most careful experimental observations the limit is determined at which just that excess of toxin becomes manifest which causes death of the animal in the first four days. The amount of toxin thus obtained represents the immediate test-dose. By means of the same dose of serum, for the more exact characterization of the toxin, the determination of a second limit is made, for the purpose of learning the dose of toxin that is just neutralized by admixture with the amount of serum named. IV. The determination of the strength of a diphtheriaantitoxin is made by means of the test-dose of toxin (see paragraph III) as follows: The test-dose of toxin in question —for instance, 0.355 cu. cm. of tested toxin at the time present in the laboratory-is mixed with 4 cu. cm. of antitoxin corresponding to the test-figures given. As the testdose of toxin is estimated for 1 cu. cm. of antitoxin of normal strength, or for 4 cu. cm. of antitoxin 4 the normal strength, an antitoxin of x-strength will have to be diluted 4x, and in testing an antitoxin 100 times the normal strength, V. The mixture obtained is injected unmodified subcutaneously into guinea-pigs weighing from 250 to 300 grams. If the animals die in the test-experiments made by two observers in the laboratory within the first four days, the antitoxin does not possess the required strength. Should death occur within five or six days, the antitoxin is close to the required strength, and in order to avoid the early withdrawal to be anticipated an improvement of from 5 to 10 per cent. is recommended the manufacturers. durations that occur in the animals experimented upon do not, however, constitute sufficient ground for objection. In the case of the dead animals an autopsy should be held, and careful attention directed to complications with previously existing disease (tuberculosis, pseudo-tuberculosis, and pneumonia) that may induce undue susceptibility on the part of the test-animals. In VI. Both liquid and solid toxins may be employed for test-purposes, if the limits defined in paragraph III can be accurately estimated, and the difference between them does not exceed fifteen simple lethal doses. If liquid toxins preserved in toluol are employed, this should be done only if as a result of preliminary investigation the permanency of the test-constants is demonstrated, if the test dose does not exceed 1 cu. cm. The examination with regard to the qualities of the test-toxins should be continued. VII. The test-toxins, if liquid, are to be examined monthly with regard to their sterility by means of culture methods. VIII. The test-poison is to be redetermined at intervals of six weeks by means of the test-dose of serum, the testdose and the net-valuation being estimated anew. If, on reexamination, any considerable deviation in the test-dose should be detected, the toxin must be considered to be in process of decomposition, and it should be replaced by fresh toxin. IX. The manufacturers are to be informed that the test-toxin in small amounts decomposes readily, and that even brief exposure to light may induce considerable attenuation. It is therefore to be recommended that a new supply of toxin be obtained from the laboratory every three weeks.* Diphtheria-antitoxin is prepared in Germany in four establishments-namely, the Höchst Works, the Factory of Schering, in Berlin, the Pasteur Institute in Stuttgart, and the Factory of Sthamer, Noack & Company in Hamburg. The Höchst Works manufacture the following preparations: ANTITOXIN OF 250 TIMES THE NORMAL STRENGTH. Number o, yellow,. Number 1, green, Number 2, white, Number 3, red, 66 600 1,000 1,500 66 66 66 * Dr. Jos. McFarland and Dr. Chas. T. McClintock, to whom these regulations were submitted, kindly describe, as follows, the method of testing pursued by the largest manufacturers of diphtheria-antitoxin in the United States: "In order to secure uniformity in the toxin the same culture of the diphtheria-bacillus is always employed. This is grown for seven days at 37° C. (98.6° F.) in an accurately prepared alkaline two per cent. peptone-bouillon. The same degree of alkalinity is always secured (phenolphthalein being used as the indicator in titration). After addition of 0.4 per cent. trikresol, this toxic bouillon is filtered through unglazed porcelain and stored in a dark, cold place. The amount of this toxin that will kill a guinea-pig weighing 250 grams on or before the sixth day is considered the minimum fatal dose. Should this toxin deteriorate ten per cent. from its original strength, it must be discarded. "In testing antitoxin, a series of guinea-pigs, weighing from 240 to 270 grams, are injected with ten times the minimum fatal dose of toxin previously mixed with varying amounts of antitoxin. Those pigs are considered protected that do not die or lose more than 20 per cent. of their original weight in seven days."-A. A. E. ANTITOXIN OF 600 TIMES THE NORMAL STRENGTH. Number 6 E, blue, .. 5 cu. cm. contain 3000 immunity-units. The Pasteur Institute, of Stuttgart, manufactures only one preparation of 50,000 Roux immunity-units. The Schering factory produces the following antitoxins : Strong antitoxin of 500 times the normal strength, 500 immunity- The Hamburg establishment dispenses diphtheria-antitoxin prepared by Ruete and Enoch and containing 300 immunity-units per cu. cm. In injecting the serum the rubber-ball syringe of Koch may be employed, and this is easily manipulated by one who is experienced. (Fig. 36, 2.) The antitoxin-syringe of Roux is to be recommended as especially practical, and it can be sterilized with the greatest ease. (Fig. 36, 1.) It is either boiled in a one per cent. solution of soda, or it is cleansed with a five per cent. solution of carbolic acid, after which it is rinsed with a 0.5 per cent. solution in order that the antitoxin may not be injured in consequence of too great concentration of the antiseptic. The number of immunity-units to be injected in the individual case will depend entirely upon its severity, and upon the day of the disease that the patient comes under treatment. In a mild case, coming under observation on the first day, 600 immunity-units will suffice. Severe cases, seen as late as the third day, will require 1000 immunity-units. In still more severe cases, seen at a late stage, 1500 immunity-units and more are employed. The results that have thus far been obtained in the treatment of diphtheria with the antitoxin have been exceedingly favorable. The mortality from diphtheria has been reduced about half under the influence of the antitoxin. In support of this statement we may quote the results of the collective investigation with regard to the diphtheriaantitoxin for the period from April, 1895, to March, 1896, conducted by Dieudonné in the Imperial Healthoffice. There were treated with antitoxin in hospitals 9581 cases, among which death occurred in 1589-15.5 per cent. If the cases still under treatment at the conclusion of the report be excluded, the proportion of deaths to recoveries was 3 to 16. In the eleven years preceding the introduction of the antitoxin (from 1883 to 1893) the proportion of deaths to recoveries averaged 6 to 16. Nearly one-half of the cases included in this study were designated in the reports as severe. The mortality among children under two years old (1189) equaled 39.1 per cent., and that of those subjected to tracheotomy (2744) 32.3 per cent. * In the cases submitted to treatment with antitoxin the disease pursued, in general, a milder and more favorable Existing manifestations of stenosis improved in a large number of cases, so that tracheotomy was avoided. Serious sequela with certainty attributable to the antitoxin have thus far not been observed. In rare cases unpleasant complications of a transient character occur-viz., infiltration at the point of injection, pains in the joints and extremities, urticaria, exanthemata, possibly also albuminuria. These symptoms are not at all dependent upon the antitoxin itself, but are to be attributed exclusively to the serum of the horse. Like amounts of antitoxic serum induce the same results in this respect, whether they contain a larger or a smaller number of immunity-units. For this reason it is desirable to obtain as highly concentrated an antitoxin as possible in order that as small amounts of serum as possible need be injected. Behring succeeded in preparing an antitoxin of 1200 times the normal strength, but, unfortunately, it was soon found that such highly concentrated solutions could not be preserved for as long a time as less concentrated solutions, and that in the course of time the immunizing activity diminished considerably. Behring was, therefore, led to the preservation of serums of greater than 500-strength by converting them into a dry form by a special procedure. The dry powder, containing *These results have been amply confirmed not only on the Continent of Europe, but also by observations in England and in America, with reference to both nonoperative and operative cases.-A. A. E. sodium chlorid and albumin, can be preserved for an indefinite time without any addition, and it is readily soluble in water. One gram contains at least 5000 immunity-units, and in some preparations as much as 10,000 immunityunits. From to % gram thus represents a simple curative dose; from 1% to 4 gram, twice as much, for severe cases; and from 4 to 1⁄2 gram, four times as much, for advanced cases. Behring considers it desirable that the preparation of these solutions should be undertaken by pharmacists, and he is hopeful that the dried serum may in due time be incorporated into the Pharmacopeia. Prophylaxis. It is important to know that after complete disappearance of the membrane virulent diphtheriabacilli may persist in the mouth and pharynx of children until the fifth week, and sometimes even much longer. The children must, therefore, be isolated for from five to six weeks after recovery from the disease, and, above all, be kept from school during that time. Those who surround the patient should also be kept under medical observation. Careful disinfection of the sick-room is a matter of importance, as it is known that diphtheria-bacilli will persist for a long time and most tenaciously in a house in which there has been a case of diphtheria. With regard to personal prophylaxis by means of injections of antitoxin, this has not yet been employed upon a large scale. An injection of 250 normal antitoxin-units confers protection lasting three or four weeks. This will suffice in most cases, but if the danger of infection continues for a longer time, a second prophylactic injection will become necessary. The main objection that has hitherto stood in the way of this form of diphtheria-prophylaxis was the fear of disagreeable complications due to the serum. Such fear would, however, be removed by the use of a highly concentrated antitoxin in powder-form, as 0.025 gram of the powder with a strength of 10,000 contain the requisite 250 immunity-units. TETANUS. The exciting agent of tetanus was recognized by Nicolaier, of Göttingen, in 1885, as a bristle-like rod with a terminal bulbous spore, but it was first isolated by Kitasato in 1889 from the foreign bacteria always associated with it |