OPSONINS 287 



of bacterial growth. In making vaccines with poorly growing organisms, 

 large surfaces must be inoculated. Organisms are best grown for this 

 purpose upon the surface of agar or glucose agar (the enrichment of the 

 agar with sugar or acetic fluid, etc., depending upon the cultural require- 

 ments of the organism in question), in square eight-ounce medicine 

 bottles laid upon their sides. This furnishes a large area for inoculation. 

 After sufficient growth has taken place upon the agar, two or three cubic 

 centimeters of sterile normal salt solution are introduced into the 

 bottles with a sterile pipette. With this the growth is gently washed 

 off the surface of the agar, more salt solution gradually being added as 

 necessary. The emulsification may be facilitated by gently scraping 

 the growth off the medium by means of a flexible platinum loop. This 

 thick bacterial emulsion is then pipetted out of the bottles, during which 

 process an equalization of the emulsion can be attained by repeated 

 sucking in and out with the pipette. The emulsion is then placed in a 

 sterile test tube which may then be drawn out at its open end into a 

 capillary opening. It is a point of practical importance that, in pre- 

 paring such capsules out of a test tube, a few inches of air space should 

 be left above the surface of the emulsion, so that expansion during 

 heating may not blow out the top of the glass tube. A dozen or so of 

 sterile glass beads may be put into these tubes in order to aid in emul- 

 sification. Shaking the beads in such a tube will help in breaking up 

 small clumps of bacteria. 



The emulsion is then standardized ; that is, a numerical estimation 

 of bacteria per cubic centimeter must be made. This standardization 

 is best done before sterilization, because during the latter process a 

 number of bacteria may be broken up, and, while unrecognizable 

 morphologically, are, nevertheless, represented in the emulsion by their 

 products. The standardization may be accomplished by highly diluting 

 a definite volume of the emulsion, planting plates with definite quan- 

 tities of the dilution, and counting colonies. Wright prefers, as more 

 exact, an enumeration of the 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 



