148 BIOLOGY AND TECHNIQTJE 



China, Blue Indicator. Make up 1 per cent solution of China blue, 

 heat almost to boiling in the water bath, add, drop by drop, normal 

 NaOH until completely decolorized. Use 1.5 per cent concentration 

 in medium. This indicator seems to be much less toxic than some 

 of the others, and has been used with success particularly by Teague, 

 to whom we are indebted for these practical details. 



Neutral red an indicator formerly used extensively by the Ger- 

 mans in the proportions of 1 c.c. of staturated aqueous solution to 

 100 c.c. of culture medium. It has been particularly used in con- 

 nection with the colon-typhoid group, in that typhoid bacilli do 

 not change the color, whereas B. coli decolorizes it. 



The list of indicators might be very much increased, but there 

 would be little point in it since most of these indicators are not 

 being used at the present time. 



ACTUAL STEPS IN THE PREPARATION OP NUTRIENT 



MEDIA 



Meat Extract Broth. To 1000 c.c, of distilled or clear tap water, add 

 5 grams or 0.5 per cent of Liebig's meat extract, 10 grams or 1 per cent 

 of Witte or any other reliable brand of pepton, and 5 grams or 0.5 per cent 

 of common salt, NaCl. 



The ingredients are mixed together in a suitable vessel and heated with 

 stirring over a free flame. When the pepton and meat extract are completely 

 dissolved, the vessel is removed from the flame. The medium is titrated 

 by the colorimetric method described above and adjusted to the desired 

 reaction. It is advisable to make the reaction about two points (on the 

 hydrogen ion scale) more alkaline than the final reaction should be, since 

 the heat of the autoclave usually increases the acidity of the medium. Using 

 Liebig's meat extract and Digestive Ferment's pepton, we have found that 

 the addition of 10-12 c.c. of normal NaOH to every liter of extract broth 

 usually brings the reaction, before autoclaving, to 7.6, making it about 7.4 

 after autoclaving, which is the optimum for most pathogenic bacteria. After 

 the addition of the alkali, the broth is autoclaved for thirty minutes at 

 fifteen pounds pressure. The medium is then filtered, the reaction checked 

 and after tubing and sterilization, is ready for use. It is not necessary in 

 most cases to add eggs to extract broth, since it is easy to obtain clear 

 without this step. 



Meat Infusion Broth. Infuse 500 grams 8 of lean meat (veal or beef) 

 in 1000 c.c. of distilled or tap water for twelve to twenty -four hours in the 

 ice-box. Strain through wet cheese cloth, squeezing the meat as dry as 



"Koughly 1 pound (1*4 Ibs.). 



