8 



General Bacteriology. 



tube and stopcock (fig. 1), from which it can be run into sterile vessels. Test-tubes 

 should contain 6-10 cc. of medium (about 3 cm. deep). Flasks are to be filled about 



three-fourths full. 



SPECIAL DIRECTIONS. Fill 15 test-tubes . and preserve remainder 

 of bouillon in larger flasks. 



EXERCISE VI. STERILIZATION OF CULTURE MEDIA. 



O f 



FIG. 1. Appara- 

 tus for filling test- 

 tubes. 



EXPLANATORY. To accomplish this steam is used almost exclusively 

 either as streaming steam or under pressure. The unconfined steam is 

 applied in an apparatus known as a steam sterilizer. Of the various 

 patterns the Arnold is perhaps the most satisfactory. It is effective, 

 economical in the use of gas, and does not allow the escape of large 

 quantities of steam into the room, as a large part is condensed to be re- 

 converted into steam. For student use the form shown in fig. 2 is very 

 convenient. The method of using these different forms is identical. 

 Always have plenty of water present before heating. The discontinuous method is most 

 frequently employed. Exposure is made on three consecutive days for 20 minutes, be- 

 ginning to count time when the material reaches the temperature of the steam, which will 

 vary with different substances and the volume treated. Between successive steamings 

 culture media should be kept under conditions favorable to bacterial development (room 

 or incubator temperature). 



For the employment of steam under pressure the 

 autoclave is essential. The lid should contain a thermom- 

 eter as well as a steam gauge, safety and outlet valve. A 

 thermo-regulator is also desirable. The following table 

 gives the temperature corresponding to atmospheres of 



pressure : 



Atmospheres. Degrees C. 



1 100 

 1.5 112.2 



2 121.4 



2.5 128-.8 



3 135.1 



This table is only true when all of the air in the apparatus is replaced by steam, 

 and hence the steam must be allowed to escape freely before the outlet valve is closed. A sin- 

 gle exposure of 20 minutes at a temperature of 120 C. (one additional atmosphere) is 

 sufficient to kill all. germ life. After the proper exposure, care must be taken not to allow 

 the steam to escape too rapidly, otherwise the culture media may be forced against the 

 plugs owing to the unequal pressure. 



GENERAL DIRECTIONS. Ordinary media may be sterilized by either method. Sugar 

 media cannot be sterilized in the autoclave as it must not be heated above 100 C. The 

 solidifying property of gelatin is impaired if submitted to a temperature of 120 C. 

 longer than 15 minutes, and at a temperature above 120 C. momentarily. 



REFERENCES. A. 55-73; M. & R. 37; McF. 109; N. 161; P. 213. 



SPECIAL DIRECTIONS. Sterilize bouillon prepared in IV. for 20 miuutes in a 

 steam sterilizer oa three consecutive days. 



FIG. 2. Simple sterilizer consisting 

 of a galvanized iron pail with a cover 

 a and a false bottom b. 



