wall. A vascular injection of the omentum or peritoneum is usually a result of the introduction of some of the fluid into the abdomen. When such reddening was noted at the autopsy, the test was repeated upon another animal, since death is hastened somewhat when this Guinea-pigs weighing between 300 and 350 grammes were used whenever possible. When larger ones had to be used, the increase in weight was duly taken into account. From the results of such inoculations the minimum fatal dose upon a guinea-pig weighing 300 grammes was calculated. The calculation when such had to be made was based upon the fact that the minimum fatal dose usually kills a guinea-pig in from three and one-half to six days. If x represents this dose, then a guinea-pig which succumbed in two and one-half days, or sixty hours, received 181, and one which succumbed in thirty-six hours, 20. Guinea-pigs of greater weight do not necessarily bear an exact equivalent increase of toxin, but usually somewhat less. In general, it may be said that the values given as the minimum fatal doses may err within 10 per cent., owing to various factors which cannot be controlled. Among these is a slight variation among guinea-pigs in their tolerance of the virus, the darker (black, or black and red) animals being able to stand about 10 per cent. more toxin than the white animals. Even if we allow a variation of 10 per cent. in the values given in Table I., the general outcome of the comparative study is not made in any sense untrustworthy. COMPARATIVE STUDY OF FORTY-TWO CULTURES OF DIPH THERIA BACILLI AND OF FOUR CULTURES OF PSEUDO- By THEOBALD SMITH and E. L. WALKER. MORPHOLOGY. The following description of the morphology and the staining peculiarities of the bacilli studied is based on microscopic preparations from cultures of twenty-four hours' growth at 35° to 37° C. on Löffler's blood-serum mixture, uniformly fixed and stained. The cover-slip preparations were dried in open air at room temperature, fixed by heating twenty minutes in a dry-air sterilizer at the temperature of 120° C., and stained eight minutes with Löffler's alkaline methylene blue solution. It may be remarked, however, that experiment shows that the method of fixation has little if any effect on the outline of the bacillus or upon the aggregation of its chromatin, and consequently upon the irregularity of its staining. μ In length the diphtheria bacilli vary from 1.5 to 13 μ, and for the purpose of description it is convenient to distinguish three groups short bacilli, including all bacilli under 2 in length; bacilli of medium length, including all bacilli between 2 μ and 4.5 μ ; and long bacilli, including all bacilli over 4.5 μ in length. Bacilli in culture No. 33 are rather remarkable for their length, averaging 7.5 to 10 μ, μ while a few were found as long as 13 u. It may be said of diphtheria bacilli in general that there appears to be a tendency for the shorter bacilli to become swollen at the middle and for the long bacilli to become swollen at the ends; and that the short bacilli are usually straight, while the long bacilli are usually curved or bent at an obtuse angle. Comparison on the basis of length, outline and manner of staining allows the bacilli of the forty-two virulent cultures to be divided into three types, of which the following description may be given : Type I. Bacilli of medium length, straight, cylindrical or slightly swollen in the middle, with blunt ends, and with intensely stained granules in an otherwise uniformly but less deeply stained cell. In the shorter bacilli of this type these granules are usually situated at the ends of the rod, one at each end; but in the longer bacilli there may be, in addition to these polar granules, one or more interpolar granules. These deeply stained bodies are usually of less diameter than the thickness of the bacillus, but may be of greater diameter, swelling the bacillus at the points where they are situated. Type II. - Bacilli long, slender, curved, more or less swollen at one or both ends, and with alternating stained and unstained (or faintly stained) cross-segments. Type III.This includes seven of the forty-two cultures. Bacilli are of various lengths, swollen in the middle, with tapering ends, and with broad, unstained terminal and intermediate segments. These unstained terminal segments may be so extensive that a body simulating a nucleus in the middle of the cell is the only stained portion. More often the cell may consist of two stained and three unstained cross-bands. The staining of this type differs from that of Type II., in that the alternating segments of Type II. are narrow and numerous and the terminal ones are always stained. Modifications of these types and intermediate forms occur even among bacilli of the same culture, but in nearly every case one form predominates sufficiently to allow the culture to be ranged under one of these three types. In the routine work of bacteriological diag nosis of diphtheria, as carried on under the direction of the State Board of Health, Type I. and its modifications are found in about 90 per cent. of the positive cases and bacilli of Type II. make up the greater part of the other 10 per cent. Bacilli of Type III. are very infrequently found. This classification holds good for young cultures on Löffler's serum mixture only. Bacilli belonging to these three types have so far proved virulent to guinea-pigs when tested according to the methods given in another part of the text. But besides these a certain number of bacilli (Nos. 3, 4, 39 and 44 of the tables) have been isolated which are nonpathogenic, and which belong to the class of pseudo-diphtheria bacilli described farther on. TOXIN-PRODUCING POWER. The toxicity of the culture fluid of the forty-six cultures after an incubation at 35° C. for ten to twelve days ranged as follows, the 300-gramme guinea-pig being the basis of the computations: Leaving aside for the moment the non-pathogenic forms, we notice in this summary, first of all, a considerable uniformity in the toxinproducing power. It is true the strongest toxin producer accumulates three times as much toxin as the weakest, but only one of such strength was found. It will be noticed also that the greater number of bacilli studied produce an 0.08 cubic centimeter toxin. If we group the cultures as follows, .036-.06 cubic centimeter toxins, the predominance of the middle group is better brought out. Cultures of much greater toxin-producing power have been isolated by Park and Williams. Of these, the minimum fatal dose is reported to range from 0.002 to 0.01 cubic centimeter. It is not stated whether these cultures produced this amount of toxin at the outset, or after periods of artificial cultivation. By comparing these figures with the results of earlier observers, the greater efficiency of the method described appears in striking relief. Experimenters when first preparing antitoxin had some difficulty in finding bacilli whose toxin would yield a minimum fatal. dose of 0.08 to 0.1 cubic centimeter. In the series here recorded only five out of forty-two fell below this mark. Although the clinical records of the cases from which the bacilli came are very meagre, they suffice to show that any direct relation between toxin production and severity of the disease is not obvious. This has been the inference of observers before us (Wright, Park and very recently Timaschew *), and we are able to confirm it after the application of more uniform and exact methods. This is what might be expected when we contemplate the complex nature of the disease process, the many factors which may enter into it, both on the part of the patient and the invading bacilli. There is one factor, for instance, which may modify the course of the disease, and therefore make any present-day estimates untrustworthy, — namely, antitoxin. If applied early enough, it may convert a potentially serious case into a mild one, in spite of a virulent organism. Antitoxin was used in nearly every case from which bacilli were studied, but the time of administration and the number of units injected were not reported excepting in a few cases, so that the facts on hand are not worth any serious study. All that can be said is that the toxinproducing power of bacilli from mild and from severe cases varies but little, and that all throat affections must be regarded equally dangerous if diphtheria bacilli are present. THE TOXIN-PRODUCING POWER OF BACILLI PERSISTING IN THE THROAT AFTER RECOVERY. Much interest has been aroused by the patients in whose throats diphtheria bacilli may be found a variable length of time after subsidence of all symptoms of disease. Löffler, in his investigation of the etiology, found diphtheria bacilli in the throat of a healthy child. Roux and Yersin first called attention to the persistence of diphtheria bacilli after recovery, but they disseminated the impression that there was a gradual attenuation going on which eventually made them harmless. That this may be true in certain cases is not disputed, otherwise it would be difficult to account for the presence, in the mouth of some healthy persons, of bacilli in no way distinguishable from those associated with disease processes except by an absence of virulence. This attenuation has not been observed by subsequent investigators, however, and no reliance can be placed upon it to purge the throat of the recovered case of its infectious character. Among the forty-six cultures studied there were eleven made from the throat fifteen to sixty-two days after the appearance of the dis "Centralblatt f. Bakteriologie," XXI. (1897), page 623. + Park and Beebe, loc. cit., page 37. |