8 IVAN C. HALL 



principal factors of decolorization and return of color in methylene 

 blue solution. 



Considering each of the ingredients of magnesium carbonate 

 glucose broth as possible single factors in the decolorization of 

 the dye, it was first shown that only those solutions sUghtly 

 alkaUnized, as by means of magnesium carbonate or sodium or 

 potassium hydroxide, lose color on boihng. The use of magne- 

 sium, carbonate referred to involves addition of an excess and the 

 removal of the undissolved residue by filtration after boiling: 

 only a trace of magnesium goes into solution and the reaction is 

 made faintly alkaline (pH = about 8). With such adjustment 

 it was found possible to dispense with the salt and any two of 

 the other three factors (meat infusion, peptone, and glucose) 

 without interfering with decolorization. But the clearest cut 

 results were obtained with glucose present. Furthermore, rather 

 prolonged boiling is required for decolorization if the glucose be 

 added to the filtrate from a heated MgCOs suspension in water, 

 i.e., is not heated in the presence of an excess of MgCOs; a 

 sUghtly alkaline solution of 2 per cent agar was also decolorized 

 easily. Further experiments were then undertaken to determine 

 the effect of variation in reaction upon methylene blue solutions 

 in the presence of these various organic substances. 



The following facts stand out as a result of many experunents. 



Neutral aqueous solutions of Griibler's Methylene blau fiir 

 Bacillen containing 0.0001 gram or more per cubic centimeter 

 are not decolorized in a water bath boihng hard for twenty 

 minutes. Neither the inorganic acids, HCl, H2SO4, HNO3, nor 

 the organic acids, oxalic, acetic, lactic, citric, butyric, succinic, 

 formic, and propionic, in a concentration of n/10, have any 

 visible effect when heated in weak solutions of methylene blue. 

 Yet methylene blue is decolorized slowly in a solution of HCl 

 acting on zinc in the presence of platinum, n/10 NH4OH has 

 no visible effect while the equivalent concentrations of BaOH, 

 NaOH and KOH produce a violet lavender color only — indicat- 

 ing, no doubt, the formation of methylene azure. 



Neutral glucose solutions ranging from 1 to 10 per cent and 

 faintly or deeply colored with methylene blue are not decolorized 



