im-ESTIGATION OF AMERICAN GENTIAN VIOLETS 531 



Ciriiblcr's, but they were distributed to the collaborators desig- 

 nated by luiniber only, and were in most cases thought to be 

 American samples. 



The collaborator,s were instructed to use these samples of 

 stain in staining; bacteria with the Gram technic. To make the 

 results more constant if possible than in the earlier work, direc- 

 tions for making the tests were furnished and cultures were dis- 

 tributed for use as test organisms. The cultures furnished were: 

 Bacillus cereus, as a strongly gram-positive organism; a small 

 short rod of the fluorescent group, as a decidedly gram-negative 

 organism; and lastly an unidentified micrococcus which had 

 been found to be weakly Gram-positive although often negative 

 with poor technic. The directions for making the stains which 

 were furnished were as follows: 



Twenty-four hour cultures should be used in the test, the coccus 

 incubated at 37°, the other two at room temperature (or 25° if available). 



To make results strictly comparable the procedure should be care- 

 fully controlled and timed as follows: 



Stain thirty seconds. 



Drain but do not wash. 



Iodine thirty seconds. 



Drain but do not wash. 



Ninety-five per cent alcohol until no more stain is removed; not over 

 two minutes. 



Keep in agitation, and transfer without washing to 



Counterstain thirty seconds. 



Wash and dry. 



Stain formulae 



The gentian, crystal or methyl violet may be made up according 

 to one or both of the following formulae. If only one formula is used, 

 select formula A; but if convenient it is hoped that formula B may also 

 be used for the purpose of comparison. 



A. Regtdar formula B. Optional formula 



cc. cc. 



Saturated alcohol solution of Saturated alcohol solution of 



stain 6 stain 10 



95 per cent alcohol 5 1 per cent ammonium oxalate 



Anilin water (1 :49, filtered). . . 50 solution 40 



