METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 191 



is added as an indicator. The solution usually remains colorless, 

 because ordinary media are acid in reaction; j$ NaOH is added slowly 

 from a burette until a faint pink color appears and persists after one 

 minute's boiling. From the amount of ^ alkali required to neutralize 

 5 c.c. of medium, the reaction of the entire amount is readily com- 

 puted. Thus: 



5 c.c. media are neutralized by 3 c.c. ^ NaOH. 

 100 c.c. media would be neutralized by 3 c.c. normal (y)NaOH. 

 1000 c.c. media would be neutralized by 30 c.c. normal (j)NaOH. 



To reduce the reaction of a liter of medium whose initial reaction 

 is + 3.0 to + 1.0, 20 c.c. of normal NaOH would be required. It is 

 necessary to heat the medium after adding the alkali, in order to 

 promote the reaction between the acids of the medium and the neu- 

 tralizing solution and a redetermination of the reaction should be 

 made to make certain that the desired change in acidity has taken 

 place. Frequently a second addition of alkali is necessary to create 

 the desired final reaction. 



A satisfactory reaction for cultural media designed for most patho- 

 genic bacteria may be created by adding ^ NaOH solution a few 

 drops at a time, to the entire volume, using filter paper dipped in 

 phenolphthalein solution, and dried, as an indicator. When the paper 

 shows a faint pink color the addition of alkali is discontinued. The 

 reaction is practically neutral under these conditions. 



The Clarification of Media. It is desirable, in the preparation of 

 culture media, to remove all insoluble substances. This is accom- 

 plished by filtration methods, with or without preliminary treatment, 

 to flocculate the substances in suspension. The addition of non-heat- 

 coagulable proteins, as gelatin, frequently requires clarification with 

 a coagulable protein, as egg-albumen, to remove the finely divided 

 suspended matter. 



Clarifying with Eggs. For each liter of medium to be clarified, two 

 eggs thoroughly whipped in a small amount of water are added. The 

 temperature of the medium should not exceed 50 C. The eggs are 

 thoroughly stirred in and the entire mixture is slowly heated to 1QO 

 C., either in a double boiler or in the Arnold sterilizer. A firm coagulum 

 forms during the heating process, which enmeshes the suspended par- 

 ticles it is desired to remove. The medium should never be disturbed 

 during the coagulating process. The clear underlying medium is 

 drawn off and filtered through cotton. 



