SHAKE-TUBE METHOD 



353 



The gelatin tube can be inoculated directly with a drop of the diluted culture ; 

 the agar tube should be cooled to 45° C. before inoculation. The culture is then 

 thoroughly mixed by rotation of the tube, and the mixture is poured out into a 

 Petri dish and allowed to set. Gelatin sets at about 25° C, agar at 38° C. In 

 warm weather the gelatin plates should be set on ice. 



C. Shake-tube Method.- — This is used chiefly for anaerobes. A test-tube con- 

 taining about 15 ml. of solid medium, generally made up with a reducing agent 

 such as glucose, is heated till the medium is melted ; a drop of culture is delivered 

 into the medium, which has been cooled to a suitable temperature, and thoroughly 

 mixed by gentle shaking and rotation. The medium is then allowed to set in the 

 tube in a vertical position. Incubation is carried out aerobically or anaerobically. 

 The great value of this method is that it affords a simple means 



of grading the oxygen pressure in the medium ; on the surface the 

 pressure is atmospheric ; at the bottom of the tube, particularly 

 if a reducing agent has been added, the conditions are completely 

 anaerobic. Thus the aerobic bacteria grow at or just below the 

 surface ; the anaerobic bacteria grow near the bottom ; and the 

 facultative aerobes and anaerobes are distributed throughout the 

 medium. The single colonies, which appear, are often fairly 

 characteristic. To pick them off, the test-tube is cut round with 

 a diamond at the middle of the column of medium, the two 

 halves of the tube drawn apart, and the medium allowed to fall 

 gently into a sterile Petri dish. A stout platinum wire is then 

 used to fish the colonies ; it is stabbed into the medium over 

 the particular colony desired, taking care that no other colony 

 is touched on the way ; and as soon as it has come into contact 

 with the colony, it is withdrawn and inoculated into broth. Some- 

 times it is advisable to cut the medium into pieces with a sterile 

 scalpel before attempting to pick off the colonies. This method 

 was used at one time for purifying anaerobes ; but now that the 

 technique of obtaining anaerobiosis has improved, it has been 

 largely replaced by the more reliable surface plating method. In 

 order to avoid breaking a test-tube every time a colony has to 

 be picked off, it is better to use a Veillon tube instead of the 

 ordinary test-tube. This consists of a piece of glass-tubing, about 

 1 cm. in diameter, and 8 or 10 inches long. One end is fitted 

 with a rubber cork, the other with a cotton-wool plug. The medium is poured 

 into the tube till it reaches about half or two-thirds of the way up. Inocula- 

 tion is performed in the usual way. When it is desired to pick off a colony, 

 the rubber cork and the woollen plug are removed, and the whole column of 

 medium expelled by a stout glass rod into a Petri dish. The tubes and corks 

 can be used over and over again. 



D, Motility. — Various methods have been devised for making use of motility 

 to separate motile from non-motile organisms. Rovida's (1925) tube, which is 

 a modification of that invented by Carnot and Gamier (1902), may be used for 

 this purpose. It consists of a large test-tube to the bottom of which is fused a 

 glass tube of 7 mm. internal diameter. This inner tube has a constriction near 

 the lower end, below which are three small holes to afford a communication between 

 the inside of the inner and the outer tubes. Above the constriction there is a 



Fig. 56.— 



Veillon 



Tube. 



