A Sugar Bacterium. 



67 



always results in the destruction of the sugar, and the production of 

 enormous quantities of carbon dioxide. Obviously, also, these fermen- 

 tations are anaerobic. 



In order to put this last point beyond all cavil, however, we placed 

 a clump of the compound organism into a mixture of a 15 per cent, 

 solution of sugar to which 10 per cent, gelatine had been added, and 

 kept the tubes at 30 3 C, while the air was pumped out from the fluid 

 mass and pure carbon dioxide allowed to filter in, and this process was 

 repeated four or five times, and the de-oxygenated gelatine, still in an 

 atmosphere of carbon dioxide, was then allowed to set. In a fortnight 

 the solid gelatine in all these tubes had visible submerged colonies 

 throughout the mass, and examination showed these to consist of the 

 bacterium and yeast found in the original clumps. 



Similarly, streak cultures on the same sugar-gelatine medium, grew 

 normally on the sloped surface in tubes filled with carbon dioxide, and 

 in these again were observed the same bacterium and the same yeast as 

 had been foimd predominating in the original clumps. 



These cultures — still preliminary in nature and only dealing with 

 the composite organisms of the clumps as a whole — suggested an 

 obvious method for separating at least this prominent bacterium and 

 yeast from the clumps. 



Sugar-gelatine, made as before, was infected with a small piece of a 

 clump, rubbed up by a platinum loop in sterile water, and plates made 

 in the ordinary way in Petri dishes ; the dishes were then placed trader 

 a receiver, attached to the pump and exhausted, and then filled with 

 carbon dioxide, with proper precautions as to the purity of the gas, 

 filtration through cotton-wool plugs, &c., and the cultures put aside in 

 an atmosphere of carbon dioxide at the ordinary temperature. In a 

 week the solid gelatine showed two kinds of colonies, one consisting 

 entirely of a yeast, the other of a bacterium, and closer investigation 

 showed them to be identical with the prevailing yeast and bacterium 

 in the original clumps. 



Repeated plate-ciiltures made in this way gave consistently the same 

 results, and there was no room for doubt that these are the two essential 

 organisms of the clumps, though they are not the only species found 

 in the original material, there being at least one other yeast-like 

 organism, so common that for some time it was thought it must play 

 an essential part. Since this latter — and certain much rarer forms 

 occasionally found — will not grow in the atmosphere of carbon dioxide, 

 however, there can be little doubt that the two anaerobic microbes 

 isolated by the above process are the essential constituents in the 

 fermentations referred to. 



Their further separation by means of repeated plate-cultures, as 

 above, was comparatively easy, the yeast especially being readily 

 picked out and further cultivated in sugar-gelatine tubes. 



