METHODS USED IN CULTIVATION OF BACTERIA 



149 



growing anaerobic bacteria upon plates by simply dropping upon the 

 surface of the inoculated agar or gelatin a fiat piece of sterile mica. This 

 method, however, rarely succeeds in sufficiently excluding the air. 



LiBOBius' Method.' — This method consists in the use of deeply filled 

 tubes of agar or gelatin, from which all oxygen has been removed by 

 boiling for fifteen minutes or more. It is advantageous, as has been 

 pointed out in the section on anaerobiosis, that 

 media used for this purpose should contain carbo- 

 hydrates in some form, perferably glucose. After 

 boiling, the tubes are rapidly transferred to ice 

 water so that as little oxygen as possible may be 

 absorbed during the hardening of the medium. 

 The tubes are then inoculated by deep stabs. After 

 inoculation, the medium may be covered with a 

 thin layer of agar, gelatin, or oil (albolin), and 

 further sealed with sealing-wax to prevent oxygen- 

 absorption. 



This method may be utilized for the isolation 

 of anaerobes (as in the original method of Libo- 

 rius) by inoculating the medium just before it 

 BoUdifies. The tubes may be gently shaken in 

 order to distribute the bacteria throughout the 

 medium and then rapidly cooled. In this case 

 colonies which develop may be scattered through- 

 out the deeper layers of the agar or gelatin, and 

 may be "fished" after breaking the tube. 



Esmaech's Method.^ — Von Esmarch has applied 

 the principles of his roll-tube to the cultivation of 

 anaerobic bacteria. Gelatin tubes are inoculated 

 as above and roll-tubes prepared. The tubes are 

 then set into cold water to prevent melting of the 

 thin gelatin layer and the interior of the tube is 

 filled with melted gelatin. 



RoxDc's Method.^ — Anaerobic bacteria are culti- 

 vated by sucking the inoculated gelatin or agar into narrow tubes, 

 which are then closed at both ends by fusing in the flame. After 

 growth has taken place the tubes are broken and the organism re- 

 covered by "fishing." 



'1 lAbonus, Zeit. f . Hyg., i, 1886. ^ Van Esmarch, loc. cit. 



3 Roux, Ann. Past., i, 1887. 



Fig, 33.— Deep 

 Stab Cultiva- 

 tion OF Anaero- 

 bic Bacteria. 



