274 Eacterio-vaccines 



of sodium chloride and 0.5 per cent, of phenol is added to each, for the purpose 

 of washing off the bacteria that have grown. This is done by tilting the bottle 

 and permitting the solution to wash over and over the surface. If the culture 

 does not detach, it may be necessary to remove it with a sterilized glass rod, or 

 by means of a sterile swab made by fastening a small plgdget of cotton batting 

 upon the end of a wire. ' 



When the growth is detached and thoroughly mixed with the salt solution, 

 it is removed to a sterile receptacle by means of a sterile pipet. 



What is next done, will depend upon the theory upon which the 

 treatment is based. The culture washings contain: (A) sub- 

 stances derived from the culture-medium that certainly cannot be 

 regarded as useful or beneficial and may be harmful; 



(B) bacterial products, of soluble quality, eliminated from the 

 cells during the hfe activities, some of which may be useful; 



(C) the bacteria themselves, which with their contained prod- 

 ucts — endo-toxins, etc. — are commonly • regarded as the essential 

 immunizing agents. 



If one's theory is that the bacterial cells are essential, and there 

 seems to be a growing tendency toward this view, further treatment 

 is necessary before actually preparing the vaccine for administra- 

 tion; if, however, the collected products of their growth are thought 

 to be of partial or equal value, and are to be preserved, this cannot . 

 be done without also retaining the less desirable matters from the 

 culture-medium. 



Let us suppose that only the bacterial ceUs are to be employed. 



The suspension of bacteria, under these circumstances, is transferred to 

 appropriate sterile tubes, plugged, and whirled in a powerful centrifuge until 

 the bacteria are thrown down to the end of the tube, leaving the supernatant 

 fluid fairly clear. The fluid is then removed by decantation or with a pipet, 

 and replaced by an equal volume of 0.5 per cent, phenol in 0.85 per cent, sodium 

 chloride solution in distilled water. In this the sediment is thoroughly mixed by 

 stirring. As the bacteria are often in masses, groups or chains, it is now necessary 

 to separate them. This is best done by adding a few small glas beads to the 

 contents of the tubes, changing the cotton stopper for a sterile rubber cork, and 

 shaking either in a shaking machine or by hand, until it can be supposed that the 

 micro-organisms are all separated. This is easily accomplished by the aid of the 

 shaking machine but is tedious to effect by hand. The tube is then returned to 

 the centrifuge and again whirled until the bacteria are again sedimented, after 

 which the fluid is again removed and again replaced and the bacteria again dis- 

 tributed. A few turns in the centrifuge now throw down particles of culture- 

 media and contained flakes of the culture and leave a uniformly clouded fluid 

 above. 



If it be desired to conserve all of the bacterial products, the wash- 

 ings from the culture bottles are immediately transferred to the 

 appropriate tube, shaken with the glass beads, given a few turns 

 in the centrifuge to throw out flakes of culture and culture-media, 

 and we again arrive at the point of having a uniformly cloudy fluid 

 with which to continue the preparation of the vaccine. 



If the vaccine is to be of scientific value, it should be made in 

 such manner that its composition represents what is desired — 

 bacterial cells only, or bacterial cells with their collected products — 

 and some means should be provided by which a reasonably accurate 



