CULTIVATION OF BACTERIA. 483 



method, beef -extract is inoculated with Bact. coli, allowed to stand 

 twelve hours in the incubator, and sterilized after the addition of pep- 

 tone and sodium chlorid. Such a non-saccharine bouillon, which is 

 also, after so short a time, free from indol, is better for testing indol 

 production than that prepared in the ordinary manner. 



(B) Albuminous. 



1. Peptone Water. In a liter of water 10.0 gm. of dry peptone 

 and 5.0 gm. of sodium chlorid are dissolved and it is then sterilized. 



2. Milk. Fresh, preferably freshly centrifugated, milk is placed in 

 test-tubes and sterilized in the steam chamber for one-half hour on 

 two successive days. Milk which contains the spores of the subtilis 

 group (see p. 53) can often not be sterilized in this way. 



3. Litmus Whey (Petruschky). The casein is cautiously precipi- 

 tated from milk by producing a very feeble acid reaction with dilute 

 hydrochloric acid. The filtrate is boiled, filtered and neutralized, and 

 mixed with some litmus. The preparation is not very easy. It can 

 be bought. 



4. Hay Decoction. About 10 gm. of dry hay are boiled in a liter 

 of water. The filtrate is filled into tubes and sterilized for two hours 

 on three successive days, the tubes being kept overnight in the incu- 

 bator, in order to destroy the very resistant spores. 



5. Beer wort (not neutralized) is allowed to settle for some time, 

 preferably several weeks, after being sterilized, and then it is poured 

 off clear into test-tubes and again sterilized. 



6. Nutrient Bouillon. (a) From meat: 500 gm. of lean beef are 

 boiled in 1000 c.c. of water in an enamelled pot over a flame for one- 

 half hour and filtered. The filtrate (meat infusion) is brought up to 

 1000 c.c., and to this is added 10 gm. peptone and 5 gm. sodium 

 chlorid^ This is placed in the steam chamber until dissolved, and the 

 whole then carefully neutralized with normal sodium hydroxid (phe- 

 nolphthalein being used as indicator 1 ) (see p. 36). It is then filtered, 

 filled into tubes, and sterilized. 



(b) From extract of beef: 10 gm. of beef-extract are dissolved in 

 1000 c.c. of water, and 5 gm. of sodium chlorid and 10 gm. of peptone 

 added. The solution is then neutralized and well sterilized several 

 times. 



7. Potato Water for Tubercle Bacilli. Five hundred gm. of 

 peeled potatoes are rubbed upon a grater and allowed to stand over 

 night in a refrigerator in 500 c.c. of water. The fluid is then decanted 

 and brought up to 1000 c.c., boiled for an hour in the water-bath, fil- 

 tered, 4% glycerin added, filled in tubes, and sterilized. 



8. Gelatin Nutrient Media. (a) Meat infusion-peptone gelatin 

 (ordinary " gelatin " or u nutrient gelatin " of laboratories) : To 1000 

 c.c. of meat infusion (see nutrient bouillon) are added 100 gm. gelatin, 



1 For example, 10 c.c. of bouillon required 2.2 c.c. of T V normal 

 sodium hydroxid solution for saturation; 1000 c.c. of bouillon requires 

 220 c.c. of the same, or 22 c.c. of normal solution. We usually add 

 only 20 to 21 c.c., t. e., a little less, so as to be sure of having no 

 free sodinm hydroxid in the nutrient medium. 



