401 



NA TURE 



[February 21, 1S95 



all accurately, the number and strength of the bacilli. Not so. 

 however, with the product? of the micro-organisms, the activity 

 of which could of course be more accurately measured, and the 

 do<e more accurately graduated. The bacilli might multiply 

 and continue their action on the tissues, but the poisons when 

 injected alone would not alter in quantity or activity. As may 

 be readily imagined, the fluid constituents of the blood can 

 only contain tho-e substances that are introduced into it from 

 without, either through the vital activity of the cells of the 

 body, the products of which must be thrown into this fluid 

 before they can be excreted, or through artificial injection. The 

 antitoxic substances then found in the blood of an immunised 

 .laimal, must in the case of natural immunity following an attack 

 of diphtheria be the result of the activity of the tissue cells, 

 especially of the connective tissue and white blood cell groups 

 which have been "stimulated " by the toxines introduced from 

 without, from the false membrance in the throat. Where it is 

 desired to produce an artificial immunity, and an "artificial" 

 antitoxic serum, the cells are stimulated by the introduci ion into 

 the body of ariificially prepared toxine. The cells acted upon 

 by the toxine elaborate ihe protective fluid, which is thrown into 

 and accumulates in the blood. This substance may act in one of 

 several, or even in several ways. (|> It may directly antagonise 

 the diphtheria toxine, and may thus prevent the paralysing 

 action of these poisons on thescavangering cells or phagocyles.as 

 they are called ; these, left free to perform their proper functions, 

 can deal with the foreign elements that have got into the blood, 

 and also with the bacilli at the seat of the local attack, for, as has 

 been pointed out by several foreign observers, and by Ruffer in 

 this countiy, immediately beneath the layer of bacilli in the 

 false membrane there is usually a very considerable accumula- 

 tion of leucocytes, especially in those cases in which recovery 

 ultimately takes place. (2) The antitoxic sub-tances may act on 

 the bacilli, inhibiting their growth and interfering with their 

 power of producing toxines. This of course can only be a local 

 action should it play any part in ;he process. (3) These sub- 

 stances may act directly on the cells of the blood lymph 

 and tissues, so stimulating and strengthening them that 

 they are able to perform those functions above mentioned. 

 It is at present difficult to state which of these pro- 

 cesses is the one, or the most important, in protecting or 

 curing the patient, and it may be that all play a part. It may 

 be that the tissue cells, when acted upon by the specific 

 diphtheria poison, become so modified that they are enabled to 

 produce or secrete a sulistance which directly antagonises the 

 action of that poison. This substance, thrown into the blood, 

 remains there for some lime, rapidly accumulates as larger and 

 larger doses of the poi-on are thrown in, neutralising the poison, 

 whose power of doing damage to the tissues is thus held in 

 check, but remaining (or some time after the toxine has disap- 

 peared ; or thi^ autitoxic substance, reacting upon the cells, 

 may render them Ic-s susceptible to the action of the toxine. 



The earlier immunising experiments were naturally performed 

 upon the smaller animals, such as rabbits. Then lichring used 

 sheep, and after various other animals h.nl been tried, the horse 

 was selected by Roux and Nocaid as perhaps the best of all 

 animals from which to obtain antitoxic serum. In the first 

 place, be is comparatively insusceptible to the action of the 

 diphtheria bacillus— even considerable doses of living bacilli 

 may be injected under the skin without producing anything 

 more than a slight local. swelling and a rise of temperature. 

 It ha» also been found that horse serum, when injected, 

 produces little or no change in the healthy human sub- 

 ject — that is, the -trum seems to mix ptrfectly well with 

 human blood plasma, and there is comparatively little danger 

 of the extra serunti being excreted by the kidneys in the form of 

 albumen. This is a most important point, and one that no 

 doubt influenced Koux and Nocard in their selection of the 

 horse a.* an animal from which to obtain immunised scrum. 

 beyond this, however, the blood, (when drawn from the vessels, 

 separate' very perfectly into two porliuns— a firm clot, which if 

 the blood be caught in a cylindrical gla.s jar. forms a kind of 

 column in the centre, anri a clear slian.colNured scrum which 

 accumulates around the clot, and lorms a layer often several 

 inches deep alrave it. This serum contains the antitoxic sub- 

 stances. Lastly, considerable <|uantitits of blood can be 

 obtained from such a large animal n.s the liorse, and if he be 

 well fed, grr>omcd and exercised, the process 01 bleeding may 

 be repeated pretty frcrjuenlly without causing any inconvenience 

 10 the animal ; in fact, as one observer said, he stands bleeding 



NO. I32I, VOL. 51] 



as well as did our forefathers, who thought as little of being bled 

 as we think of going to Aix or liuxton. 



Let us now turn for a moment to the method of treating the 

 horses that we wish to render immune, in order that they may 

 supply the antitoxic scrum that is to be used for the treatment 

 of cases 01 diphtheria. Roux's method, which is that that h.is 

 been most carefully described, and which is the one used in 

 this country first by Dr. Ruffer at the liritish Institute of Pre- 

 ventive Medicine, and then by Prof McFadyean at the Royal 

 Veterinary College, consists in introducing diphtheria toxine of 

 a given strength in gradually increasing doses, until the blood 

 of the animal so infected is found to contain a sufficient quantity 

 of the antitjxine. 



Preparation of Diphtheria Toxine. 



The toxine with which the animal is to be injected is prepared 

 as follows : — A broth is piepaied by soaking a pound of finely- 

 minced beef in water. This is allowed to stand for twenty four 

 hours in the cold. To the fluid expressed from tlie meat fibre 

 at the end of that time is added A per cent, of common sail 

 and 2 per cent, of peptone (meat artificially digested by pcpsine). 

 This broth is then rendered faintly alkaline by ihe addition of 

 soda salts or caustic soda. This is done because it is found 

 that the diphtheria bacillus cannot grow at all vigorously, 

 or form its poisons rapidly in an acid solution, and such 

 poison as is formed is neutralised, or is unable to act 

 in the presence of even a faint trace of acid. It is found 

 that even in Koux's solution, which is always faintly alkaline 

 to begin with, an acid reaction soon appears, but, after 

 about ten days, this is replaced by an alkaline reaction, and .is 

 soon as this takes place, the growth of the bacilli takes on new 

 activity, the quantity of toxine is increased, and it becomes 

 much more virulent. Roux found that he obtained his most 

 virulent toxines after a month's growth. If the growth is 

 allowed to go on longer than this, a process of oxidation appears 

 to take pl.ice, and I have found that the toxine from a culture 

 carried on for two months had already lost much of its toxic 

 activity. It should be noted that a virulent bacillus should 

 always be taken in the first instance, otherwise the results may 

 be very disappointing. 



This nutrient material is placed in a layer of not more than 

 half an inch thick in a flat-bottomed flask, which is plugged 

 with cotton wadding, and then closed with an indiarubber cork 

 or cap. Through this composite plug three tubes are passed 

 into the flask ; the two lateral tubes are bent at right angles, 

 both inside and outside the flask ; whilst the centre tube 

 is fitted with a small thistle-head, which may be plugged 

 with cotton wadding, and then closed with an indiarubber 

 cap. The outlets of the lateral lubes ire also plugged 

 with cotton wadding, and the whole apparatus is kept for 

 an hour or two in steam maintained at a temperature of 100° 

 C. (flasks so treated may be preserved for years without any 

 change, beyond some slight evaporation, taking place in the 

 broth.) A small quantity of a pure broth culture of the virulent 

 diphtheria bacillus is now drawn into a long thin pipette, the 

 indiarubber cap and the cotton wadding plug are removed from 

 the thistle-head, and the contents of the pipette arcintroduced ; 

 the pipcite is withdrawn, the cotton wadding is replaced, the 

 indiarubber cap is fitted in jiosition, and the flask is placed in 

 an incul:)ator which is maintained at the temperature of the body 

 igSV J'-' <^' 3S"'2' C). As soon as the grosvth is well started 

 (usually at the end of aliout twenty-four hours), a current of moist 

 air is made to pass continuously over thcsui face of this cultivating 

 fluid, the air being first warmed and saturated with moisture, in 

 order as far as possible to prevent evaporation. A fine flocculent 

 deposit soon makes its appearance on the bottom of the vessel, 

 the supernatant fluid remaining clear. This deposit increases in 

 thickness, much moie luxuriant growth going on alter the first 

 ten days. Toxine is formed by the diphtheria bacilli so long as 

 they can grow freely — that is, so long as they can obtiiii sufli- 

 cient nutrient material from the fluid and Irom the air that is 

 continually passing over the surface. At the end of a month, 

 if all these precautions are taken, the toxine should be of such a 

 strength that i/iolh of a c.c. (about two or three ilrops) injected 

 into a guinea-pig weighing 500 grammes (over 17 ounces) will 

 kill it within forty-right hours. The strength of the toxine or 

 poi.son may be a little greater or a little less than this, but it is 

 a compaiaiively e.asy matter to measure the strength, and there- 

 fore to graduate the dose to be used in immunising the horse. 

 This only holds good, however, if the active diphtheria b.icilli 



