468 THE PLAGUE BACILLUS 



a saturated solution at 37 C. The bacterial bodies are filtered off through hardened 

 paper at 37 C and suspended in water. This suspension constitutes solution B. 



[The lethal dose for rats is an amount of solution corresponding to about 0*1 mg. 

 of contained nucleo-protein. Heating to 55 C. lowers its toxicity.] 



4. Vaccination. 

 A. Animals. 



(i) With toxins. (a) The inoculation of even highly active toxins does not 

 result in the production of an absolute and lasting immunity and does not 

 lead to the formation of an anti-plague serum (Roux ; Yersin ; Calmette 

 and Borrel). 



[(&) A single injection of even minute quantities (O'02-O'OOOl mg.) of 

 Rowland's toxin confers a substantial immunity upon rats.] 



(ii) With dead cultures. Inoculation with bacilli killed by heat gives 

 better results. Yersin, and Calmette and Borrel scraped the growth from 

 a 48-hour old culture on agar, mixed it with a little broth, put the mixture 

 into tubes, sealed them and heated them to 58 C. for 1 hour, and in this 

 way obtained a product which killed rabbits when inoculated in large doses 

 into the veins or into the peritoneum. If, however, one or two inoculations 

 of sub -lethal doses were given into the veins or peritoneal cavity the animal 

 was protected against the subsequent sub-cutaneous inoculation of a living 

 virulent bacillus, provided that at the time of the test inoculation the animal 

 had completely recovered from the effects of the vaccine. 



Rabbits can also be immunized by sub-cutaneous inoculation of heated cultures 

 but the method requires time : as a rule it is necessary to give three or four inocula- 

 tions at intervals of a fortnight. 



The guinea-pig is not so readily immunized by this method. 



(iii) With living cultures. Horses are difficult to immunize. The inocula- 

 tion of bacilli killed by heat produces little reaction and is very slow in its 

 results : the most efficient method is to inoculate into the veins first heated 

 cultures, then progressively increasing doses of living bacilli (Roux). The 

 method of immunizing horses for the supply of therapeutic serum actually 

 in use at the Pasteur Institute, Paris, is described later (vide Serum therapy). 



B. Man. 



(i) With dead cultures. Haffkine was the first to prepare a vaccine for 

 human prophylaxis by killing virulent cultures by heat at 60 C. 



The bacillus was sown in large flasks half-filled with a special broth (a maceration 

 of goat meat peptonized with hydrochloric acid and neutralized) or ordinary broth,, 

 the surface of 'the latter being covered with a thin layer of sterilized oil to obtain 

 a growth of stalactites (p. 473). The flasks were incubated [in large room?] at 

 27-30 C. [the average temperature in Bombay] for 2 months, being shaken from 

 time to time in order to break up the stalactites. After verifying the purity of the 

 culture the contents were distributed into tubes which were sealed and heated at 

 60 C. for a quarter of an hour ; a little carbolic acid (0'5 per cent.) was subsequently 

 added. The vaccine was used 2 months after preparation. 



For purposes of human vaccination 5 c.c. of the vaccine are inoculated 

 beneath the skin of the arm in the neighbourhood of the insertion of the 

 deltoid. (Inoculation in the region of the shoulder or of the abdomen is 

 less painful.) A few hours later a painful swelling occurs around the site of 

 inoculation accompanied by a rise of temperature (38 C.) and a slight swelling 

 of the glands ; the temperature is again normal in about 36 hours and the 

 symptoms have disappeared. Immunity is established as soon as the tem- 

 perature falls. The vaccine exerts no prophylactic properties during the 



