SEA-FISHERIES LABORATORY. 245 



From each secondary agar culture a tertiary sub- 

 culture is made in a tube of each of the following media. 

 Bile-salt glucose broth (McConkey's well-known medium), 

 litmus glucose, lactose, mannose, sucrose, and glycerine 

 broths, and litmus milk. These tertiary sub-cultures are 

 then incubated for 48 hours at 42° C. and the results, 

 production of acid and gas, and in the case of the milk, 

 acid and clotting, noted. The secondary agar cultures 

 are also examined for the motility of the microbes. 



By Bacillus coli is understood a microbe giving the 

 following reactions : — 



1. Deep red colonies (surrounded by a haze usually 



if deep) in neutral-red, bile-salt, lactose agar. 



2. Acid and gas in bile-salt glucose broth. 



3. „ ,, glucose broth. 



4. ,, ,, lactose broth. 



5. ,, ,, mannose broth. 



6. „ ,, or no change in sucrose broth. 



7. x^o change, or simple acid in glycerine broth in 



48 hours. 



8. Acid and clotting in milk. 



9. Well-marked motility in young agar cultures. 



It will be seen then that the process of analysis is 

 laborious and cannot be hurried. 



In the case of the examination of a dozen mussels 

 24 primary cultures have to be made. When the neutral 

 red primary plates are examined the colonies resembling 

 those produced by B. coli are counted and recorded as 

 " colon-like colonies." From the twelve plates so counted 

 perhaps 18 (at the least) separate colonies are sub-cultured 

 on agar and each of these is then sub-cultured T times — 

 that is 126 tertiary sub-cultures may be made. Of the 

 18 colonies thus examined in detail, perhaps 9 will prove 

 Q 



