Sterilization and Protection of Culture-media 173 



pressure, in the steam sterilizer or autoclave before they can be 

 pronounced sterile. 



It must always be borne in mind that after sterilization has been 

 accomplished it is necessary to protect the sterilized objects and 

 media from future contamination. 



To Schroder and Van Dusch belongs the credit of having first 

 shown that when mouths of flasks and tubes are closed with plugs 

 of sterUe cotton no germs can filter through. This discovery 

 has been of inestimable value, and has been one of the chief means 

 permitting the advance of bacteriology. If, before sterilizing, 

 flasks and tubes are carefully plugged with ordinary (non-absorbent) 

 cotton-wool, they and their contents will remain free from the 

 access of germs until opened. Instruments may be sterihzed 

 wrapped in cotton, to be opened only when ready for use; or instru- 

 ments and rubber goods sterihzed by steam can subsequently be 

 wrapped in sterile cotton and kept for use. It is of the utmost 

 importance to carefully protect every sterihzed object, in order that 

 the object of the sterihzation be not defeated. As the spores of 

 molds falling upon cotton sometimes grow and allow their mycelia to 

 work their way through and drop into the culture-medium, Roux 

 has employed paper caps, with which the cotton stoppers can be pro- 

 tected from the dust. These are easily made by curling a small 

 square of paper into a "cornucopia," and fastening by turning up 

 the edge or putting in a pin. • The paper is placed over the stopper 

 •before the sterihzation, after which no contamination of the cotton 

 can occur. 



n. Sterilization and Protection of Culture-media. — As almost 

 all of the culture-media contain about 80 per cent, of water, which 

 would evaporate in the hot-air closet, and so destroy the material, 

 hot-air sterihzation is inappropriate for them, sterilization by stream- 

 ing steam being the only satisfactory method. The prepared 

 media are placed in previously sterihzed flasks or tubes, carefully 

 plugged with cotton-wool, and then sterilized in an Arnold's steam 

 sterilizer. 



The temperature of boiling water, ioo°C., does not kill the spores, 

 so that one exposure of the culture-media to streaming steam is of 

 little use. The sterilization must be applied in a systematic manner 

 — intermittent sterilization — based upon a knowledge of sporulation. 



In carrying out intermittent sterilization the culture-medium 

 is exposed for fifteen minutes to the passage of streaming steam or 

 to some temperature judged to be suflftciently high, so that the adult 

 micro-organisms contained in it are killed. As the spores remain 

 uninjiu-ed, the medium is stood aside in a cool place for twenty-four 

 hours, and the spores allowed slowly to develop into adult organisms. 



When, the twenty-four hours have passed, the medium is again 

 exposed to the same temperature until these newly developed 

 bacteria are also killed. Eventually, the process is repeated a 



