BUTYRIC ACID BACTERIA 289 



Sodium phosphate . . . . . i gr. 

 Magnesium sulphate . . . . . i ,, 

 Sodium chloride . , : , . .t 



Water. .... . , .' 200 c.c. 



Boil briskly for two or three minutes, and while boiling add 

 10 to 15 grams of fresh garden soil. 



Plug loosely with cotton wool and transfer while hot to an 

 incubator kept at 35 C. 



The heating destroys moulds and non-sporing bacteria and 

 drives out dissolved air : the spores of the butyric acid bacteria 

 resist boiling, and, as the liquid cools, germinate and grow, the 

 necessary anaerobic conditions being maintained to a certain 

 extent by resistant aerobic bacteria introduced in the soil. 



Examine the sediment at the bottom of the flask daily for a 

 week as follows : 



(a) Tilt the flask gently and remove a small portion of the 

 slimy deposit with a platinum loop. 



Add it to a drop of a dilute solution of iodine in potassium 

 iodide on a glass slide : put on a cover-slip and examine with a 

 high power. 



The butyric organisms containing granulose are stained a 

 violet or violet-blue colour. 



Look out for clostridial forms with spores in them ; the latter 

 appear as unstained, bright, oval or round bodies in the clostridial- 

 cells. 



(^) When spores are found mix a small amount of the slimy 

 deposit with two or three drops of water in a watch-glass : then 

 spread a drop or two of the mixture on two or three cover-slips 

 Allow to dry : then stain spores by method given in Ex. 12. 



Ex. 140. Half fill a small yeast flask with the solution men- 

 tioned in the previous experiment. 



Close the neck with a rubber cork through which passes a 

 narrow bent glass tube arranged as in Fig. 47. 



Now boil the solution for two to three minutes : and while 

 boiling add 10 to 15 grams of fresh garden soil 

 T 



