INVESTIGATION OF AMERICAN STAINS 135 



Griibler's methylen blue for bacilli was apparently mostly 

 zinc salt, this being the cheapest form of methylen blue available. 

 It was not pure, however, and thanks, apparently, to the fact 

 that small amounts of the zinc-free compound were present in 

 it, it could be used in the Loefller formula, which calls for 3 parts 

 saturated alcoholic solution to 10 parts water. Unfortunately 

 some of the preparations now on the market called "methylen 

 blue for bacilli" are so nearly pure zinc salt as to be unsatis- 

 factory for the LoefRer solution. On the other hand, some 

 American stains labelled exactly the same are apparently nearly 

 zinc-free, and are adnoirable for staining purposes, even in the 

 Loeffler formula. 



In the present series of tests, samples of both the zinc salt 

 and the medicinal (zinc-free) dye were used. Fourteen in- 

 vestigators took part in the tests, and the samples were tested in 

 several different ways. Four men, Healy, Hunter, Macy and 

 Eobertson, used the samples for staining dried milk smears 

 according to the Breed method of examining milk for bacteria; 

 Macy and Robertson used aqueous solutions of the stain samples, 

 Healy and Hunter the Loeffler solution. Healy and Robertson 

 also used their samples for staining ordinary bacterial cultures, 

 and with the exception of Levine, all the other investigators used 

 the stains for this purpose only, all employing the Loeffler solu- 

 tion, except Robertson who used a saturated aqueous solution. 

 Levine tested the samples by using them in his eosine-methylen- 

 blue medium for colon-aerogenes difTerentiation. 



The test made by the writer was a special test made at the 

 suggestion of Mr. Bell of the Coleman and Bell Company. 

 Only those samples were selected for it which had already been 

 found to give the best results. Three solutions of each were 

 prepared: a 1 per cent aqueous, a 3:10 alcoholic aqueous (i.e., 

 3 parts saturated alcohohc solution to 10 parts distilled water), 

 and a Loeffler solution (i.e., a 3:10 mixture using 0.0001 per cent 

 NaOH instead of distilled water). Smears of a diphtheria culture 

 were stained with each, allowing only about three seconds for 

 staining and then washing under a tap. j\Ir. Bell suggested 

 this as the most severe test he knew, and one which his methylen 

 blue for bacilli was required to meet. 



