470 THE PLAGUE BACILLUS 



The exudate from one guinea-pig is made up to about 50 c.c. with the following 

 solution : 



Water, - - 100 c.c. 



Sodium chloride, - 0'70 gram. 



Sodium carbonate, - 0*25 



Carbolic acid crystals, - - 0'50 



The dose of vaccine for a man is 2-2'5 c.c. 



(iv) With bacillary extracts. (a) Lustig and Galeotti have introduced the 

 use of bacterial extracts as a method of immunization against plague. The 

 technique is complicated and the results do not show that the method has any 

 advantage over the preceding : the immunity is of but short duration. Some 

 200 persons were inoculated at San Nicola de la Plata. Tavel of Berne 

 has made some modifications in the technique. 



In Lustig and Galeotti's method the bacilli are sown on agar plates and incubated 

 for 3 days ; the growth is then scraped off and allowed to macerate in a 1 per cent, 

 solution of caustic potash for 24 hours. The solution is diluted, filtered and then 

 precipitated with dilute acetic acid and the precipitate washed and finally dried 

 in vacuo. 



The precipitate consisting of nucleo-proteins is used for human vaccination in 

 doses of 0'003 gram dissolved in a 0'5 per cent, solution of sodium carbonate. The 

 inoculation is painful and is accompanied by a sharp reaction. 



(v) Sero- vaccination. With the various methods of vaccination described 

 above there is a delay of a few days before the person inoculated becomes 

 immune, and in some cases the person so inoculated is more susceptible during 

 that time to the plague bacillus : in other words, a negative phase follows 

 vaccination. To overcome this difficulty Calmette and Salimbeni devised 

 a method of sero-vaccination. Either a mixture of vaccine and antiplague 

 serum is inoculated, or a dose of serum (5 c.c.) followed 2 days later by a 

 dose (2-3 c.c.) of Haffkine's vaccine. In this way not only is the person 

 immunized at once but the reaction following the inoculation is less marked. 

 On the other hand, unfortunately, the immunity is only short-lived and 

 the results " are not much better than those obtained by the use of serum 

 alone " (Besredka). 



To prepare the vaccine the bacillus is sown on agar in Roux bottles and incubated 

 for 48 hours. The growth is then scraped off, mixed with normal saline solution, 

 and the emulsion filtered through filter paper. The organisms retained on the filter 

 are made into an emulsion with a little normal saline solution, heated at 70 C. for 

 an hour, and dried in vacuo. The product mixed with antiplague serum constitutes 

 the vaccine. 



Shiga prepares a vaccine in the following manner : 



Agar cultures three days old are scraped off, made into an emulsion with normal 

 saline solution (1 c.c. for each loopful of growth), heated at 60 C. for half an hour 

 and carbolic acid added in the proportion of 0'5 per cent. Just before use, 

 equal parts of the emulsion and antiplague serum are mixed and inoculated in doses 

 of O'6-l c.c. for a man : a few days later an inoculation of emulsion without serum 

 is administered. This sero-vaccine has been used in Japan. 



Besredka's vaccine. Besredka attributed the poor results obtained by 

 Calmette's method to the presence of an excess of serum in the mixture. By 

 reducing the amount of serum to a minimum this observer was able to induce 

 a rapid, powerful and lasting immunity in animals (p. 382). 



Bacilli from a two-day-old culture on agar are made into an emulsion with a very 

 little normal saline solution, heated at 60 C. for an hour and then mixed with anti- 

 plague serum. At the end of 24 hours the bacilli after being washed repeatedly 

 to remove all traces of free serum are made into an emulsion with normal saline 

 solution, distributed into tubes, sealed and then heated again for an hour at 54 C. 

 to ensure sterility. The vaccine is now ready for use. 



