iii^rlO MANUAL OF METHODS FOR PURE CULTURE STUDY 



air normally present within the container. The method has been 

 modified by Mueller and Miller (1941) and their report forms the 

 basis of the description below. 



Materials: (1) A suitable container (see below); (2) fruit jar 

 rubber ring moistened with glycerol or plasticene; (3) chromium 

 powder^; (4) H2SO4, 15% by volume (3 vol. cone, acid to 17 vol. 

 distilled water); (5) Na2C03. 



Note: In this method a desiccator equipped with a stopcock may be used if 

 available; or, for tube cultures a 2-quart Ball fruit jar, prepared as follows, can be 

 recommended: Have a metal casting of the glass cap made. Solder a short length 

 of brass tubing into a hole drilled through the cap. Attach a short LT-tube of 5 or 

 7 mm. glass tubing by a rubber connection. Dip the other end of the U-tube below 

 the surface of mercury (about 2 ml.) in the bottom of a small tube about 2 inches 

 in length. Plug the open end of this tube with cotton to prevent spattering of 

 the mercury. Tie this latter tube to the brass tubing or hold in place by a rubber 

 band. 



Method: Place inoculated tubes in jar. Add tube of methylene 

 blue solution (see p. 11143-5). Add 3 g. of chromium powder and 1 g. of 

 Na2C03. Using a funnel, introduce 30 ml. of 15% H2SO4. Clamp 

 lid on jar immediately; if plasticene is used, prepare the seal around 

 the lid, and allow the hydrogen and CO2 to escape through the mer- 

 cury trap tube. As soon as the bubbling subsides, place the jar in 

 the incubator. 



Advantages: Quick method of obtaining anaerobiosis for tube culture. With 

 other containers the system may be used for plate cultures. Relatively inexpensive 

 chemicals are employed, though the powdered chromium may not always be avail- 

 able. Disadvantages: Necessity of securing metal castings of jar top. Outsides 

 of tubes become covered with chemicals necessitating rinsing when they are removed 

 from container for examination. 



Oxygen Removal by Combustion Using Laidlaw Principle 

 For laboratories which are engaged in problems where anaerobic 

 plating is to be done frequently, it is advisable to plan for this and 

 to purchase equipment accordingly. Although the systems discussed 

 above may be adequate for this purpose, it is well to consider one 

 of the jars which utilize, on the Laidlaw (1915) principle, combustion 

 as a means of securing the anaerobic environment. These methods 

 were designed especially for incubation of plates, but other culture 

 vessels (flasks, tubes, bottles, etc.) may be used. Jars using this 

 principle are those of Brewer (Brown and Brewer, 1938) and Mc- 

 intosh and Fildes (Fildes and Mcintosh, 1921). 



BREWER ANAEROBIC JAR^° 



Materials for method of Brown and Brewer (1938): (1) Brewer 

 jar complete with electric cord; (2) source of illuminating gas or 



^Chromium powder — 98% pure; e.g., from Fisher Scientific Company, Pittsburgh, 

 Pennsylvania or Eimer and Amend, New York, New York. 



^"Brewer jar. Baltimore Biological Laboratory, Baltimore, Maryland and Fisher 

 Scientific Company, Pittsburgh, Pennsylvania. 



