CULTIVATION OF ANAEROBIC BACTERIA. 83 
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 anaérobic culture will appear as shown in Fig. 52. 
To isolate anaérobic bacteria in pure cultures it is well to make a 
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ay 
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Fie. 52, Fie. 53. 
series of dilutions as heretofore described for aérobic 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 stab 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 aérobic 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 
