EXPERIMENT STATION BULLETINS. 559 



Coo ledge and Wyant (1) obtain results which check well with the actual 

 condition of the milk by adding 0.1 cubic centimeter of the milk to be tested 

 to a tube of broth containing the indicator Brom-thymol blue and incuba- 

 ting at 37 °C. pH readings are made hourly and the condition of the milk 

 is judged by the rate of change. Results are thus obtained in from 1 to 8 

 hours which enable the writers to divide the milk into 17 classes depending 

 upon the ability of the bacteria and enzymes present to cause the changes 

 in readings. 



METHODS 



Preparation of Media. The only medium necessary for this work is the 

 bouillon cube broth recommended by Wyant (4) and containing the indica- 

 tor Di-bromthymol sulphonepbthalein (Brom-thymol blue). One bouillon 

 cube, 1 percent Witte's or other peptone, 0.5 percent salt are added to each 

 liter of water and after the usual heating and filtering 80 cc. of a 0.04 of 1 

 percent aqueous solution of brom-thymol blue added for each hter of broth. 

 The broth is then cooled to 20 '^C. and the color adjusted to compare with 

 tube 6.8 of the standards which are mentioned later. A compensating blank, 

 broth without indicator, must be placed in front of the standard and the 

 readings must be made at room temperature. 



The broth is then placed in 100 or 2C0 cc. flasks sterihzcd, and stored. 

 When needed 10 cc. of the broth indicator solution is transferred to a 2.8 xl6 

 cm. sterile test tube with a 10 cc. sterile pipette and compared with standard 

 tube pH 7.0 in the comparator. A few drops of N/1 NaOH or HCl are 

 then added to the flask of indicator solution if needed to make the color of 

 the broth solution compare with tube pH 7.0. Broth without indicator 

 must be used as a compensating blank in front of the standard tube. The 

 broth is now ready to use and is transferred in 10 cc. portions to 2.8x16 cm. 

 sterile test tubes using a sterile 10 cc. pipette. 



Preparation of Samples. In taking samples the same precautions should 

 be observed as when sampling for other bacteriological tests. Preliminary 

 experiments, however, indicate that pipettes may be re-used with safety 

 after rinsing in hot water. 



It is desired to add 0.1 cc. of milk to each tube of brom-thymol blue broth. 

 To reduce the error due to measuring direct as small an amount of milk as 

 0.1 cc, sterile 9 cc. water blanks in test tubes are prepared and 1 cc! of the 

 milk to be tested added to these. After thorough mixing 1 cc. of each di- 

 luted sample is transferred to a tube of the broth. 



Incubation. After the 0.1 cc. portions of milk have been added to the 

 tubes containing brom-thymol blue broth they are placed in a water bath or 

 incubator at a temperature of 37 °C. Hourly observations are made to 

 detect the first change in color of the indicator due to the development of 

 acidity resulting, from the activity of the bacteria and enzymes present. As 

 eoon as pronounced changes are noticed, hourly readings are made by placing 

 the tubes containing milk — broth — indicator in the comparator and com- 

 paring with the standard tubes as mentioned below. The samples must 

 be placed in a water bath and cooled to 20 °C. before readings are made. In 

 this work readings should be made at a constant temperature. The readings 

 of the unknown and standards vary somewhat with change in temperature, 

 making this precaution necessary. 



Standards. The standards are the buffer solutions described by Clark 



