136 PHARMACEUTICAL BACTERIOLOGY. 



the equipment to prepare the homologous or autogenous bacterins. The 

 method of preparing a homologous bacterin may be outlined as follows: 



a. A tube, flask or plate with the suitable culture medium (agar or 

 gelatin) is inoculated with the germs taken from the patient and incubated, 

 until a maximum development has taken place, about twenty-four hours. 



b. The growth is separated from the culture medium by means of a 

 sterile physiological salt solution and a platinum wire loop. The salt 

 solution with the bacteria is transferred to a sterile test-tube which is then 

 sealed in a flame. 



c. When the tube is cool, it is shaken vigorously so as to emulsify the 

 bacteria in the salt solution. 



d. The tube is opened and about one drop is removed with which to 

 make the blood-corpuscle count, to be explained later. The tube is again 

 sealed in the flame. 



e. The tube is now placed in a water bath (opsonic incubator of special 

 construction for this work) at a temperature of 60 C. for a sufficient length 

 of time to kill the germs; one hour is usually adequate. This constitutes the 

 bacterin and is ready for use as soon as it is standardized. Usually some 

 preservative is added when the tube is opened and before the bacterin is 

 injected (0.2 per cent, lysol, 0.4 per cent, trikresol, etc.). 



f. From the above it must be evident that no two preparations contain 

 the same number of germs per c.c. and hence the physician cannot know 

 how many dead microbes are injected at a dose. Therefore the necessity 

 of standardizing the bacterin, which is done as follows: 



g. Mix one part of freshly drawn blood with one part of the bacterin 

 (taken from the tube in d.), add two or three parts of physiological salt 

 solution, and spread evenly on a slide. Examine under the microscope and 

 determine the number of microbes per c.c. in terms of the number of red 

 blood-corpuscles per c.c. This is done by making numerous (10 to 20) counts 

 of red blood-corpuscles and microbes. Knowing that there are 5,000,000,000 

 red blood-corpuscles per c.c., it is then a simple matter to compute the 

 number of microbes per c.c. in the bacterin under consideration. The 

 count thus determined divided by the number of bacteria desired for one 

 dose, indicates the number of times the bacterin is to be diluted. This is 

 very clearly illustrated in a chart prepared by Hough ton, shown in Fig. 61. 



The number of bacteria administered per dose depends upon the thera- 

 peutic effects to be produced, the kind of bacterin used, the nature of the dis- 

 ease and the condition of the patient. The rule is to start with small doses, 

 gradually increasing them in such a manner as to secure a maximum of 

 positive opsonic phases with a minimum of negative opsonic phases. In 

 round numbers the dosage ranges from 5,000,000 to 50,000,000 bacilli, rep- 

 resented by varying quantities of the bacterins. 



