204 



BOTANICAL GAZETTE [September 



gelatinous coating and revert to megalococci. This "cycle recalls a 

 little the cycle followed by the organisms of Java soils, as related by 



WiNOGRADSKY (l2, flgS. lO, 1 2). 



The staining methods adopted in this stud\- are similar to 

 the methods recommended by Winogradsky. Although several 

 of the ordinary staining solutions were tried, none seemed to give 

 as good and clear pictures as the malachite-green and gentian- 

 violet method. The salts, which are generally placed on the slide 

 with the bacterial preparations, especially when solutions are the 

 origin of the material studied, are not stained by this method. 

 The technic is as follows: The coverglass preparation, flame 

 iixed, is mordanted for i minute in the cold with a 0.25 per 

 cent solution of malachite-green in distilled water, washed with 

 cold water, and stained cold with a 0.25 per cent water solu- 

 tion of gentian-violet for i more minute. Washing is then done 

 rapidly with water previously heated to 50-60° C. This washing 

 takes out any coloration of the salts which might cloud the micro- 

 scopic field. Preparations are thus obtained which stain the 

 jelly of the megalococci a deep purple and the small cocci of the 

 (8 type a purple-black color. Treatment with acid is not neces- 

 sary to dissolve the salt formations. In the fresh unstained state 

 the cells are not easily visible, and a search for them often proves 

 unsuccessful. The color differentiation mentioned, obtained in 

 Meissner solution, constitutes what seems to be the best and most 

 rehable one for a stud}' of the organisms in hanging drop prepara- 

 tions. 



Temperature relations 



The thermal death point of the organism studied was found 

 to lie between 50 and 55° C, when the vitality of the organism, 

 after heating 5 . 5 minutes at the required temperature, was tested 

 at rest in Omelianski's solution containing basic magnesium car- 

 bonate. An additional study of the resistance of the organism 

 to heat was also made. One cc. portions of a strong nitrifying 

 culture were placed in sterile vials, heated at given temperature 

 for given lengths of time, and then quickly cooled in stone cold 

 water, the depth of the solution layer being 4-5 mm. over a 

 surface of =^2.8cin\ Heating was done in ovens, and tests for 



