After these preliminary remarks we will now consider shortly some of the methods employed for the production of antitoxins. An animal is required from whose body a considerable quantity of blood can be drawn without injurious effect. Moreover, it must be an animal that can stand 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 (b) dead cultures, or (c) filtered cultures containing no bacteria and only the toxins, we are able to produce in the blood of the horse first the toxins and then the antitoxins of the disease in question. The non-poisonous doses of living organisms can be weakened, or, as we say, 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 the 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-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
Flask used for the Preparation of the Toxin of Diphtheria 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.
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.
3. To Obtain the Antitoxin. During this period of rest the interaction between the living body cells of the animal and the toxins results in the production in the blood of an antitoxin. By means of a small sterilised cannula, five, or eight, or even ten litres of blood are drawn from the jugular vein of the horse into sterilised flasks or jars. The top of the jar is closed by two paper coverings before it is sterilised. Then it is again covered with a further loose one. Before use the loose one is removed and replaced by a metal (zinc) lid, which has been separately sterilised. This metal lid contains an aperture large enough for the tube which conveys the blood from the cannula to pass through. The tube, therefore, passes through the metal lid and two paper covers, which it was made to pierce. When enough blood has passed into the vessel the tube is withdrawn, and the metal lid slightly turned. Thus the contained blood is protected from the air.[87]
The jar containing the blood (which contains the antitoxin) is next placed in a dark, cool cellar, where it stands for two or three days. During this time the blood naturally coagulates, the corpuscles falling as a dense clot to the bottom, and the faintly yellow serum rising to the top. The serum, or liquor sanguinis, averages about 50 per cent. of the total blood taken. Sometimes antiseptics are added with a view to preservation. It is generally filtered before bottling for therapeutic use, and sometimes examined bacteriologically as a test of purity.
4. The Use of Antitoxins. The antitoxins are now ready for injection into the patient who has contracted diphtheria, and in whose blood toxins are in the ascendency and under which the individual may succumb. They are injected in varying doses, as we have already pointed out.[88] The general result is that mortality has been greatly lessened, and that in fatal cases there has been a considerable lengthening of the period of life. Moreover, the whole clinical course of the disease has been greatly modified, and suffering lessened.[89]