448 HILDA HEMPL HELLER 



produce anaerobiosis should be avoided whenever possible for 

 routine work, as it interferes with the making of satisfactory- 

 smears; for long incubation and under certain circumstances de- 

 manded by technical considerations, vaseline will be found very 

 useful. Ghon and Sachs recommend the use of agar for strati- 

 fication; liquid media should be frozen before the agar is poured. 



Heating. To free sporulating organisms from non-sporulating 

 organisms heating is always resorted to. Heating of inoculum 

 may be performed in one of two ways. Heavily inoculated media 

 may be heated to 80° in a water bath for fifteen to thirty minutes. 

 This method is highly inaccurate, especially in case pasty media 

 are used, but it serves on occasion. Or the material to be in- 

 oculated may be heated in a Pasteur pipette after the following 

 fashion: 



Sera, exudates, and muscle extracts should be diluted with 

 sterile saline. Cut the end of a Pasteur pipette off square with a 

 file, flame it, then draw up the inoculum for about two inches by 

 capillary attraction, and seal the pipette with less than a quarter 

 of an inch of air space between the tip and the liquid. To kill 

 non-spoi ulating organisms heat in a waterbath for ten minutes 

 at 70° to 72°. Then flame the pipette above the inoculum to kill 

 organisms that may have been above the water-line, mark the 

 tip in several places with the file or diamond, slowly flame the tip, 

 insert it in the tube of fresh medium, flame a pair of light forceps 

 and with them break the tip of the pipette against the inner wall 

 of the tube and expel the material. 



If a worker is certain that the type of sporulating anaerobe de- 

 sired is always highly resistant to heat, he may use higher tem- 

 peratures, in the neighborhood of 100°C. for heating his cultures. 

 Dr. K. F. Meyer informs me that he has repeatedly employed 

 this method with success in the isolation of Bacillus botulinus. 

 Von Hibler sowed mixtures containing such organisms, and even 

 less resistant ones, directly into hot agar. Some strains of B. 

 hotuUniis and of Novy's bacillus are highly resistant to heat. 



