SEROLOGY MANUFACTURE AND USE OF SERA AND VACCINES 269 



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 cc. 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 cc. in terms of the number of red 

 blood-corpuscles per cc. 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 cc., it is then a simple matter to 

 compute the number of microbes per cc. in the bacterin under considera- 

 tion. 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 Houghton, 

 shown in Fig. 67. 



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 

 disease 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, 

 represented by varying quantities of the bacterins. 



b. Sensitized Bacterins. The ordinary bacterins effect protection 

 against disease in two ways. a. Stimulating the formation in the body 

 cells and in the blood serum, the specific amboceptors which will increase 

 phagocytosis, b. Stimulating the development of the specific antibodies 

 which will neutralize the specific bacterial toxin (endotoxin). It takes 



