66 INFECTION, PHAGOCYTOSIS, OPSONINS 



leukocytes, bacterial emulsion) are mixed by being several times 

 drawn up and again expelled from the pipette into a watch crystal 

 or glass slide. The mixtures in the little pipettes, after these have 

 been sealed in the frame, are finally placed in the incubator or a 

 special opsonic oven. After half an hour the mixtures are blown on 

 a slide, spread out, air dried, stained, and then the bacteria taken 

 in by 200 leukocytes are counted. 



FIG. 31 



Opsonic pipette after mixture of the three constituents; the tip has been fused and the 

 pipette is ready for the opsonic incubator. (Miller.) 



Preparation of Vaccines. Considerable care has to be exercised in the 

 preparation of the bacterial vaccines or bacterines used for therapeutic 

 injections. Unless an autovaccine, that is, a vaccine, from the bacteria 

 infecting the patient it wanted the vaccines are generally procured from 

 one of the pharmaceutical houses which prepare them in their biologi- 

 cal laboratories. The autovaccine must be prepared directly from the 

 infecting organism. As a first step, a pure culture must be obtained, 

 which may be raised in bouillon or on slanted agar; the latter is prob- 

 ably the better. The growth is then removed with a platinum loop 

 or spatula, mixed with physiologic salt solution, shaken long and 

 thoroughly, and then heated to kill the bacteria. The remaining 

 coarser particles are removed by centrifuging and the uniform emul- 

 sion is standardized by estimating the number of bacteria present per 

 cubic centimeter. This is done by collecting and mixing one part of 

 the vaccine, three of physiologic salt solution, and one of normal 

 human blood in a blood pipette. After these ingredients have been 

 thoroughly mixed, drops are blown on a clean slide, spread, air dried, 

 fixed, and stained. Then 200 red blood corpuscles are counted and 

 the number of bacteria in the same number of fields is ascertained. 

 Since it is known that each cubic millimeter of normal human blood 

 contains five million erythrocytes, the number of bacteria present 

 in the same amount of fluid can easily be calculated from the number 

 counted in the same spaces which contained the 200 erythrocytes. 

 After the calculation has been made the vaccine can be so diluted 

 that it contains from 50,000,000 to 300,000,000 bacteria per cubic 

 centimeter. These are the average doses used in the vaccine treat- 

 ment in man. A small amount of lysol or some other antiseptic 

 should be added to the vaccine, unless it is used at once. In count- 

 ing the blood corpuscles and bacteria, it is advantageous for accurate 

 work to have the microscopic field of vision limited by placing horse- 

 hairs in the form of a rectangle in the eyepiece or by the use of 3 

 square diaphragm. 



