OPSONINS AND VACCINE THERAPY 343 



process a number of bacteria may be broken up, and, while un- 

 recognizable morphologically, are, nevertheless, represented in the 

 emulsion by their products. The standardization may be accom- 

 plished by highly diluting a definite volume of the emulsion, planting 

 plates with definite quantities of the dilution, and counting colonies. 

 Wright prefers, as more exact, an enumeration of tfte bacteria 

 against red blood cells. This is done in the following way : 



A little of the emulsion is placed in a watch glass and from it, 

 with a pipette as used in the estimation of the opsonic index, one 

 volume is taken and is mixed with an equal volume of blood from 

 the finger and two or three volumes of salt solution. The salt 

 solution is added in order to dilute the red cells so that they can 

 be conveniently counted and to prevent clotting. These substances 

 are thoroughly mixed in a pipette and spread upon a slide as in 

 making a blood smear, and as even and uniform a smear as possible 

 should be made. They are then stained either by Jenner's or 

 Wright's blood stain. 



The preparations are examined with an oil-immersion lens. In 

 order to limit a definite microscopic field, it is convenient to use an 

 Ehrlich diaphragm, or else, in lieu of this, to mark a circle with 

 a blue pencil upon the lens of the eye-piece. The red blood cells 

 and bacteria, in a number of these fields, are counted and the ratio 

 between them is estimated. Knowing the number of red blood cells 

 to the cubic millimeter in the particular blood employed, by previous 

 blood count, and knowing that equal volumes of blood and of bac- 

 terial emulsion have been used in the mixture, it is easy from this 

 ratio to ascertain the number of bacteria contained in a cubic milli- 

 meter of the original emulsion. Thus, for instance, if in an average 

 of twenty fields bacteria are to red blood cells as two is to one, 

 and the blood employed contains five million red blood cells to each 

 cubic millimeter, then a cubic millimeter of our emulsion contained 

 ten million bacteria, and a cubic centimeter one thousand times 

 as many. 



Special centrifuge tubes with graduated narrow tips at the bot- 

 tom have been suggested by Hopkins for vaccine standardization. 

 Bacteria centrifugalized up to a certain mark represent a definite 

 number per cu. mm. wnen taken up in a given volume of salt solution. 



The vaccine, thus standardized, is sterilized at 60 C. for one 

 hour for several days. Its sterility is then controlled by culture 

 and animal inoculation. 



