MEMOIRS OF TIIF. NATIONAL ACADEMY OF SCIENCES. 



Fig. 1. 



Fig. 2. 



anaerobes. Their study presents considerable technical difficulties. Of the various methods 

 proposed for the cultivation of these bacteria, two appear to be of special value. These are the 

 method of Buehner (Fig. 1), in which the oxygen is absorbed by means of pyrogallic 

 acid, leaving a residual atmosphere of nitrogen; and, second, the employment of an 

 atmosphere of hydrogen. Buchner's method has the objectionable feature of causing 

 a negative pressure, which puts the bacteria under unnatural conditions. All things 

 considered, the use of an atmosphere of hydrogen seems the most practical method we 

 possess, and is favorable to the growth of the most important anaerobic organisms 

 known to us. 



For plate cultivation, an apparatus similar to that described by Liborius in the 

 Zeitschrift fur Hygiene, Vol. I, page 128, is most useful. It consists of a bell jar having 

 openings at the top and near the bottom, in which perforated rubber stoppers, bearing 

 glass tubes with stopcocks, are inserted. The jar and plate on which it rests are put 



in a frame by which they can be tirruly clamped 

 together. A ring of soft rubber is put between 

 the jar and plate, and it is well to supplement this 

 by several coats of melted paraffin applied outside. 

 The air is pumped out from the bottom and hydro- 

 gen introduced at the top, the process being re- 

 peated three times, at least, in order to insure the 

 removal of all oxygen. It is well to put a vessel 

 containing about 50 c. c. of an alkaline solution 

 of pyrogallic acid under the jar also, as it shows 

 whether the apparatus is leaking or not. and at 

 the same time serves to absorb any oxygen which may have 

 remained. (See Fig. li.) 

 For test-tube cultures the method of Friinkel (Fig. 3), or, better, the method of Sternberg 

 (Fig. 4) may be employed. In the former an ordinary tube is closed by a soft-rubber stopper, 

 through which two glass tubes pass, one reaching nearly to the bottom 

 of the test tube, for the introduction of the hydrogen; and the other, a 



short one, for the escape of the gas. The method 

 of Sternberg is essentially a modification of this. 

 After inoculating the tubes the cotton plug, or 

 a part of it, is pushed down into the neck of the 

 tube. Above this a section of a rubber stopper, 

 carrying two glass tubes, both short, is inserted 

 for about a half inch. The space above is coated 

 with melted sealing wax, which does not con- 

 tract ou cooling. The tube is then attached to 

 the hydrogen generator and inverted, the gela- 

 tin or agar being solidified. The hydrogen 

 being light rises into the tube, displacing the 

 air. After a sufficient time the outlet tube is 

 sealed in the flame, then the inlet also. Both 

 of these methods are useful mainly for the study 

 of colonies in roll tubes, and the latter can be 

 used only for solid cultures. The employment 

 of the air pump attached to the outlet tube 



facilitates matters greatly and enables Sternberg's method to be applied 

 to liquid cultures. Should Sternberg's method be used the tubes should 

 be freshly sterilized and the top of the plug of cotton burned off, as 

 otherwise the culture will soon be contaminated with moulds in abundance, many of which thrive 

 in an atmosphere of hydrogen. 



The most simple method of test-tube culture is the employment of a jar or bottle, in which a 

 number of tubes may be placed at the same time. The apparatus of Liborius above described 



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Fig. 4. 



