180 BIOLOGY AND TECHNIQUE 



upon the oxygen-absorbing qualities of alkaline solutions of pyro- 

 gallol. 



Cultivation by the Mechanical Exclusion of Air. Koch succeeded in 

 growing anaerobic bacteria upon plates by simply dropping upon the surface 

 of the inoculated agar or gelatin a flat piece of sterile mica. This method, 

 however, rarely succeeds in sufficiently excluding the air. 



LIBORIUS' METHOD. 3 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 carbohydrates in some form, preferably 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 Liborius) by inoculating the medium just before it 

 solidifies. 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 throughout the deeper layers of the agar or gelatin, 

 and may be "fished" after breaking the tube. 



ESM ARCH'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. 



Roux's METHOD. 5 Anaerobic bacteria are cultivated by sucking the inocu- 

 lated 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 recovered by "fishing." 



FLUID MEDIA COVERED WITH OIL. Erlenmeyer flasks or other vessels are 

 partially filled with glucose-bouillon over which a thin layer of albolin or 

 other oil is allowed to flow. The oxygen is driven out of the liquid by 

 vigorous boiling for fifteen minutes or more. 



It should be remembered whenever using this or similar methods that 

 a layer of fluid oil does not form an impermeable seal. By covering an 

 alkaline pyrogallol solution with oil it can easily be shown that oxygen 



'Liborius, Zeit. f. Hyg., i, 1886. 

 4 Von Esmarch, loc. cit. 

 Koux, Ann. Past., i, 1887. 



