300 METHODS OF IDENTIFICATION AND STUDY 



METHOD. 



1. Prepare tube cultivations on solid media of opti- 

 mum reaction; incubate forty-eight hours under opti- 

 mum conditions as to temperature and atmosphere. 



2. Examine preparations from the cultivation micro- 

 scopically to determine the absence of spores. 



3. Pipette 5 c.c. salt solution into each of twelve 

 capsules. 



4. Suspend three loopfuls of the surface growth 

 (using a special platinum loop, vide page 316) in the 

 normal saline solution by emulcifying evenly against 

 the moist walls of each capsule. 



5. Transfer emulsion from each capsule to sterile 

 250 c.c. flask, and mix. 



6. Pipette 5 c.c. emulsion into each of twelve sterile 

 test-tubes numbered consecutively. 



7. Adjust the first tube in the water-bath, regulated 

 at 40 C., by means of two rubber rings around the 

 tube, one above and the other below the perforated 

 top of the bath, so that the upper level of the fluid 

 in the tube is about 4 cm. below the surface of the water 

 in the bath, and the bottom of the tube is a similar 

 distance above the bottom of the bath. 



8. Arrange a control test-tube containing 5 c.c. sterile 

 saline solution under similar conditions. Plug the 

 tube with cotton-wool and pass a thermometer through 

 the plug so that its bulb is immersed in the water. 



9. Close the unoccupied perforations in the lid of 

 the water-bath by means of glass balls. 



10. Watch the thermometer in the test-tube until it 

 records a temperature of 40 C. Note the time. Ten 

 minutes later remove the tube containing the sus- 

 pension, and cool rapidly by immersing its lower end in 

 a stream of running water. 



11. Pour three gelatine (or agar) plates containing 

 respectively 0.2, 0.3, and 0.5 c.c. of the suspension, 

 and incubate. 



