1. _To Obtain the Toxin._ First grow a pure culture of the Klebs-Löffler bacillus of diphtheria in large flasks containing "Löffler's medium," or a solution made by mixing three parts of blood serum with one of beef broth and adding one per cent. of common salt (Na Cl) and one per cent. of peptone. An alkaline medium is preferable. The flask was thoroughly sterilised before use, and is now plugged with sterile cotton-wool and incubated at 77° C. for three or four weeks. Pure 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 into sterilised flasks, and some favourable antiseptic added to ensure that nothing foreign to the toxin shall flourish, and the flasks are kept in the dark. Here, then, we have the product, the toxin, ready for injection into the horse. [Illustration: Flask used for the Preparation of the Toxin of Diphtheria] 2. _Immunisation of the Horse._ It is evident that only healthy horses are of service in providing healthy antitoxin, even as healthy children are necessary in arm-to-arm vaccination. To provide against any serious taint the horse is tested for glanders (with mallein) and for tuberculosis (with tuberculin). The dose of the injection of toxin is at the commencement about 1/10 cc., or a little more. The site of the inoculation is the apex of the shoulder, which has been antiseptically cleaned. A mere prick is the whole operation. After the first injection there is generally a definite febrile reaction and a slight local swelling. From 1/10 or 1/2 cc. the dose is steadily increased, until at the end of two or three months[86] perhaps as much as 300 cc. (or even half a litre) may be injected without causing the reaction which the initial injection of 1/10 cc. caused at the outset. This shows an acquired tolerance of the tissues of the horse to the toxic material. After injecting 500 cc. into the horse without bad effect, the animal has a rest of four or five days.