4 



Mr. J. Thompson. Action of 



[Aug. 27, 



Throughout the experiments detailed below, the air was circulated through 

 the apparatus every 24 hours, readings being taken to check the course of the 

 experiment. At the conclusion of each experiment, the residual gas in the 

 apparatus was examined gasometrically for marsh gas and hydrogen, which 

 were in each case found to be absent. 



II. For the examination of the products resulting from the action of 

 B. cloacae, on citric and malic acids, the general method described below was 

 employed. The media were prepared from the acids by drying these to 

 constant weight in vacuo over sulphuric acid, dissolving in distilled water, 

 and, after neutralising with ammonia to convert them into their ammonium 

 salts, adding sufficient normal sulphuric acid to give the finished product an 

 acidity of +15. Acid potassium phosphate was added in the proportion of 

 0"1 grm. per litre of medium. The malic acid was present in the proportion 

 of 6 "7 grm. per litre, while, for the citric acid medium, the proportion used 

 was 7 grm. of crystallised acid per litre, this quantity corresponding to 

 6 - 4 grm. of the anhydrous acid. 



One litre of the medium was placed in a 2-litre flask, fitted with leading 

 and delivery tubes plugged with cotton-wool. The delivery tube was 

 connected outside the incubator with an absorption flask provided with a 

 soda-lime tube, to allow of exit of the excess of air or nitrogen, and containing 

 200 c.c. of a 10 per cent, solution of caustic potash for the absorption of the 

 carbon dioxide produced. After sterilisation, the medium was inoculated 

 from a pure culture of B. cloacae, on agar, and a slow current of air, free from 

 carbon dioxide, was bubbled through the apparatus during the course of 

 growth. In the case of the experiments conducted in the absence of 

 oxygen, the leading tube of the incubation flask was fitted with a three-way 

 tap, by means of which a stream of nitrogen could be passed through the 

 flask, after first displacing the air in the leading tube. The nitrogen was 

 prepared by the action of ammonia solution on copper turnings, and passed 

 through wash-bottles containing sulphuric acid, chromous chloride solution, 

 and, finally, caustic potash. Growth in the incubation flask having ceased, 

 which was found to be the case after about four weeks, the amount of carbon 

 dioxide was determined by double titration of the caustic potash solution, and 

 the contents of the incubation flask were examined as follows : The liquid 

 was made up to 1 litre with distilled water, 500 c.c. were taken, made acid 

 with 40 c.c. of normal sulphuric acid, and carefully distilled. The first 

 200 c.c. of distillate were collected, titrated with normal potash, using 

 phenolphthalein paper as indicator, and redistilled. One hundred cubic 

 centimetres of distillate were collected, and its specific gravity determined. 

 In each experiment this was found to be equal to that of water, proving the 



