of 

 these organisms prefer a slightly alkaline environment [27, 

 18], and for this reason CaCOg , K2CO3 or MgC03 are fre- 

 quently added to the medium [34, 20, 12]. Such substances 



