108 

 APPENDIX B. CULTURE MEDIA 



Numerous mediums have been utilized in the bacteriological examina- 

 tion of water. The preparation of the more important is described here. 



I. ADJUSTMENT OF REACTION OF CULTURE MEDIA 



(A. P. H. A. 1920) 



1. Phenol Red Method for adjustment to a hydrogen- 

 ion concentration of PH+ = 6.8-8.4. Withdraw 5 c. c. of the 

 medium, dilute with 5 c. c. of distilled water, and add 5 drops of a solu- 

 tion of phenol red (phenol sulphone phthalein). This solution is made 

 by dissolving 0.04 grams of phenol red in 30 c. c. of alcohol and diluting 

 to 100 c. c. with distilled water. 



Titrate with a 1:10 dilution of standard solution of NaOH (which 

 need not be of known normality) until the phenol red shows a slight but 

 distinct pink color. Calculate the amount of the standard NaOH solution 

 which must be added to the medium to reach this reaction. After the 

 addition check the reaction by adding 5 drops of phenol red to 5 c. c. of 

 the medium and 5 c. c. of water. 



2. Titration with phenolphthalein. (For the convenience of 

 those who wish to retain the use of this method for the present it is given 

 here, but it is recommended that as soon as possible the more accurate meth- 

 od of determining the hydrogen-ion concentration be substituted.) 



In a white porcelain dish put 5 c. c. of the medium to be tested, add 

 45 c. c. of distilled water. Boil briskly for one minute. Add 1 c. c. of 

 phenolphthalein solution (5 grams of commercial salt to one liter of 50 

 percent alcohol). Titrate immediately with a n/20 solution of sodium 

 hydrate. A faint but distinct pink color marks the true end point. This 

 color may be precisely described as a combination of 25 percent of red 

 (wave length approximately 658) with 75 percent of white as shown by 

 the disks of the standard color top made by the Milton Bradley Educational 

 Co., Springfield, Mass. 



All reactions shall be expressed with reference to the phenolphthalein 

 neutral point and shall be stated in percentages of normal acid or alkali 

 solutions required to neutralize them. Alkaline media shall be recorded with 

 a minus ( ) sign before the percentage of normal acid needed for their 

 neutralization and acid media with a plus ( + ) sign before the percentage 

 of normal alkali solution needed for their neutralization. 



The standard reaction for culture media for water analysis shall be 

 + 1.0 percent, as determined by tests of the sterilized medium. As ordin- 

 arily prepared, broth and agar will be found to have a reaction between 

 +0.5 and +1.0. For such media no adjustment shall be made. The re- 

 action of media containing sugar shall be neutral to phenolphthalein. 

 Whenever reactions other than the standard are used, it shall be so stated. 



II. STANDARD STOCK MEDIA 



A. Nutrient Agar (A. P. H. A., 1920). 1. Add 3 grams of beef 

 extract, 5 grams of peptone and 12 grams of agar, dried for one-half hour 



