WATER 439 



5 . To each tube of litmus milk add a measured quan- 

 tity of the suspension corresponding to the amounts 

 employed in isolating the coli group (vide page 437). 



6. Incubate each tube anaerobically at 37 C. 

 Anaerobic conditions can be obtained by putting the 

 cultures up in Buchner's tubes or in Bulloch's appa- 

 ratus. If, however, whole milk has been used in making 

 the litmus milk the layer of cream that rises to the sur- 

 face will be sufficient to ensure anaerobiosis ; whilst 

 if separated milk has been employed it will be sufficient 

 to pour a layer of sterile vaseline or liquid paraffin on the 

 surface of the fluid. 



7. Examine after twenty-four hours' incubation. 

 Note (if B. enteritidis sporogenes is present) 



(a) Acid reaction of the medium as indicated by 

 the colour of the litmus or its complete decolourisation. 



(b) Presence of clotting, and the separation of clear 

 whey. 



(c) Presence of gas, as indicated by fissures and bub- 

 bles in the coagulum, and possibly masses of coagulum 

 driven up the tube almost to the plug. 



8. Replace the tubes which show no signs of growth in 

 the incubator for a further period of twenty-four hours 

 and again examine with reference to the same points. 



9. Remove those tubes which give evidence of growth 

 from the Buchner's tubes and carefully pipette off 

 the whey ; examine the whey microscopically. 



10. Inoculate two guinea-pigs each subcutaneously 

 with 0.5 c.c. of the whey and observe the result. 



Vibrio Choleras. 



1. Number ten tubes of peptone water consecutively 

 from i to 10. 



2. To each of the tubes of peptone water add a meas- 

 ured quantity of the suspension, corresponding to 

 those amounts employed in isolating the members of 

 the coli group (vide page 43 7) . 



