TECHNIQUE OF BACTERICIDAL TEST-TUBE EXPERIMENTS. 351 



plate sown must contain many thousands or an innumerable number 

 of colonies. The bactericidal effect will then be distinctly shown by 

 the reduction in the proper plates of this large number of colonies 

 to zero or almost zero. 



The test-tubes most advantageously employed are the little 

 tubes 9-10 cm. long and 1.3 cm. diameter. The cotton stoppers are 

 removed and all the different components filled into the tubes. Then 

 the stoppers are replaced after being flamed. If the air is at all 

 still one need not fear keeping the tubes open for this length of time. 



In testing an immune serum one commences by examining the 

 immune serum in the fresh active state, and, of course, in the same 

 manner that the serum of the animal in question was examined pre- 

 vious to immunization. For this purpose a number of test-tubes are 

 filled with 1.0, 0.3, 0.1, 0.03, 0.01 cc. of the fresh active serum. Finer 

 gradations are useless in view of the lack of sensitiveness of the test- 

 tube method. This we have already pointed out. The amount of 

 culture to be sown is then added and all tubes filled up to 2 cc. with 

 physiological salt solution. Finally three drops of bouillon are added 

 to each tube. The addition of bouillon has proven to be of consider- 

 able value, for it suffices to balance disturbing variations of the 

 osmotic pressure. It is important to make the total volume of fluid 

 the same in all the tubes by the addition of fluid. Besides this it is 

 important to have a number of controls, namely, a control of the 

 culture sown, second, a control testing the sterility of the maximum 

 amount of serum employed, and third, a control, or better a series 

 of controls, containing the culture sown plus the serum in an inactive 

 form. By means of this last control one can see whether a thermostable 

 complement is present or not. It also serves to show that the bacteri- 

 cidal action is not simulated by the agglutinating power of the serum. 



The tubes are now kept in the thermostat for at least three hours, 

 having previously, however, been carefully shaken. On being taken 

 out of the thermostat they are again carefully shaken and then 

 worked up into plates. For this purpose 5-10 drops are taken from 

 each tube by means of uniform pipettes and made into plates in the 

 usual way. The plates are placed in the thermostat upside down, 

 and kept there until the following day. The growth is best and 

 most rapidly described by means of approximate estimates, using a 

 scheme somewhat as follows: or almost 0, about 100, several hun- 

 dreds, thousands, very many thousands, infinite number. A distinct 

 bactericidal action is only then present if the controls result as they 



