426 THE QUESTION OF IMMUNITY AND ANTITOXINS 



an attack of such diseases as diphtheria and tetanus. Such an 

 animal is the horse. Now, by injecting into the horse (a) living 

 organisms of the specific disease, but in non-fatal doses, or 

 (&) dead cultures, or (c) filtered cultures containing no bacteria 

 and only toxins, we are able to produce in the blood of the horse 

 first the toxins and then by natural processes the antitoxins of the 

 disease in question. The non-poisonous doses of living organisms 

 can be attenuated, by various means. Dead cultures have not 

 been much used to produce immunity except by Pfeiffer. In actual 

 practice the third method is much the most general, viz., filtering a 

 fluid culture free from bacteria, and then inoculating, this in ever- 

 increasing doses. The preparation of diphtheria antitoxin may be 

 taken as an example, but what follows would be equally applicable 

 to other diseases, such as tetanus : — 



1. To obtain the Toxin. — First grow a pure culture of the Klebs- 

 Loffler bacillus of diphtheria in large flasks containing "Lofiler's 



medium," or a solution made by mixing three 

 parts of blood serum with one of beef broth, 

 and adding 1 per cent, of common salt 

 (NaCl) and 1 per cent, of peptone. An 

 alkaline medium is necessary, and a free 

 supply of oxygen and the presence of a 

 large proportion of peptone in the medium 

 favours a high degree of toxicity. The 

 bouillon must be glucose-free. The flask, 

 thoroughly sterilised before use, is now 

 plugged with sterile cotton-wool and incu- 

 bated at 37° C. for three weeks. Sterile air 

 may be passed over the culture periodically, 

 thereby aiding the growth. After the lapse 

 of about a month a scum of diphtheria 

 growth will have appeared over the surface of the fluid. This is 

 now filtered through a Chamberland filter into sterilised flasks, and 

 some favourable antiseptic added to ensure that nothing foreign to 

 the toxin shall flourish. The flasks are kept in a cool place in the 

 dark. Here, then, we have the product, the toxin, ready for injection 

 into the horse. 



The power of the toxins is estimated by subcutaneous injection 

 of varying amounts into a number of guinea-pigs, and the minimum 

 lethal dose (M.L.D.) is obtained. The standard M.L.D. is the 

 smallest amount which will kill a 250-gram guinea-pig in four days. 

 According to Behring, a normal M.L.D. (expressed as D.T.N.^) is 

 ■01 c.c. ; a toxin of which '02 is M.L.D. will therefore be expressed 

 as D.T.N. -^ 



2. Immwiisation of the Horse. — The general principle is that 



Fig. 37 Flask used for Prepara- 

 tion of the Toxin of Diphtheria. 



