APPENDIX. 409 



is thus placed under favourable (anaerobic) conditions for its growth. 

 To obtain inoculating material from such a tube it is necessary to break it at 

 the bottom when the growth is easily taken from the lower part of the 

 medium. Salomonsen uses a modification of this method, placing one 

 tube within another. A small inner tube containing gelatine or agar 

 is inoculated with the anaerobic organism, the outer tube, the neck 

 of which is sealed, or the opening of which is closed with paraffin, 

 contains bouillon which is inoculated with a strongly aerobic organism, 

 such as the Bacillus subtilis. In place of the bouillon and Bacillus 

 subtilis a solution of one part of pyrogallic acid to ten parts of a ten 

 per cent, solution of caustic potash may be used. Buchner, in making 

 use of this method, plugged his inner tube in the ordinary fashion with 

 cotton wadding and supported it on a kind of stage, closing the orifice of 

 the larger tube with an indiarubber cork. This is a very convenient 

 plan, as it enables the observer to gain access to the cultures very readily 

 indeed. All these methods may be used with both solid and fluid media ; 

 and now that potatoes can be used within test tubes they also may be used 

 for the cultivation of anaerobic organisms. Here all that is necessary is a 

 strong test tube with a lateral stem. The inoculation is made through the 

 open mouth of the tube which is then sealed, after which, by means of an 

 exhaust apparatus, the air is extracted from the potato and then from the 

 tube ; the lateral tube is then sealed. 



To Separate Bacteria from their Prodticts. 



To obtain the products of bacteria apart from the bacteria, and, there- 

 fore, in a sterile condition, several methods of filtration have been suggested, 

 all of them depending on the close porous nature of unglazed porcelain and 

 baked clay. When fluid that originally contains bacteria is aspirated or 

 forced through such a filter, all the organisms are kept back, and a perfectly 

 clear sterile fluid comes through on the opposite side. The best form of filter 

 is a tube of this unglazed porcelain, one end of which is closed, and the 

 other so constricted that a thick walled indiarubber tube may be affixed. 

 The Chamberland filter consists of a wide tube, as above described, the 

 upper piece of which is composed of a glazed funnel-shaped end with a 

 nipple on which a piece of indiarubber tubing can be fastened. The other 

 end of this tubing is fitted to the long glass tube of an ordinary strong wash 

 bottle or flask which acts as a receiver ; the second or short tube of the wash 

 bottle is attached to an aspirating apparatus, either in the form of a siphon 

 bottle placed at a lower level, or of a Geissler water exhaust pump. The wash 

 bottle, the filter, and the glass tube, are all carefully plugged, covered with 

 paper or cotton-wadding and sterilized for an hour at 150 C. The 

 indiarubber stopper of the wash bottle and the tubing are sterilized by 

 being well soaked in a I per 1,000 solution of bichloride of mercury, 

 washed with sterilized distilled water, and then boiled for twenty minutes 

 in the steaming apparatus. As soon as everything is cool the hands are 

 thoroughly cleansed, and the apparatus is put together as above described, 

 a small cotton-wadding plug being left in the short tube of the wash bottle 

 to prevent the return of unfiltered air when the exhaust apparatus is re- 

 moved. When all is ready, the filter is lowered into a tall glass jar with 

 a firm base, which contains the fluid to be filtered. The aspirator or other 

 exhaust apparatus is set to work (care being taken that the exhaust is not 

 too great), and the fluid is drawn into the flask which is gradually filled. 



