February 21, 1895J 



NATURE 



405 



are removed or destroyed ; the^e, if left in the fluid, would be a 

 complicating and inconstant factor in the equation. In order to 

 kill these bacilli the Germans recommend the addition of \ per 

 cent, of carbolic acid to the cultiirL- ; the dead bacilli fading 

 to the bottom, leave a perfectly clear supernatant fluid. The 

 French, on the other hand, recommend the separation 

 of the bacilli from the fluid by means of a Pasieur- 

 Chamberland filter. By this means a clear virulent poison 

 which does not contain any diphtheria bacilli is obtained. 

 With this fluid a horse with a good constitution, and 

 which has been proved to be free from tubercle and 

 glanders, is injected under the skin of the side of the neck in 

 front of the shoulder. Small doses are first injected, either 

 pure or with the addition of i '3 of the volume of weak solu- 

 tion of iodine of potassium. II the fluid is of full strenjjth, only 

 about 2C. c. can be given at the first injection. This is followed 

 within twenty-four hours by a local swelling at the seat of in- 

 jection, about the size of the palm of the hand, and the temper- 

 ature may rise l°or2' F. (.i°to i' C), otherwise the general health 

 of the horse does not seem to suffer. He eats well, and unless 

 regularly exercised may become very lively; of this we have 

 had ample evidence during the recent frost and snow, when it 

 has been unsafe to give much exercise to horses that are not 

 very sound in limb, and as a result Ihey have been very fresh 

 indeed. As soon as the swelling has disappeared and the 

 temperature has receded to the original level, a somewhat larger 

 dose is given ; the same process is repealed time after time (the 

 dose being gradually increased to bring about the same amount 

 of swelling and lise of temperature) for about three months, or 

 until such lime as the requisite amount of immunity is acquired, 

 i.e. until the antitoxic action of the blood is sufficiently marked. 

 That there is a gradually increasing immunity is evidenced by the 

 fact that enormously large doses of the toxine in the later stages 

 of the treatment produce even less local and constitutional dis- 

 turbance than was observed after Ihe first few injections of 

 comparatively small quantities. 



The blood is now drawn off' from the jugular vein of the 

 immunised horse by means of a metal cannula or tube to which 

 is attached an indiarubber tube ; these are first thoroughly 

 boiled, in order that no living micro-organisms of any kind may 

 remain on or in them, and ihe skin of the horse is carefully 

 cleansed with antiseptic lotions. The indiarubber tube leads 

 the blood into a flask or vessel which has also been carefully 

 sterilised, and provided with a well-fitting cotton-wadding plug 

 or glass-stopper. The vessel when filled is placed in an ice- 

 safe until the solid part, llie clot, is completely separated from 

 Ihe fluid— the serum. From each gallon of blood about one 

 quart of serum is expressed, thjugh this varies considerably in 

 different cases, and according to the time that the separation is 

 allowed to continue (24 to 48 hours). This serum, a limpid 

 straw-coloured fluid, is carefully decanted under strict antiseptic 

 precautions, and, mixed with carbolic acid or camphor, is 

 stored in small phials, each of which contains about a sufficient 

 quantity for the treatment of a single patient. In the Pasteur 

 Institute, and in the Uritish Institute of Preventive Medicine, 

 the antitoxic serum is apparently brought up to such a strength 

 that , i,r of a c.c. injected into a medium-sized guinea-pig (500 

 grammes, or over 17 ounces) will protect it against an injection 

 twenty-four hours later of \ c.c. ot a culture of living 

 diphtheria bacilli strong enough, if given by itself, to kill the 

 guinea-pig in twenty-four hours. It is usually recommended 

 that 20 c.c. of this serum should be given at ihe first dose, and 

 that if necessary a second 10 c.c. should be given half an hour 

 later. The method of testing the strength ol the setum .adopted 

 by the Germans is that devised by Ehrlich, who takes ten 

 limes the lethal dose of diphtheria loxine, and in a test-tube 

 adds a definite and known quantity of the blood to be tested. 

 This mixture is then injected into a guinea-pig, and if the anti- 

 toxic power of the blood has been gauged aright, the animal 

 does not suffer in the slightest degree from what under ordinary 

 circumstances would kill ten guinea-pigs. The addition of less 

 or weaker serum, or of more toxine would leave the mixture 

 still toxic. 



In order to obtain a definite standard with which to compare 

 the antitoxic power of any serum, and to determine the dose of 

 such serum, Behring and Ehrlich have described what they 

 term a normal antitoxic serum — that is, a serum of such a 

 strength thai ,',,ih of a c.c. added to ten times the lethal dose of 

 diphtheria toxine isexactlysufficient to render it innocuous. I c.c. 

 of such normal serum contains one "immunisation unit," and 



should be sufficient, when added to a hundred times the lethal 

 dose and injected, to render it innocuous. In horses wholly 

 immunised the serum may be hfty or even a hundred times as 

 active as the normal serum above mentioned and the dose 

 to be given varies according to the number of immunisation 

 units in any sample. It is not here necessary to go into the 

 j question of do>e, but it may be staled that 500 of these 

 ! immunisation units are usually necessary to produce the desired 

 j effects in cases of diphtheria, though in some cases still larger 

 quantities have to be used. Behring now supplies four strengths 

 of the serum, the weakest (marked with a yellow label) is sent 

 out for injection of cases where the disease has not already 

 been contracted. The next (marked with a green labelj is of a 

 strength of 600 antitoxine units, and is given to those cases in 

 which the treatment is commenced at the very outset of the 

 disease— that is, when the firstsymptomsof diphtheria manifest 

 themselves. The next stronger antitoxic serum (white label) 

 equals 1000 antiioxine units, and is used fcr cases somewhat mere 

 advanced in which the prognosis is at all grave : whilst in still 

 j graver cases, and where the symptoms have been developed for 

 some considerable lime, it is often necessary to give a serum of 

 1500 units ; ihis is marked with a red label, and is of course 

 highly concentrated in order that the size of Ihe dose may not 

 be unduly increased. In place of No. i. healthy children and 

 adults who are exposed to diphiheritic infection mav receive a 

 quarter of the dose of the gieen label flask, which Behring 

 considerswillprotect against diphtheria with verygreat certainty. 

 Although the>e general directions are laid down, it is strongly 

 insisted upon by Behring, Kossel, Roux, and in fact by all 

 those who have had experience of antitoxic serum, that the dose 

 must vary according to ihe severity of the di;ease, so that much 

 must be left to the discretion of the medical practitioner in 

 charge of the patient. The great error into which those who 

 first me ibis agent fall, is the administration of far loo small a 

 dose, especially in the case of children, for whom ihe dose is 

 nearly as large as it is for adults. For this reason some of the 

 statistics published in this country and abroad are far too 

 unfavouiable to the method. The great drawback with this 

 method is that the dose necessary to be injected is so large ; 

 but in the loose tissue of the side of the chest, the back, or the 

 buttock, immediately under the skin, the fluid soon disappears. 

 It is hoped that before long, however, the active principle may 

 be separated, and so obtained in smaller bulk. 



So far we have dealt principally with the antitoxic serum as 

 prepared by Behring and Roux and by Roux's method, which 

 is certainly attended with comparatively few difficulties ; these, 

 however, have the disadvantage that they take from three to six 

 months to give the desired results. In order to do away with 

 this disadvantage, Klein has carried out a series of experiments in 

 which he has been able to obtain serum of considerable activity 

 in as short a period as twenty-three days. Instead of introduc- 

 ing the poison only, he adopts the plan used by Behring and 

 Roux in their earlier experiments, of injecting living bacilli 

 which have lost a certain degree of their activity, using for Ibis 

 purpose old cultures. He afterwards introduces toxine along 

 with more virulent bacilli, and thus obtains in the animal such 

 a degree of immunity ihat it is enabled to wilhsland or to react 

 very slightly to moie than a fatal dose of diphtheria bacilli. By 

 the third week the animal will bear the injection of large 

 quantities of virulent bacilli, and by the end of twenty-three or 

 twenty-six days the serum has acquired such antitoxic proper- 

 lies that I c.c. of it will protect 40 to So guinea-pigs against a 

 lethal dose of living diphtheria bacilli. It is difficult to com- 

 pare these results with Roux's and Behring's, but Klein's serum 

 has been used with marked success in certain cases of diph- 

 theria. It appears to have a special power of causing the 

 membrane to clear away, and so to remove ihe manufactory of 

 the poison, as on this membrane the diphtheria bacilli accumu- 

 late. This method is mentioned as one that m.iy be used 

 especially where it is desired to obtain antitoxic seium quickly. 

 Smyrnow has suggested quite a diflTerent method of preparing 

 antitoxine. Under Nercki's advice he passed electric currents 

 through ihe seium of animals, and was thus able to endow it 

 with a certain immunising power. But he was still more 

 successful in obtaining powerful aniitoxire by electrolysing 

 diphlheiia bouillon cultures ; curiously enough, the more virulent 

 the culture the more powerlul was the antitoxic substance he 

 obtained. When this antitoxic substance was injected into a 

 rabbit, which Uvenly-four. hours before had received about 

 \ c.c. of a two or three days old diphtheria bouillon culture, there 



NO. 



1321, VOL. 51] 



