ANAEROBIC CULTURES 



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(A) Method I (Hesse's Method). 



1 . Make a stab culture in gelatine or agar, choosing 

 for the purpose a straight tube containing a deep 

 column of medium, and thrusting the inoculating 

 needle to the bottom of the tube. 



2. Pour a layer of sterilised oil (olive oil, vaseline, or 

 petroleum), i or 2 cm. deep, upon the surface of the 

 medium. 



3. Incubate. 



Method II. This method is only available when 

 dealing with pure cultivations. 



1. Liquefy a tube of gelatine (or agar) by heat, 

 pour it into a Petri dish, and allow it to solidify. 



2. Inoculate the surface of the medium in one spot 

 only. 



3. Remove a cover-slip from the pot of absolute 

 alcohol with sterile forceps ; burn off the alcohol in the 

 gas flame. 



4. Lower the now sterile cover-slip carefully on to 

 the inoculated surface of the medium, carefully exclud- 

 ing air bubbles, and press it down firmly with the 

 points of the forceps. (A sterile disc of mica may be 

 substituted for the cover-slip.) 



5. Incubate. 



Method III (Roux's Physical Method). 



1. Prepare tube cultures of fluid media (or solid 

 media rendered fluid by heat) in the usual way. 



2. Aspirate some of the inoculated media into capil- 

 lary pipettes. 



3. Seal both ends of each pipette in the blowpipe 

 flame. 



4. Incubate. 



Method IV (Roux's Biological Method). 



1. Plant a deep stab, as in method I. 



2. Pour a layer, i or 2 cm. deep, of broth cultivation 

 of a vigourous aerobe e. g., B. aquatilis sulcatus or B. 



