330 TRANSACTIONS LIVERPOOL BIOLOGICAL SOCIETY. 



sterilised, tightly closing paint tin. They were taken 

 back to Liverpool on the same evening and the unopened 

 tins were at once placed in a sink and packed round with 

 fragments of ice. The lids of the tins were tightly 

 closed to prevent ingress of the water resulting from the 

 melting of the ice. Every precaution was thus taken 

 to prevent multiplication of the micro-organisms con- 

 tained in the shellfish during the interval between 

 collection and analysis. When the tins were opened 

 next day the valves of the mussels were still tightly 

 closed ; in the meantime not all the ice had melted. 



The primary inoculations were made in the forenoon 

 of June 7th, within 20 hours after collection of the 

 sample. 'Ten mussels were selected at random from the 

 entire sample. The outside of the shell of each was 

 rapidly washed with tap water (Liverpool water is free 

 from sewage bacteria). The mussel was then opened 

 with a sterile instrument ; a slit with a sterile knife was 

 made through the visceral mass into the stomach, and 

 then about l/10th cc. of the liquid in the latter cavity 

 was withdrawn in a recently-drawn-out glass pipette. 

 This was evenly spread over the surface of a recently 

 poured plate of Grrtinbaum's neutral-red, bile-salt, lactose 

 agar. At the same time a similar quantity of liquid was 

 taken up in the same pipette and inoculated in a recently 

 boiled tube of sterile milk ; a different pipette was used 

 for each mussel. The plates were incubated for 24 hours 

 at a temperature of 42°C. The milk tubes were heated 

 to 78°C. for 20 minutes to destroy vegetative forms of 

 bacteria, and they were then incubated for 24 hours 

 at 42°C. under anaerobic conditions (in an atmosphere of 

 hydrogen). 



The results of the primary surface inoculations on 

 the neutral-red agar are given below: — 



