METHODS USED IN CULTIVATION OF BACTERIA 187 



oil at the top, placed into anaerobic jars, or incubated without jars 

 according to purpose. 



For anaerobic jar methods, we hardly ever use hydrogen, em- 

 ploying one of the two following : 



Jars may be of any size or shape, provided they are well constructed 

 strong museum jars with well fitting lids, preferably ground glass surfaces 

 on lid and top of jar, and with one perforation in the top. Stoppers and 

 glass tubing are fitted into the top of the jars with Major's cement, and 

 recemented every time the jars are used. Glass tubing which communicates 

 with the exterior through the cemented joints is drawn out in the flame, 

 along its course connecting with the suction pump, and closure is effected 

 by sealing in these narrow places with the flame. 



The fitting of the lid for closure is accomplished by a thick layer of 

 sculptors' plastocene, a smooth layer of which is placed on the top of the 

 jar, and another on the lips of the lid before the lid is put in place. It is 

 useful to have a vacuum gauge cemented into the lid so that the functioning 

 of the suction pump can be controlled. This, however, is not absolutely 

 necessary. A small tube containing a weak methylene blue solution in 1 per 

 cent glucose broth is placed inside the jar with the cultures. Decolorization 

 of this on the following morning will prove anaerobiosis. 



I. One method which can be used for Petri plates or tubes consists in 

 placing a suitable amount of dry pyrogallic acid into the bottom of the 

 jar. The cultures are then inserted and next to them is placed an envelope 

 of thick brown paper, such as used by commercial houses, which is torn so 

 as to be open at the top and to form a sort of elongated bag or cornucopia. 

 The glass top of the lid is then connected with the suction pump before 

 it is placed on the jar, the suction pump started while an assistant holds 

 the lid ready for placement on the jar. A suitable amount of 20 per cent 

 KOH is then poured into the envelope and the lid immediately placed 

 in position on the top of the jar, pushed down on all sides so that the 

 plastocene flattens out between the lid and the jar. The suction immediately 

 begins to draw the air out of the jar, before the KOH solution works its 

 way through the envelope and begins to dissolve the pyrogallol. The suction 

 is then continued for a suitable length of time, and while the pump is still 

 going a Bunsen burner is placed under a narrow place in the connection 

 glass tube and this is sealed in the flame. This method if successfully carried 

 out, gives almost complete anaerobiosis. 



II. The second method is that of Mclntosh and Fildes, 11 which 

 is, in our opinion, the best anaerobic method for application to the 



"Mclntosh and Fildes, Lancet, 190, 1916, 768. Methylene blue added in 

 sufficient quantity to 10 c.c. of a 2 per cent dextrose alkaline broth to give a 

 distinct blue color, is the most convenient anaerobic indicator. It depends on 



