CHEMICAL CRITERIA OF ANAEROBIOSIS 9 



during thirty minutes in a boiling water bath. The same is 

 true of 2 per cent Witte's peptone solutions and of 2 per cent 

 agar solutions. Neither does the addition of 0.5 per cent glucose 

 to any of the acid solutions mentioned above result in decolori- 

 zation on heating; likewise 2 per cent agar and 2 per cent Witte's 

 peptone in graded hydrochloric acid solutions up to n/10 for 

 agar (which fails to solidify), and up to n/2 for peptone, refuse 

 to decolorize on heating. The neutral sodium salts of the above 

 acids formed by adding equivalent amounts of standardized 

 NaOH do not alter the result; none decolorize on heating. 



On the other hand an extremely small excess of alkali causes 

 the heated glucose, agar or peptone solution of methylene blue 

 to lose its color completely. Even such traces of alkali as may 

 be dissolved from the glassware may cause the decolorization of 

 methylene blue in glucose solutions on heating. Incidentally 

 we recall that Laird (1913) found the reaction time for Fehling's 

 solution reduced by boiling glucose, laevulose, galactose, maltose 

 and lactose in various makes of German glassware, owing to the 

 abstraction of calcium hydroxide from the glass. All experi- 

 ments reported herein were made with glassware carefully cleaned 

 with chromic acid cleaning fluid and rinsed in distilled water. 

 The use of such weak concentrations of alkali, which were approxi- 

 mated only by dilutions of standardized n/1 solutions, involves 

 the possibility of other factors of error, as for example, atmos- 

 pheric CO2 and non-neutral distilled water, which do not enter 

 so fully with higher concentrations. Repeated tests of the 

 distilled water by colorimetric tests with phenolsulphonephthalein 

 showed the limits of pH value to be 6.8 and 7.0; thus this possible 

 factor of error was excluded. The CO2 factor of error was 

 reduced as far as possible by the use of freshly boiled distilled 

 water for the preparation of solutions and checked as a disturbing 

 factor in the interpretation of results. The great difficulty in 

 adequately and exactly controlling the very shght alkalinity of 

 the solutions in different experiments without the use of buffer 

 substances may account for some nonsignificant discrepancies 

 between the results with high dilutions of alkali in different 

 tests. It should be made quite clear that decolorization of 



