METHODS OF CULTURE AND OF EXAMINATION. 101 



the inner space is cut off completely from the outer air. 

 (Fig. 32.) Novy's apparatus may also be connected with 

 an air-pump, and be exhausted of air ; or, finally, it may be 

 freed of oxygen with the aid of an alkaline solution of 

 pyrogallic acid. (See method of Buchner, below.) 



Anaerobic pure cultures in test-tubes may be prepared in 

 the following manner : 



1. In a High Layer. Stab-cultures are made in tubes 

 that are filled with nutrient material to a higher level than 

 usual, and that are boiled again shortly before being used. 

 In the lower portions of the culture, free from oxygen, to 

 which the needle has penetrated, development takes place, 

 whereas the upper portions of the medium containing 

 oxygen remain sterile. The anaerobic bacteria thrive more 

 vigorously when reducing substances are added to the 

 nutrient media e.g., two per cent, of grape-sugar or from 

 0.3 to 0.5 per cent, of formic acid. 



2. In an Atmosphere of Hydrogen. The test-tubes are 

 closed with sterilized rubber stoppers doubly perforated, 

 through which two glass tubes bent at a right angle lead into 

 the interior. (Fig. 28.) Through the longer 



tube, which reaches almost to the nutrient 

 medium and is closed with a cotton stop- 

 per, hydrogen is passed, while through the 

 shorter tube atmospheric air is expelled. 

 As soon as gas in pure state escapes 

 through the shorter arm, both tubes are 

 sealed by heat, or closed by means of rub- 

 ber tubes. Inoculation must, of course, 

 have been effected before the introduction 

 of the hydrogen. 



j. With Complete Exclusion of Air. A 

 test-tube with a long-drawn-out tenuous 

 neck is filled with nutritive material and 

 inoculated in the usual manner. .The neck 

 is then connected with an air-pump, and 

 when the air has been completely ex- 

 hausted, the tube is sealed by heat. 



4. According to the Method of Buchner. 

 The culture-tubes, closed by means of a 

 loosely introduced cotton stopper, are in- 

 troduced into a large, hermetically sealed tube, whose floor 

 is covered with alkaline solution of pyrogallic acid ( I gram 



j. 33 E 



method of making 

 anaerobic cultures. 



