CULTIVATION OF ANAEROBIC BACTERIA. 



81 



for the admission of hydrogen, which passes through the liquefied 

 culture medium ; and the other a short tube for the escape of the gas. 

 The outlet tube is sealed in the flame of a lamp while the gas is 

 freely flowing, and after sufficient time has elapsed to insure the 

 complete expulsion of atmospheric oxygen which, when the hydro- 

 gen flows freely, requires about four minutes (Frankel) melted 

 paraffin is applied freely to the rubber stopper to prevent leakage of 

 the hydrogen and entrance of oxygen. A roll tube may then be 

 made after the manner of Esmarch, and, after colonies have de- 

 veloped, the anaerobic culture will appear as shown in Fig. 52. 



To isolate anaerobic bacteria in pure cultures it is well to make a 



FIG. 52. 



^sV 

 hJji 



-t 



FIG. 53. 



series of dilutions as heretofore described for aerobic cultures ; we 

 will then usually obtain isolated colonies in tube No. 2 or No. 3 of a 

 series, and by removing the rubber stopper we may transplant bac- 

 teria from these colonies to deep stick cultures in nutrient gelatin or 

 agar. 



The Writer's Method. The following simple method has been 

 successfully employed by the writer: 



Three Esmarch roll tubes are prepared as is usual for aerobic cul- 

 tures. The cotton air filter, or a portion of it, is then pushed down 

 the tubes for a short distance, as shown at a, Fig. 53. A section of 

 a soft rubber stopper carrying two glass tubes is then pushed into the 



