BY R. GREIG SMITH. 71 



To obtain a medium free from the disturbing influence of 

 phosphoric acid, bouillon was shaken up with slaked lime for 

 several hours and filtered ; washed carbon dioxide was passed 

 through the filtrate for some time and the precipitated carbonate 

 filtered off. The filtrate was then boiled to decompose the dis- 

 solved bicarbonate and the fluid again filtered to separate the 

 precipitated carbonate. The resulting neutral medium contained 

 no phosphoric acid and no lime, which was shown by testing with 

 ammonium molybdate and ammonium oxalate. Neither did it 

 give a precipitate with calcium salts, even with the addition of 

 small quantities of sodium hydrate. 



The three experimental bacteria grew slowly in the phosphate- 

 free bouillon. When they had made some headway portions were 

 tested, and the bacteria were found to be entirely unaffected by 

 the addition of calcium chloride, potassium, ammonium or sodium 

 salts. When the phosphoric acid was restored to the medium by 

 the addition of traces of potassium phosphate, calcium chloride 

 resumed its flocculating power. On treating the phosphate-free 

 cultures with calcium chloride and alkali (0-5 c.c. tenth-normal 

 sodium hydrate to 2 c.c. culture) a fine precipitate was obtained 

 which very slowly gravitated. The deposit when examined 

 microscopically was found to contain no bacterial floccules, and 

 the organisms were free in the supernatant liquid. Accordingly 

 it seems probable that the precipitate was the calcium salt of an 

 organic acid elaborated by the bacteria. The precipitate is more 

 marked when the bacteria are killed and partly disintegrated by 

 boiling. 



These experiments have shown that a pure flocculation of 

 bacteria by means of the usual flocculating agents cannot be 

 obtained. The reason for this is undoubtedly because the salt 

 diffuses quickly through the bacterial cell and no surface pressure 

 is occasioned. In working with dilute solutions of calcium 

 chloride or better calcium bicarbonate and cultures containing 

 phosphates, a flocculation of the bacteria is noticed, and this is 

 quite apart from the entangling action of the calcium phosphate. 

 It is worthy of emphasis that the amorphous particles of recently 



