242 



MICROBIOLOGY 



of -f-2.9 to phenolphthalein). In order to have a suitable culture on 

 liand, subcultures should be made daily. A culture more than 72 

 Jiours old should not be used in preparing the test fluid. 



Test-fluid. The test-fluid is prepared by washing the growth 

 from the agar culture by the aid of a sterile wire loop into distilled 

 water containing 0.85 per cent sodium chloride and 0.5 per cent car- 

 bolic-acid crystals. This suspension is then placed in a thermostat 

 at 60 C. for two hours, which kills the bacteria. A temperature 

 higher than 65 C. or lower than 60 C. should be avoided. After 

 lieating, the suspension is thoroughly triturated and filtered through 

 sterile cotton. Thorough trituration of the emulsified growth is 

 S3ential before filtering. The filtrate thus prepared is diluted with 

 the carbolized-salt solution until it is of a faintly clouded appearance. 

 The proper dilution of the filtrate can only be determined by experi- 

 ence. The test-fluid gives the best results when made with freshly 

 prepared carbolized-salt solutions. 



Procuring the serum. The serum is easily obtained. At least 

 10 cc. of blood are drawn from the jugular vein, under aseptic pre- 

 cautions, into a small sterile bottle, and sent to the laboratory. As 

 soon as the clot forms, the supernatant serum is placed in a centri- 

 fuge and all the sediment thrown down, leaving the liquid perfectly 

 clear. One cc. of the serum is then added to 39 cc. of a physiologi- 

 cal salt solution, which makes a dilution of 1-40. It is desirable that 

 the serum should be secured as soon as possible after the blood is 

 drawn. If necessary to delay the test, the serum has given the best 

 results if kept at about 10 C. until used. 



Making the test. Three cc. of the "test-fluid" are placed in 

 .each of several small test-tubes. With a sterile pipette, the diluted 

 tserum is added to the tubes of test-fluid and thoroughly mixed. In 

 making the different dilutions, the amount of diluted serum to be 

 used is readily ascertained by the following table: 



TABLE 



