CCLTIVATIOX nr BACTERIA 65 



from time to time to the outer vessel the temperature can readily !>e 

 kept between 34 and 38 C., which is sufficiently uniform for bacteria 

 such as the diphtheria bacilli to grow. 



As a temporary expedient during the night, when haste is necessary, 

 it is possible, when the culture medium is solid and within a strong 

 glass tube or metal case, to make use of the body heat by putting it 

 under the clothing next to the body or sleeping upon it. Naturally, 

 this should only be done when other means fail. Several times, when 

 in the country, this method has enabled the writer to obtain a growth 

 of diphtheria bacilli over night, and thus get important information, 

 when otherwise it would have been impossible. 



Methods for Obtaining Anaerobic Conditions for Bacteria. Pasteur 

 excluded the oxygen by pouring a layer of oil on the culture fluid. A 

 simple device is that of Koch, who placed a thin strip of sterile mica 

 upon the still fluid agar or gelatin in the Petri dish, which had already 

 been inoculated. After the solidification of the media the portion 

 under the mica is excluded from the air and anaerobic growth can 

 develop. 



A second simple method (Liborius) is to fill the tubes with media 

 fuller than usual and to inoculate the bacteria deep down to near 

 the bottom of the tubes while the media are still semisolid. An 

 anaerobic growth will take place in the lower part of the tube. In a 

 similar way the closed arm of the fermentation tube will suffice for 

 anaerobic growth, if the opening connecting it with the open bulb is 

 quite small. Wright devised the following procedure: A short glass 

 tube with constricted ends is used. Each end has a piece of rubber 

 tubing attached. One of these is connected with a glass tube, which 

 projects through the cotton plug of the test-tube. The test-tube con- 

 tains bouillon. The whole is sterilized and then the test-tube inocu- 

 lated. The bouillon is then drawn up into the constricted tube, which 

 is sealed by simply pushing down the tube so that both rubber ends 

 are sealed by being bent on themselves. When spores are present, a 

 simple method suggested, I believe, by McFarland, can be success- 

 fully employed. Vessels plugged with stoppers perforated by glass 

 tubes drawn to a point are filled to such a height that when the fluid 

 is heated to 80 C. it will just fill them. They are inoculated when 

 the bouillon is at about 60 C., heated to 80 C., and then sealed by 

 closing the tube's point by means of a flame. After inoculating and 

 heating, instead of sealing the glass tube a sterile rubber cork can be 

 inserted. 



If much fermentation is expected, the cork should be clamped or tied 

 to the bottle, so that it will not blow out. One advantage of this method 

 is that any contaminating organisms which have no spores will be 

 killed. 



When sealed the bottles should be cooled and then placed in the 

 incubator. 



A very convenient modification of Pasteur's method for the growth 

 of bacteria in fluid media is to cover the fluid with albolene or paraffin. 



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