268 CHEN CHONG CHEN AND LEO F. RETTGER 



after some preliminary attempts to obtain colorless peptone 

 media, a method was followed by which the peptone-phosphate- 

 glucose medium could be prepared and sterilized so that it re- 

 mained as free from color as the synthetic. Previously sterilized 

 test tubes are filled with the required amount of medium, and the 

 final sterilization carried out by placing the tubes in containers 

 which have impervious bottoms and sides, as for example canning 

 tins or glass beakers, instead of the usual wire baskets, and steril- 

 izing in the autoclave for ten minutes at an extra pressure of 

 10 pounds. During the entire course of the investigation very 

 few tubes were found not to be sterile. We made it a common 

 practice not to employ the tubed medium until its sterility had 

 been tested by proper incubation for at least twenty-four hours. 



The colorimetric standards were prepared according to the 

 descriptions of Clark and Lubs. Three color indicators were 

 employed, namely methyl red, brom thymol blue and brom 

 cresol purple. Brom thymol blue is particularly well adapted 

 for registering the hydrogen ion concentration of aerogenes cul- 

 tures. The brom cresol purple was selected because it gives more 

 distinguishable shades of color than the methyl red at the less 

 acid end of the methyl red range where the troublesome neutral 

 tints occur. The following technique was adopted for the test. 



Five cubic centimeters of the bacterial culture fluid were trans- 

 ferred to test tubes of uniform diameter, and 0.2 cc. of the indi- 

 cator added which by the preliminary drop method on a watch 

 glass, or better still on a sheet of glazed paper or cardboard, was 

 found to cover the indicated hydrogen ion range. The tubes 

 were sorted out into three groups, with a fair knowledge of the 

 approximate range of hydrogen ion concentration of each group. 

 The pH value of each culture was recorded by comparison in the 

 comparator with the previously prepared color standards. 



A total of 3725 individual hydrogen ion concentration deter- 

 minations was made in the three media with the two types (coli 

 and aerogenes) isolated from soils and feces. The results are 

 shown in the accompanying charts, in which the frequencies of 

 occurrence are plotted as ordinates, and the corresponding pH 

 values as abscissae. The middle line in the charts is an arbitrary 



