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Herbert E. Durham 355 agglutinating actions are concerned, the word "specific " is inapplicable. In my previous paper ' it was suggested that the word "special" would be a better one to employ. I t will be remembered that Prof. Gruber and I found that within the " species " Vibrio choler~e asiatic~e the serum reactions were not uniform, in that the serum obtained by immunising with one race did not necessarily give more than a trace of reaction in vitro and none whatever in vivo when tested with another race, although it was capable of giving complete clumping and positive Pfeiffer reaction when tested upon the first race. At that t ime we ascribed thi~ to differences in virulence of the races, for the serum obtained by the use of non-virulent cultures had little or no effect upon the most virulent stocks; on the other hand, the serum obtained by the use of virulent cultures affected both the non-virulent and the virulent stocks of cholera vibrios. Since then, however, I have found that certain less virulent strains of Bacillus enteritidis derived from the same source were less affected by a given serum (both in vitro and in vivo) than the more virulent strain. The matter is one which possesses considerable complexity, and we (and Pfeiffer in following us) were probably incorrect in ascribing the difference to a mere difference in virulence. Further, in discussing the " specific " value of the test, I found that two clearly differentiable vibrios--the "Massowah," with its four flagella (not the "]YIassaouah" used by Bordet and others in the Institut Pasteur, which reacts as a cholera vibrio with serum, as well as in its cultural characters), ~nd the " phosphorescent Elwers" vibrio, with its single flagellum and other special characters--were, so to speak,: serum-identical, that is, the serum of either affected the other not only by the clumping test, but also by the Pfeiffer bacteriolytic test and the protection afforded to animals. I t seemed from these observations that the serum test, both in the test-tube and in the animal, could not be considered a final criterion for the diagnosis of species; for, first, there was some want of uniformity in the action of serum tests within the "species" cholera vibrio (especially when the serum H. E. Durham, On a special action of the serum of highly immunised animals. Journal o f .Pathology and JBacteriology, 1896, iv, p, 13. 356 A g g l u t i n i n s , B . t y p h i , B . e n t e r i t i d i s a n d all ies identity of V. " Berolinensis" of Rubner and V. " Versailles" of Sanarelli and V. " Iw~noff " are remembered), and secondly, 'there was mutual serum identity when apparently perfectly distinct " species" (such as V. " ~Iassowah " and V. " Elwers phosphorescent ") were investigated. Since then I have made a number of agglutination tests upon merehers of the B. enteritidis group. So far as cultural and morphological tests are concerned, I could not certainly diagnose these races from one another, it being understood that comparative observations were made not only at a single time, but now and again in some cases over a period of years and in others of months. Temporary variations do occasionally occur, but these are not constant and, after all, are insignificant in that they do not affect main characteristics. So far as these characters go we have a well-defined group, yet, when tested by serum for agglutination, marked differentiation may be found. Thus the races " Giirtner" and " 3/[orseele" are both strongly affected by " G a r t n e r " serum, whilst the races " H a t t o n , " " m o r bificans boris," "psittacosis," " A e r t r y c k e , " " Calmpthoult," " Gand," " S i r a u l t , " "hog cholera," " typh i murium," " Sheffield" (kindly sent to me by Dr. Robertson), are not very markedly affected by this serum. To some extent the race " G i i n t h e r " would appear to be intermediate; still it is more markedly affected by the serum of the Hatton type. Taking the reverse view, we find that the serum of " I ta t ton" has comparatively slight effect upon the races " Giirtn e t " and " ~orseele; " thus a serum which ~ave good reaction at 1 in 200,000 upon " H a t W n , " practically gave but a minimal reaction upon " Giirtner" and "5Iorseele" at 1:2000. I t may also be mentioned that Giirtner serum, though efficient against " G'f irtner," has practically no protective effect against the living Hatton bacilli; so that here within a group of bacilli, the characters of which can be more closely studied than is the case with the vibrios, the serum test does not give material aid in defining its limits. At the same time the Giirtner type has a distinct tendency to be affected by the serum derived by the use of typhoid bacilli. Here again we find differences between the typhoid bacilli, for instance the serum of the race Herbert E. Durham 357 "Weichse lbaum" has much more effect than that of my race " H S , " though here it is far less than that produced upon any of the typhoid races (29) with which I have directly compared them. To put the matter in a short way, it might be said in the formula of a proportion sum that " HIatton " is to " Giirtner " as " Giirtner" is to typhoid "Weichselbaum." Two very interesting cultures from the Pathological Laboratory of the Johns Hopkins University, " Gwyn " and Baeillns " 0 , " for which I have to thank Prof. Flexner and Dr. Harvey Cushing respectively, have also yielded evidence that the clumping test has only a limited value in the diagnosis of species. Both of these were obtained from eases clinically resembling typhoid fever.' They are both distinguishable from the typhoid bacilli and also from the enteritidis group in their cultural reactions, but I hardly think that I could distinguish one from the other with certainty, so much do they resemble each other. Both of them fail to give the slightest clumping reaction with typhoid serum (potency 20,000-100,000) or with various enteritidis sera (potency 50,000-500,000); of the latter, " Giirtner," "Hat ton ," " ' Giinther" and " morbitlcans boris" have been tried. I prepared a " Gwyn " serum, active up to 1:20,000 upon " Gwyn," but this has not the slightest effect upon Dr. Cushing's Bacillus " O " at 1:100 dilution, t tere the serum reaction confirms the notion obtained from cultural tests that these two bacilli are different from the typhoid and the enteritidis bacilli, but from their similarity it might be expected that they would shew some mutual reaction; this, however, is not the ease. One more example may be given, and it is of some value in shewing that distinctly differentiable bacilli of the colon group may simulate one another by their mutual serum reaction. Bacillus " W," a member of the B. coli communis verus race (vide, p. 371), has no power of fermenting sucrose (cane-sugar), Bacillus " G " will readily ferment this sugar; both bacilli have been under my observation for fully four years, and they have retained these characters and can be differ4 Gwyn, Bulletin of the Johns Hopkins Hospital, 1898, ix, p. 54. Cushing, ibid., 1900, xi, p. 156. 358 Agglutinins, B. typhi, B. enteritidis and allies entiated from each other by culture with ease. ~-ow, both " W '" and " G " are mutually susceptible to the clumping serum test, so much so indeed that one sample of " ~ V " serum acted on a culture of ~' G " up to about 50,000 dilution, whilst upon " W " itself it fell out about 30,000. lgext it was found that apparently indistinguishable true t~. coli communis races were not mutually affected by highly potent sera (none of which was less than 50,000 potency). This is a point which was foreshadowed in my easier paper, and which, indeed, has been worked at by a number of observers with a like resist. I think, however, that they have not worked with such highly potent sera, and that they thereby have saved themselves the time and patience which in some respects I have wasted. So far as my experience goes, it is rather unusual to find two otherwise indistinguishable colon bacilli, from different sources, which give any mutual serum reaction in test dilutions. I have now tested a good many different cultures during the past four years and have almost always obtained completely negative results, although the sera were highly potent for their own races. In the light of the oft-repeated statements of Prof. Baumgarten, that the serum of rabbits readily agglutinates colon bacilli, I may state clearly that this is not my experience. I rarely test in lower dilutions than 1 in 200, and at this or higher dilutions of rabbit's serum (whether normal or immunised) the results are practically always negative. I t is possible that his results may be partly due to the use of cultures in broth containing muscle sugar, which, when mixed with fresh broth or with serum, may give some precipitation of proteid matters and thus carry down the bacilli, giving rise to their apparent agglutination. In order to avoid such fallacies, I use agar cultures rubbed into a suspension with sterile I per cent NaC1 solution; this is a standard method when a given parallel-sided loop and equal amounts (say about 2 mgrm.) of bacilli per 1 cc. are used. In sedimentation tubes, I find that the reading after 18-20 hours is generally the same as that taken after 48 or 72 hours, there being little or no multiplication of the bacilli. Herbert E. Durham 359 THEOI~ETICAL CONSIDERATIONS UPON T t t E COh 'STITUTIO~ OF SERUM OF

the journal of experimental medicine/the journal of experimental medicine

SOME THEORETICAL CONSIDERATIONS UPON THE NATURE OE AGGLUTININS, TOGETHER WITH FURTHER OBSERVATIONS UPON BACILLUS TYPHI ABDOMINALIS, BACILLUS ENTERITIDIS, BACILLUS COLI COMMUNIS, BACILLUS LACTIS AEROGENES, AND SOME OTHER BACILLI OF ALLIED CHARACTER