ZOOLOGY AND BOTANY, MICROSCOPY, KTC. 305 



white-hot and an explosion will result. The haemoglobin in the super- 

 ficial layers of the medium becomes reduced as the oxygen is given up, 

 and the surface is rendered suitable for anaerobic growth in a few 

 minutes. 



By this method the organisms of tetanus, botulismus, and malignant 

 oedema grow with great freedom, and nearly the whole surface of the 

 medium is covered with growth in forty-eight hours. Plating methods 

 may be adapted by use of the Roux bottle, or similar contrivance, using 

 larger pieces of platinum and plenty of hydrogen, as explosions in such 

 bottles might be dangerous. When taking samples with a warm platinum 

 loop several minutes should be allowed to elapse after opening the bottle, 

 in order to avoid risk of explosion. 



The method can be applied to broth. As the tube cannot be held 

 upside-down, a very brisk stream of hydrogen is bubbled through the 

 medium and the tube corked up as soon as possible after the capillary 

 has been withdrawn. The method is rapid and simple in theory and 

 practice, and is quite cheap, as the pieces of platinum can be used again 

 and again. A tube or bottle can be inoculated, rendered anaerobic, and 

 put in the incubator in a few minutes. 



Method 2. Colloidal Platinum.- — In this method, test-tubes which have 

 been cut short are introduced upside-down into the lumen of somewhat 

 larger tubes. These tubes are filled with broth to which a trace of 

 methylene-blue has been added. The tubes are then plugged and 

 sterilized ; 2 c.cm. of platinum sol are added to each tube, the tube 

 inoculated, and a stream of hydrogen passed through the upper layers 

 of the medium by means of a fine capillary. The methylene-blue is 

 gradually bleached, the colloidal platinum acting as a catalyst, and the 

 hydrogen gradually destroying all the oxygen present in the medium. 

 The capillary is then passed down to the bottom of the tube, and the 

 inner tube filled with hydrogen. The capillary is then withdrawn, and 

 the tube plugged and subsequently incubated. The hydrogen in the 

 inner tube acts as a reducing agent which destroys oxygen from the 

 surface of the medium. The method gives good results with the organ- 

 isms of botulismus and malignant oedema, but with tetanus the growth is 

 slow and poor. 



Method 3. Colloidal Platinum and Sodium Formate. — An attempt to 

 dispense with hydrogen was made by adding to broth excess of sodium 

 formate and colloidal platinum, the idea being that the free oxygen 

 would be used up in oxidizing the formate owing to the vigorous 

 catalytic action of the colloidal platinum. In broth, however, some 

 constituent interferes with the reaction. Bouillon made with Witte's 

 pepton (Douglas) did not give good results, but broth made with a 

 tryptic digest proved to be more suitable. To the latter medium was 

 added a trace of methylene-blue, sodium formate up to 1 or 2 p.c, and, 

 just before inoculating, 2 c.cm. of colloidal platinum, or platinum-black 

 suspension. The tubes were plugged with cotton-wool, and the con- 

 tained media thus given free access to the air. The tubes remained 

 anaerobic for twenty-four or forty-eight hours. B. Botulinus grew ex- 

 tremely well and regularly, the' bacillus or malignanl oedema less 

 regularly, while the Barilla's tetani either grew badly or not at all. 



