PATHOGENIC BACTERIA. 347 



also where the culture employed is a vigorous one.* On the other hand Theo- 

 bold Smith f finds that the alkalinity of the broth is not the essential point in 

 the production of strong toxin, but that it is necessary to use broth which has 

 been fermented with the colon bacillus to rid it of muscle-sugar. Smith's 

 directions are as follows : 



Beef infusion prepared in the usual way by using 500 grams of finely chopped, 

 lean beef is kept in the cold for 12-24 hours; the juice then expressed; the 

 reaction brought to 1.5 or 2 per cent, acid with normal sodium carbonate solu- 

 tion; heated to 40 C.; inoculated with 30-40 c.c. of a 24-hour bouillon culture 

 of B. coli; and placed in the high-temperature incubator for 16 hours or over 

 night; it is then clarified by adding the white of one egg to every liter of infu- 

 sion and boiling for 45-60 minutes in the Arnold sterilizer; cooled down and 

 filtered; 2 per cent, of Witte's peptone and 0.5 per cent, of common salt are 

 added and dissolved by gentle heat; the acidity is reduced to 0.8 per cent, with 

 sodium carbonate; o.i per cent, of dextrose is added; steamed for or boiled 

 for 20-30 minutes and filtered; distributed into Fern bach flasks in shallow 

 layers, 2.5 ccm. deep and sterilized in the autoclave at 110-115 C. for 30 

 minutes. The cultures of diphtheria which are employed should form mem- 

 branes over the medium promptly, leaving a clear fluid beneath. Previously 

 grown beef-broth cultures in test-tubes are used to inoculate the Fern- 

 back flasks. The cultures in these .flasks become distinctly alkaline to 

 phenolphthalein in 6-8 days at which time the maximum of toxin has 

 been formed. After having been tested for purity with the microscope and by 

 cultures they are treated as follows: "Rendered sterile by the addition of 10 

 per cent, of a 5 per cent, solution of carbolic acid. After 48 hours the dead 

 bacilli have settled on the bottom of the jar and the clear fluid above is 

 syphoned off or it is filtered through ordinary sterile filter paper and stored in 

 full bottles in a cold place until needed." The filtrate contains the toxin. 

 The toxin is injected into the animal from which the antitoxin is to be obtained 

 in small doses. The dose depends on the strength of the toxin. The aimal 

 usually employed is the horse, which should be healthy; the presence of tuber- 

 culosis and glanders should have been excluded by testing with tuberculin 

 and mallein; the possible presence of tetanus should also be considered (see 

 page 330). 



The injection is repeated at intervals of about one week, using larger and 

 larger doses, until the animal is able to tolerate a very large dose indeed as 

 much as 300 c.c., or even more. If the treatment is successful, the general 

 condition of the animal should not suffer. The injections last over a long 

 period usually about two or three months. The general condition of the 

 animal remaining good, the toleration of these large doses of toxin is presumed 

 to indicate the existence of a concentrated antitoxic substance in the blood. 



*Park. Loc. cit. p. 205-206. 



"\Journ. Exper. Med. Vol. IV. 1899. pp. 273-297. 



