1802 Journal of Applied Microscopy 



on the surface of the bouillon at (c). During the heating a large portion of 

 the air absorbed by the medium is driven out, and its re-absorption, while 

 the flask is cooling, is prevented by the paraffin. When cool inoculate the bouil- 

 lon by piercing the solid film of paraffin with a platinum needle, loop or capillary 

 glass tube charged with inoculating material. Now liquify the paraffin in the 

 lateral bulb by carefully heating it over the flame and pour the liquified paraffin 

 on the thin film of solid paraffin on the surface of the medium by inclining the 

 flask slightly. Upon hardening this additional layer of paraffin constitutes an 

 almost perfectly air tight cover which becomes even more effective by being 

 pressed into the tapering neck when the culture medium is warmed in the incu- 

 bator. This seal is made tighter yet by the pressure of the gases generated in 

 the culture. When the flask is to be emptied, warm the vessel and incline the 

 flask so that the liquified paraffin will flow over into the lateral bulb. 



This method is likewise advantageous in the preparation of toxins, as the 

 culture can be easily transferred in a very pure condition to the filter after the 

 warmed portion of the neck of the flask has cooled and the paraffin in the lateral 

 bulb has hardened. 



Park (1901) modified the above method as follows : Use tubes or flasks con- 

 taining sterile nutrient glucose bouillon. If non-spore-bearing anaerobes are to 

 be cultivated, cool the medium quickly, inoculate and cover the bouillon w'ith a 

 layer of very hot, sterile paraffin. Where absolute exclusion of oxygen is desired 

 sterilize the tubes containing the medium and the layer of paraffin in an autoclav ; 

 this renders the bouillon free from oxygen. Spore-bearing bacteria are inocu- 

 lated through the liquid paraffin before the bouillon is cooled down. In case of 

 bacteria without spores the medium is first cooled down and the inoculation is 

 made by breaking through the solidified film or by heating and melting the paraf- 

 fin in the gas flame, A pipette charged with the culture can then be carried 

 through the hot paraffin into the cool liquid medium, 



D, MICA OR GLASS PLATE. 



Kodi's method : Prepare a gelatin plate in the usual way. Before the inoc- 

 ulated gelatin is completely congealed cover it with a thin, sterile blade of mica. 

 The mica blade has the thickness of a thin sheet of writing paper. It must 

 be well flamed, and after cooling so placed upon the semi-liquid gelatin that no 

 air bubbles form beneath it. It must cover the gelatin completely and perfectly. 



Saufelice modified Koch's method by using a glass plate instead of a mica 

 plate as a cover. Pour the inoculated gelatin or agar into the sterile cover of a 

 Petri dish and when the medium is nearly congealed press the other half of the 

 Petri dish, bottom downward, gently into the medium. 



E. EXCLUSION OF AIR BY BOILING THE MEDIUM AND SEALING THE 

 APPARATUS IN THE FLAME. 



Hufner and Rosenbach use a heavy 250 c. c. flask with flat bottom and taper- 

 ing neck as shown in Fig. 40. Immediately below the tapering neck a lateral 

 tube projects horizontally. At its union with the neck it measures 3 mm. in 

 diameter. At (a) the lateral tube is constricted, then blown out into a small bulb 



