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No. 6.— On the Diplococcoid Form of the Colon Bacillus. f BY j)^eorge adami, m.a., ivid., maude i^^bott, b.a., m.d., e/jMjicmc AND fICMOLSON, B.A., M.D. o-.^-^^l With Three Plates. [Reprinted frtmi the Tnuuac^on* of the AtiooUtion of American Physicians, Montreal, 1899. QlHKiiitfrtt ■i mamtm^ I [F-eprinted from tho TrnnsnotinnR of the Assnciatinn of American I'liyBiciaiifl, IHHH.] 4- ON THE DIPLOCOCCOID FORM OF THE COLON BACILLUS. Hy J. GEORGE ADAMI, ^[.A., M.I)., F.R.S.E., MAUDE K. ABBOTT, B.A., M D., AND F. .1. NICHOLSON, B.A , M.D. {From the Patkological iMhoratnri/ of the Roi/ul Vintoria Ifo.tpita/, ^hn^re<ll.) In the course of a careful study of a long series of livers, both cirrhotic and otherwise, we have in the specimens examined, with scarce an exception, encountered larger or smaller numbers of minute bodies, and the more we have studied them the more assured we have become that these are bacterial in nature. Under the ordinary ^ immersion lens, and by the usual methods of staining these may easily be overlooked, and if recognized they may easily be mistaken for minute pigment granules present in the liver cells. But by more intensive staining and by employing a good ^j immersion lens their nature becomes more evident. The methods we have employed with the greatest amount of success have been by staining with carbol-fuchsin (one-half the ordinary strength) and subsequent bleaching in the sunlight in our earlier observations, and of late, almost exclusively, carbol-thionin, made ac- cording to the formula in Muir and Ritchie's text-book, the sections being cleared by aniline oil. Stained by either of these methods the granules resolve themselves in the main into the fine diplococci, sur- rounded often by a fine halo, as to the nature of which we shall speak later. When these diplococci are present in any numbers there may also be isolated minute spherical and ovoid bodies of the same dimen- sions, and there may also be seen occasional strings of three or four coccus-like bodies. We have recognized these in the livers of man, the cow, sheep, rabbit, and guinea-pig. At first, working with "the cirrhotic livers A DA. MI, AIJHOTT, AND MClIUl^HON, of ciittlc 1111(1 iiiiiTi, one of lis wiis iiicliiicd to Vf^riird tlifsc iis peculiar to cirrliosis, l)iit, iis aln-adv iiiiiioiinri'il,' fuller study liiiviiij^ sliowii tlicir existence in the iippiirciitly iHiiinal liver, tliey cannot ho rcjiarded as specific of any one disease. Hlth()U)i;h it is possilde tlnit they are one factor in the production of certain forms of tihrosis. Under these conditions they tend to take on a relatively deep stain, hut in the inajoritv of case,« they stain I'adly, have a characteristic hrownisli tiiifi^e and wouhl seem to he dead. From several cases of cirrhosis in which these were recoj^nizable cultures "ave either vigorous, or what wc must now rej^ard as attenu- ated. <irowths of a colon l)acilliis, while after intravencuis inoculation of adult rahl)its with forty-eij^ht-hour liroth ;,M-owths of our stock cul- tuve ol" the colon bacillus, which is in every respect ty|)ical, the liver colls showed these minute diplococcus forms in enormous nuiiil)ors. We were, therefore, led to conclude that, while it might bo that other bacillary forms may also siiow a diplococcus like appearance in the tissues, we had adeipiate evidence that the colon bacillus can show this appearance, and durinj,' the last few months we have conducted « lon<5 scries of obs "rvatioiis bearin;;^ more especially upon this diplo- coccus-liko modificaticm of tho bacillus. Our woric is divisible into two portions : I. On the production cf a diplococcoid form of the colon bacillus outside the organism. II. On tho diplococcoid form of the bacillus within the tissues. Tho former portion has been undertaken in part by Dr. Nicliolson ; tho latter portion and the studies upon growths in body Huids by Dr. Maude E. Abbott.* Part I. ^ I. ON THE PRODUCTION OUTSIDE THK BODV OF A DIPLOCOCCOID FORM OF TlIK COLON HACILLUS. While, under ordinary conditions of growth outside the body and ordinary staining by Loefflor's blue, for example, the colon is an ♦ But for the fact tlint, by my previous )>ublioation on the subject, I have miule myself pecu- liarly res|M)Usible for these observations upon the colon bacillu?, I would very gladly have left my own name oil' the titlepage, for, In consequence of prolonged absence from my labora- tories, the observations have been throughout conducted by Dr. Abbott, and Mr., now Dr. Nicholson, and I cannot sumciently acknowledge the enthusiasm which they have thrown into the work.— J. G. A. I'culiiir to >\vii their winded iis \' are one (ler tlicHc lit ill the lir()wni.s)i (•■^nizablo IS att(Mui- HU'ulatioii stock cul- tlii' liver inl)ors. t be that Miraiice in ; can show ndnctcd « liis (lipid- isible into n bacillus issues. "Jicliolson ; (Is by Dr. :ioiD FORM body and olon is an e myself iiecu- lartly have left m my labora- Mr., now Dr. ■ have thrown 1 I DlI'l.OCOUCOlI) KOKM OK T »I K COLON HACIM.US. 3 undoubted bacdliis with no rec()^iiizal)le internal orpinizatioti, it lias been a matter of fre(jiieiit observation that it nii^ht present distinct polar stainin<^, and, indeed, when stained by f'uehsiii or other strong reagent for purposes ot photoj^raphy this so-called polar stainin<: is very conspicuous. We need but refer to the various published pho- toi^raphs to contirin this statement, in tliesc! jihoto^raphic reproduc- tiotis of film preparations from cultures the majority of the bacilli are seen to be present as two rounded coccus-like bodies lyinj.; in dose apposition, a coninion enclosing or joining sheath being more or l«.'^s clearly evident. The appearance here observed is that which is generally spoken of as " polar staining." It is commcui to a largo number of bacteria, and, in not a few cases, as, for example, among the bacteria of h«m- orrhagic septiciemia, has led in the past lo not a liti!.- cjnriision iti descriptions, authorities having been divided as to wlietlier to cla.s.s bacteria, exhibiting the property in a marked degree as bacilli f>r diplococci. In certain cases, as in connecti with the typhoid bacillus, 't has been attributed to a retraction of the ' rotoplasin to the [)oles during the process of preparation and stninin-' of the film of bacteria, and thus has been regardeil as an artefact. We shall not here enter into the discussion concerning jiolai and metachromatic granules, but simply state that our observations, so fur as they go, would seem to negative this hitter supp3sition and to ren- der it evident that, in the case of the colon bacillus at least, f/un-e is a structural condition or internal orijanization of the ynicrohe under- lying and explaining such polar stainiiu/. What is more, tluy show us that the appearances .seen in the colon bacillus are closely allied to the " beading " to be made out in the tubercle bacillus under cer'-ain conditions of growth and anvironment. As already pointed out by A. Schmidt,- Ilodot,^ and others, the colon bacillus varies according to the length of time it is kept outside the body, according to the medium in which it is grown, the reaction of the medium, the temperature, and the part from Avhich it has been isolated. Rodet has found that when it is taken from the healthy intestine the individuals during the earlier generations outside the body are singularly even in their length and thickness and stain well throughout ; w hen taken from diseased tissues, from the inflamed gall-bladder, for e.vample, this is no longer the case ; they are irregu- 4 AUAMI, ABBOTT, AND NICHOLSON, lur, l)oth in length and thickness ; they stain irrcguhirly, and sliow clear spaces and deeper staining portions. He points out that a temperature of 4-1° to 45° leads, during the first fev hours, to the appearance of very long filameits, though other individual forms are of the normal length. All these filaments show refractive bodies which take up intensely the basic aniline color. After twenty-four hours these long filaments disappear. In addition, accord- ing to this author, growth upon broth containing 2,5 per cent, lactose leads to the peculiar short and small forms, almost like cocci, the majority of which are double and in the form of diplococci. These observations of Rodet have just come into our hands, and we can in the main confirm them. Indeed, in ignorance of this work, pub- lished two years ago, we have been Avorking very much along the same lines as those indicated by Rodet, ,vho, however, it may be added, has noted these appearances without studying more fully their nature. We find that the long filaments mentioned by Rodet are to be observed in cultures kept for a few hours at a high temperature. It must not, however, be thought that they are exclusively confined to this period. Similar long filaments, showing even more clearly the presence of deeper staining bodies within them, are to be gained from old cultures associated with involution forms. Thus, in a specimen of our stock colon bacillus grown for a fortnight in broth containing a trace of bile we found great numbers of these long bodies, and associated with them numerous small diplococcoid forms. Perhaps the most interesting of these long filaments were observed in an a car CD CD ^ plate culture obtained from tho liver in a case of cirrhosis, which had been subjected for a few hours to a temperature of al)out 45° or 4t)°; removed from the incubator, tliij had grown, under difficulties (brought about by the partial drying-up of the medium), for four days at the ordinary temperature. In this the disposition of the deeper staining points was remarkable. ( Vide Fig. 8, Plate I.) Seen undei- the j'g immersion lens, after staining by carbol-fuclisin and decolorizing by weak acetic acid, these fine deeper staining points were ari'anged in a succession of pairs with occasional larger single ovoid bodies in- terposed. AVe have come across one other specimen of a rather pro- longed growth, in which the same appearance was recognizable, though not quite s:, clearly. Possibly the exact extent of the staining and subsequent decolorization may have something to do with the diflfi- ■■ larly, and show .'ads, during the ts, though other 3 fihunpiits show line color. After addition, accord- per cent, lactose t like cocci, the cocci. ir hands, and we f this work, pub- h along the same ay be added, has their nature. Rodet are to be temperature. It vely confined to more clearly the J be gained from s, in a specimen jroth containing ong bodies, and forms. Perhaps erved in an agar hosis, which had !)out 45° or 4(5°; i'iculties (brought four days at the } deeper staining Seen under the and decolorizing ts were ari'anged ovoid bodies in- of a rather pro- )gnizable, though :he staining and with the difl5- DIl'LOCOCCOII) FOKM OF THE COT, OX HACILLUS. 5 culty in recognizing this particular arrangement of the contained bodies. We have also found that taking saliva, filtering and sterilizing it, and making cultures in this medium at the ordinary temperature, we obtain the production of those long filaments, which may be present in the growth not only during the first twenty-four hours, but during the continuance of the culture. Under these conditions in the saliva of one of us (F. .f. N.) the bacilli were throughout singularly slim, and in the later growths again they tended to sliow the development within the bodies of the bacilli of a succession of deeply-staining dots. A. Schmidt has noted that be obtained tlieso filamentous t'ornis of the colon bacillus by the addition of caustic soda to broth. We found that we oblained the longest forms I)y employing lactose broth rendered l.o° aeid to pbenolphtiiallein and containing 'I.-') per cent, lactose. Mere, more especially on the surface exposed to the air, at the end of twenty four hours, we ol:"ained remarkably long filaments. Indeed, we cannot agree with Jiodet that the additi(Ui of this relatively large percentage of lactose to broth results in the production of the diplococcus forms. It is a misfortune that Rodet did not state more precisely the composition and the reaction of his l)rotli. In order to obtain the diplococcus form of the bacillus we conducted a series of experiuK^nts upon growth in broth of varying degrees of alkalinity and acidity at a temperature of 4<J°. Under these condi- tions it seemed certain that after the lirst twentv-four hours we ob- taineil, more especially in slightly acid broths, a relatively incrt'asetl proportion of short forms with polar staining, but we could not con- vert all the bacilli into the diplococcoid form. It was when we attempted to grow the bacillus upon certain of the body fluids that we met with the greatest amount of success. The frequency with which we had encountered this diplococcus form in our observations on the liver made us wonder whether our method of gaining cultures might not have been, in part at least, accountable for the phenomenon. As Livingood' has shown, growth of the colon bacillus in organic juices expressed from the liver, spleen, etc., has some slight effect upon the mor[»hology of this microbe. He noted that while the colon bacillus, in general, was relatively very large when grown upon heated liver juice, u[)on milieated he obtained very short. 6 ADAMI, ABBOTT, AM) NICHOLSON, tliick, almost oval furins witli abrupt ends, tliivso occurriii- occusionally m pairs. Here in tlic ilcvolopincnt of tlicse oval fortiis there is, it may bo urged, an apj.rnach toward our diplococcoid form, but so careful an observer would have made a fuller note upon tlie subject liiid be reco-nizod constantly the development of the diplococcoid appearance. But it must be pointe.l out that there is a dineronce between inocu- lating a medium with a loopful of a culture—/. ,-., with hundreds of thousai.dsof a micro-organism, and employment of a medium in which what bacilli are present have gained an entrance through the ducts and L.rcretory channels of the organs from which the fluid lias been obtained. AVorking with bile, for example, we have frequently found tliat by making ordinary streak cultures in the usual method we ob- tained no results, whereas gaining the bile direct from the bladder by means of a pipette and adding a drop or two of this to broth, .r.-owtli^s were obtainable. " The conclusion wbicli we have reached is that in such cases the bacteria have been present in relatively small numbers, numbers so .small that the somewhat weak inhibitory action of the bile has been sufficient to prevent growth when this bile has not been diluted. All our work goes to show, in fact, that bile has a slight inhibitory effect, not necessarily destroying the micro-organisms, but permitting growth to continue under unfavonible conditions, and it is under these un- favorable conditions that we have obt.-.ined either abs.mce of -rrowth or development of the diplococcoi.l form. For example, we have noticed in .several cases that whereas with bile taken immediately from the b.dy we have obtained no cultures, when a pipette of that bile has been kept for several days in the incubator, fairly numerous fine colonies of the bacillus coli have .leveloped, in which the individuals show a tendency to assume the diplococcus form. These observations prepared us to find that the diplococcoid form of the bacillus might be a modification brought al)out by the action of the body fluid."; but more especially were we led to employ these body fluids bv two inter- esting observations 111 September, 1808, our attention was called by Dr. W. F. Hamilton to a case of what was diagno.sed as atrophic cirrhosis in the medical wards of the Royal Victoria ITospital: this diacnosis was subscpientiy fully confirmed at autopsy. Through the kindiy inter- UIl'LOCOCCOII) FORM OF THK COLON BACILLUS. )c'C!isionaIly tliorc is, it rin, Ijiit so tlio subject iplococcoid I'een iiiocu- midreds of 11 in which the (hicts 1 1 us been ntly found lod we ub- ihiddor by li, growths cases tiie umbers so has been ited. All ory effect, ng growth these un- of growth , we have iniediately te of tliat numerous ndividuals servations 1 might be uids ; but 'WO inter- r. W. F, isis in the nosis was diy inter- est of Dj. Hamilton we were present at the first tapping of the patient, and then obtained under careful antiseptic precautions sterilized flasks of ascitic fluid ; at the same time a guinea-pig was inoculated with 10 c.c. of the same fluid, iind cultures were made directly upon broth, agar, and blood-serum. A full account of this case is on the point of jiublication by one of us, and we will here only give a brief epitome of the results. Upon agar and Loefller's blood-serum there develo[)ed scattered small colonies of a form Avhicli at first was taken to be a diplococcus, but which later, in the counie of forty-eight hours, upon these media, as in the broth, showed the presenoe of definite stumpy bacilli, often arranged as short diplo-baciili in fact, the form which Ave recognize as very characteristic of the colon bacillus. Unfortunately, vacation time came on and the opportunity tc fully examine these forms passed by. However, the guinea-pig died in twenty-four days, the autopsy was performed a few minutes after death, and from all the organs we obtained a pure culture of the colon bacillus, which appeared to be quite typical. Among the organs from which cultures were made was the gall-b!".ddcr; this gave a pure culture of the colon bacillus. A pipette full of the bile of this guinea-pig, which possessed the characters dwelt upon by Welch and IJIachstein'* — /. c, was clear, abundant, and of a relatively light color — showed, even Avhen placed in tlie incubator, no apparent growth, but remained unclouded ; at most, a few fine, granular flocculi were present after some days. 15ut upon examining a film of this bile which had thus been kept, it was found to contain abundant minute diplococci. [Mde ¥ig. 12, l*Iate I.) These grew easily when transferred to agar, the colonies being minute and much smaller than those of the tyi)ical colon bacillus. In the first transfer upon broth coccus and diplococcoid forms predominated, with occasional homogeneous stumpy bacilli. {Vkh' Fig. 1, Plate 1.) Later transfers upon agar from this broth led to the development of the typical bacillary form— stumpy bacilli with rounded ends, often arranged is sliort diplo-bacilli, and showing a tendency toward polar staining. The morphological features of the cultures now became coarser, and resembled those of the ordinary colon bacillus. Evidently, therefore, the bile of the guinea-pig exercised an inhibi- tory eflect upon the growth of the colon bacillus, and this in two 8 A DA Ml, AHISOTT, AND NICIIOI-SON, (lii'cctidiis. In tlif first pliici', tlie growth wns peculiiirly slow, 80 that the hilc (hil not hocoiiic turbid; in the second, the individual bacilli wore distinctly rnodilied — they were very much smaller than normal, and stained in such a way that they might easily be mistaken for minute diiilocoeei. In fact, the rescndilance between these minute diploeocei and the minute diplococcus forms seen both in the cirrhotic and the normal liver is most strikinir. What is true of the bile would seem equally true of the ascitic fluid taken from this case of cirrhosis. The fluid obtained was slightly opalescent, and upon keeping there gradually separated out a tliin gelatinous proteid preci[)itate. I'laced in the incubator, the fluid remained clear, and for the first few d;iys appeared to be sterile; by the end of a fortnight, however, a granular deposit was distin<'iiished. and now examination of the fluid sIiowcmI the presence in it of sin"u- larly minute diplococci tending to lie arranged in chains. ( J'i</,'. Fig. l', Plate I.) It may lie remarked that tiiis chain-like arrange- ment of the colon bacillus luis been previously observed by Dunbai'," A. Sehniidt," aiul other workers. Jietween the :Sd of September an<l the l;itli of Octoiier no less than five tappings were made, of wiiich tlie tliird and fiCtli .vere subjected to examination. l>oth of these gave cultures upon broth and agar, showing diplocoeei merging into stumpy ovoid forms. Here, again, cultures when made immediately from the ascitic fluid sliowed forms of the colon liacillus, but the ascitic Huid kept in the incubator presented oidy pure cultures of an extremely Tuinute diplococcus. After kee]iing for three weeks, subcultures upon agar made from the ascitic fluiil no longer gave the typieal colon form : instead of this a modified form was obtained ; the individuals remained relatively small and very short. {Mile Kig. 5, Plato I.) Only after prolonged sub- cidture and successive iiuiculation from 1 per cent, glucose broth did the fornis become slightly larger and develop into a stumpy diplo- bacillus smaller than the typical colon. What is more, they did not induce fermentation of glucose or <lextrose broth or cause the indol reaction. It must be pointed out that by this process of successive cultivation through glucose broth the form which was a characteristic dililococcus had become converted into a small bacillus arranged as a diplo-bacillus, and this stained homogeneously. Upon passage through three guinea-pigs (the guinea-pigs being .( 1)1 I'liOUOCCOl l> KUKM OV T 11 K COLON HAOILM'S. mg killi'd fVotii twelve to twenty-four lioiirs after intraperitoneiil inocula- tion) iinil growtli upon l'.'> per cent, lactose broth the form has become still larjrer and more typical, but wc still fail to obtain gas production. {Vuh Fig. »), I'late I.) Within the last few days we have again obtained this diplococcus form from the human liody. The patient, under Dr. (Jarrow, in the surgical wards of the lloyal Victoria Hospital, suffering from marked biliary crises, was operated upon, in the expectation of finding a con- dition of cholecystitis with gallstones. Tpon opening the ahrlomen a small amount of fluid presented, and a platinum loop of tliis was smeared upon agar-agar — this remained sterile — and immediately about a drachm of the Huid was collected, under strict aseptic pre- cautions, in a sterile flask and brought over to the pathological labor- atory. Here this was added to about an e^ual (juantity of sterilized broth and placed in the incubator. Upon continuing the operation the gall-bladder and ducts were found |)orvious ; there was, however, a condition of perihepatitis, with subacute peritonitis affecting the upper half at least of the abdominal cavity, and with this was associated some thickening of the great omentum. Upon examining the above-mentioneil broth culture after twenty- four hours. Dr. IJrown, the resident surgeon, found that it contained a pure culture of minute diplococci, and immediately called our atten- tion to it. In the features of this growth upon various media this form has so far been found to resemble the minute dipiococci already mentioned as obtained from tlie case of cirrhosis, thougli the growth is sligiitly more active and free. Passage through guinea-pigs and lac- tose broth has resulted in the development of a form identical with that just mentioned. (Viih' Fig. 7, Plate I.) There is very slow development of turbidity in ordinary broth, rather more rapid in glucose broth, but absence of any sign of fermen- tation. The growths upon the surface of agar in both wei'e at first singularly fine, so that they resembled closely those of the strepto- coccus pyogenes, though possibly more transparent than the latter. Upon potato the growth was invisible ; upon blood-serum the colonies were also very fine, and were of an opaijue white fading on to a yel- low tinge. Upon gelatin there was slow growth witliout liquefaction, while litmus-milk was decolorized until it became almost perfectly 10 ADAMl, AHHOTT, A X I) NlCHOIiSON, white; tlioii slowly, in tlio course of the fifth day or ho, a fine pink color was (level. jped in the iiiediuni ; the milk is coa>;ulate(l at the end of a week. Growth upon hroth was definite, hut not ahundant, and was associated with siii;j;ularly little turbidity, a white somewhat stringy ))reeipitate being slowly formed. In the fermentation tube the open limb became opalescent or moderately turbid in the course of forty- eight hours, the closed limb remained perfectly clear, and, in a(Mition, in neither glucose nor iti lactose broth was there any production of gas ; further, there was and is no indol reaction, and if turbidity be present it is still singularly slight. It is unnecessary here to describe all the methods that we have employed in order to cause these forms to revert to type. Briefly, y\i) may say that we have obtained the greatest change by culture for twenty-four hours upon broth rendered J. 5° acid, according to the method recommended by the Committee of Bacteriologists, to which 2.0 per cent, of lactose has been added. In this medium, already at the end of twenty-four hours, there is abundant growth md well-de- veloped turbidity, and the individual forms are relatively large ani^ ovoid, frequently arranged as stumpy bacilli. ( Vide Figs. H and ^ Plate I.) When this form is inoculated into the guinea-pig intraperitoneally and cultures made from the peritoneal fluid at the end of nine hours, both upon agar and glucose broth, growth upon glucose broth in the fermentation tul)e is much more active than before inoculation ; and, whereas, previous to inoculation, only the open end of the tube had been rendered opalescent, now there is turbidity throughout both tubes. As already stated, after passage through three guinea-pigs and growth on this medium the form produced is undistinguishable from the normal colon bacillus. It is possible that this remarkable and somewhat persistent di[)lo- coccoid form, obtained both from the bile of the inoculated guinea-pig and from the ascitic and peritoneal fluids, has become attenuated dur- ing its stay in the body, and that in the case of the bile, for example, during the passage through the liver, the colon bacilli have been mark- edly modified. We have taken sterilized human bile and added to this a minute (piantity of a stock culture of the colon bacillus, and have not been able to obtain in the bile the diplococcoid form alone, although it is true that diplococcoid forms have been relatively abundant. UlPIiOCOCCOII) FORM OK THK COLON' BACILLUS. 11 Here it is interesting to note a point wliieli \vc again find observed by Rodet— namely, that tlie human bile has a distinct iidiibitory effect upon the multiplication of the colon bacillus. Bile to which a minute drop of a twenty-four-hour old culture had been adiled remained to all appearances perfectly clear, and apparently no growth had occurred during four diys; but when a drop of this bile was added to about 10 c.em. of slightly alkaline broth and placed in the incubator, that broth rapidly became turbid, and there was most abundant de- velopment of the bacilli. We are making further observations upon this modification of the bacillus by growth in bile. This, however, may be said at the present time, that jmssibly the existence of bac- teria in the bile may easily be overlooked when tlie ordinary methods of culture upon solid media are employed, the concentrateil bde inhibiting their growth. One of us (M. E. A.) has already found that human bile (three cases), which was apparently sterile when streaked upon agar-agar, gave abundant cultures of the cohm bacillus wlien a suiall drop was added to about 10 c.cm. of glucose broth. CoxCLUSloNr^. Thus far, then, our observations upon the colon bacil- lus grown outside the body have led us to the following.conclusions : 1. The sliort form of the normal colon bacillus cultivated upon the ordinary bacteriological media fre(iuently presents polar staining, the appearance given being that of two rounded bodies, staining more deeply than the rest of the bacillus, lying in and united by less deeply sti'ining material. 2. In tiie more filamentous form a succession of these more deeply staining bodies is at times to be recognized. 3. Growth outside the body under relatively unfavorable conditions renders the polar staining more prominent, so that the shorter forms may closely resemble diplococci, and the filamentous forms show a common unstained or lightly-staining sheath, in which is to be made out a succession of minute dots in pairs and of somewhat larger single ovoid dots. 4. We have so far been unable by modifying the reactions of ordi- nary media, and l)y continiieil growth at a high temperature (4f')°), to produce cultures in which the diplococcoid form alone has been present, although by these means we have gained cultures in which this form has predominated. 12 AHAMI, AHHOTT, AM) NUMIOLSON, T). On the otlicr liaiid, certain bodv HiiidsRown iiiitiinillv, if we may so term it, with tlie colon bacillus — i. c, tlie ascitic and peritoneal fluids from ii case of hepatic cirrhosis and of peritonitis respectively, and the liileof a guinea-pij^ inoculated with ( ? an attenuated form of) the colon hacdius have yielded us diplococcoid j^rowths so modified that we have not so far been able to cause them to revert completely to type. ti. It has been by the prolonjied action of these fluids that these races of the colon bacillus have l)een ))roduced : cultures made from them immediately after removal from the body have yielded us, either immediately or after one or two transfers, typicid cultures of the colon bacillus. Where the fluid has been kept from ten to twenty days the modified diplococcoid form has been produced. 7. The slight but definite inhibitory action of bile upon the growth of the colon bacillus is shown in two ways : (r/) Smear cultures of bile upon agar may remain sterile, whereas the same bile added to ordinary peptone broth may be the seat of active griiwth. {b) Simi- lar bile kept for several days in the inctibator remains clear and shows singularly little evidence of growth within it, though subcultures from this yield fairly numerous colonies of a modified diplococcoid form of the bacillus. 8. The axitic fluid from a ca.sc of hepatic cirrhosis was found to possess similar properties of modifying the colon bacillus and inhibiting its growth. 9. These modified colon bacilli are relatively minute, assume a diplococcoid form, are non-motile, form pin-point colonies upon agar- agar, cause but slight turbidity in broth and an almost invisible growth upon potato ; act but slowly upon litmus-milk, have lost the power of fermenting glucose, lactose, and dextrose broth, and do not develop the indol reaction. Part II. ox Tin-: D11'I,0C0CCU8-MKH MODIl'lCATION OK THE COLON HACILl.US IN Til 10 TISSUES. Taking a series of four young rabbits weighing from 225 to 305 grammes, we inoculated into the marginal vein of each 0.75 c.cm. of a twenty-four-hour growth of the colon bacillus, and killed the animals DIPLOCOCCOID KOKJI OF THK COLON HACILM'S. 13 iit intervals of lifteen iiiinutes, tliirtv iniiuitcs, one and two liours. The various organs were immediately jdaced in t'orniol-Miiller, and were sul)se(|uently cut in celloidin and parallin. the sections heing stained by carbol thionin. Our attention was at first es[)ecially directed to the liver. Here ah'eaily in the animal killed at fifteen minutes after intravenous inocu- lation a definite series of changes was seen to have occurred. ( KA/e Figs. 13 and 14, Plate IT.) In the bloodvessels of the liver free bacilli of normal size and appearance were occasionally to be observed, but alreaily bacilli could be recognized within the leucocytes in the blood- stream. ( Vide Fig. T3.) The number of these leucocytes was not excessive, but each contained a relatively large number of bacilli. In addition, already the endothelium lining the vessels was seen to be very prominent ; here and there these cells containeil a fairly large number of bacilli. In thirty minutes the number of bacilli in the endothelium cells and the number of endothelial cells containing bacilli were markedly increased. The bacilli, situated within the endothelial cells, already show strongly marked differences from those free in the blood-stream. The latter were of normal length and thickness, and took on a homo- geneous stain. Those within the endothelial cells were short and stumpy, sometimes almost coccus like. The appearance given is that of primitive bacilli having been broken up into shorter lengths. In the rabbit killed at the end of one hour the number of bacilli seen in the blood-stream was distinctly less, but there was a further increase of those in the endothelial cells. Occasionally, in the endo- thelial cells relatively large bacilli could be seen, but the majority of forms were, as in previous specimens, very short and stumpy, and the impression gained by a study of the sections is that the bacillus is taken up in the long form and subseijuently broken up into shorter sections. So far no well-stained bacilli could be seen in the liver cells. Already in the endothelial cells certain of these stumpy forms had the appearar'ce of diplococci of fair size. In the liver of the rabbit killed at two hours after inoculation the same appearances were to be made out as those seen in the rabbit of one hour — namely, the presence of short and stumpy bacilli in the endothelial cells; we were of the opinion that a larger proportion of these had the appearance of diplococci than in the previous sections. 14 AI>AMI, AHUOTT, AM> NICHOLSON, III st'vcnil pliiccs l)ot\v('cii tlio liver cells, as iiulciMl also in sections taken at an earlier peiioij, there were to be made out hyaline masses, apparently situated within the vessels, wliieh hyaline musses contained numerous hacilli. We have found scmie little dilliculty in eouiinj,' to a conclusion as to the nature of these masses ; the lar^e ones would seem certainly to he hyaline thrombi, l)Ut in the smaller ones it was often ditRcuUto make ipiite certain whether we were not dealinj^with some phenomenon in c(mnection with the endothelial cells; for very frequently a nucleus of endothelial type was in close connection with these smaller hyaline masses. We could not absolutely leave out of account the possibdity that we were dealing with very greatly swollen endothelial cells. I'p to this i)oint we were unable to recognize in any of the sections of this series indications that the Imcilli had been taken up by the liver cells. F.ut in a rabbit killed four hours after inoculation we came across great nuud)ers of extremely miinite brownish shadows de- finitely within the hepatic parenchyma. ( Viih Fig. 1 5.) We have been wholly unable to stain these little bodies, and, indeed, only by very careful examination with the ^^^ immersion lens have we been able to see them <listinctly ; but with this magnification there they most certainly are, ami the more carefully they are studied the more clearly they are seen to be present in general as extraordinary minute little brown- ish diplococci, at times showing a halo around them. And the more one has studied these appearances the more it seems likely that this apparent halo indicates that these small bodies lie in vacuoles, although in part also the api)earance may be due to the existence of an unstained sheath or body-substance. Evidently, judging by the sections from this stage of the inocula- tion disease, not only are the bacilli taken up in large numbers into the liver cells, but being taken up they undergo rapi<l digestion and destruction, so that they can no longer be stained by the ordinary methods, and what we see are essentially the shadows of the bacilli. We have attempted to make out the stages by which the bacilli pass from the endothelium into the liver cells, but so far without great success. Here and there in sections of the two-hour rabbit we have been able to make out that the endothelium appeared to be raised from the underlying cells, and on the inner side of this endothelium very rarely we could see in the spaces between the endothelium and am am^ r)II'IiOC0CC0II) FOKM OK THK I'OMIN HACIMil'S. 15 ill sections lino masses, es contained in coinini^ to ones would ones it was (lealinji; with lis ; for very nection with leave out of L'ally swollen ' the sections ri up by the oculution we shadows de- V'e have been only by very en able to see lost certainly clearly they ' little brown- nd the more vely that this :)les, although ' an unstained ' the inocula- numbers into digestion and the ordinary )f the bacilli, e bacilli pass without great ibbit we have to be raised } endothelium lothelium and cell well-stained coccus or diplococcus-like bodies. We are, however, unwilling to dwell too strongly upon these appearaiutes, inasinuch as the enilothelial cells showing these features were crowde<l with bac- teria, and we could not <'xclude the jiossibility that in the process of preparation the cells might have become slightly dislodged, and that the appearance of the bacilli apparently outside the main body of the cells might be due to their presence in a slightly different plane. Taking next well-developed rabbits similarly inoculated and killed at the end of twenty-four hours, wo liavv> found in thein the presence of bacilli in the endothelial cells, while ^he brown shadows, as we may term them, have been present in ene-mous numbers in the liver cells. Thus far, then, from what we have said, it would appear evident that when the colon bacillus enters into the circulation it is liable to be taken up rapidly by the endothelium lining the hejiatic vessels, and in this process undergoes <livision into smaller segments, mo that in the main one meets with stumpy forms in these cells, forms which still stain well, although often showing a tendency toward a iliplococcoid appearance. Following up this within lour hours these bacilli are discharged by the endothelial cells, and are by some means or other taken up by the hepatic cells and rapidly destroyed, so that it is only by careful examination that minute coccus or diplococcus-like bodies are discovered within the liver cells. It is interesting to note that upon examining a film of the bile taken from inoculated animals at the end of twenty four hours one can by careful preparation recogni'/e in it these very minute di[ilo- coccus-like bodies. To see them it is necessary to make a very tine film, treat with weak acetic acid, wash, and then stain with dilute carbol-fuchsin, and examine under the highest power. It would, therefore, seem evident that the liver cells are capable of discharging these modified and destroyed bacilli into the bile capillaries. But we now come to certain great difficulties in connection with the statements here made. In the first place, making a large series of control observations upon the livers of apparently normal adult rab- bits we have frequently come across these same diplococcus-like bodies, and in four instances in relatively very great numbers. Indeed, these diplococcus-like bodies would seem to be very frequently present, more often present than absent from the rabbit's liver. U; ADA Ml, AIJHOTT, ANI> N 10 IfO I,«ON , To (.J.viiitr tliis ilillicnlt.v. it smiicd to uh tliiit we ini;:lit obtain more •Iccisivc n-sulfs l.y .'inpioyin^' wry yoiiiin; nil.l)its from tliiw to six wooks ,,1(1. In our control sco'tions of tlu> livers of tlit'se very younj; nil.I.its \v(« liavt' foun.l that tlic diplococci appear to Ix- iibseiit. Upon making a like scries of inoeiilations into these very yoiinji ral)l>its, and killing at two, four, and twenty-four liours, we Imped definitely to settle the ([uestion. But here, at lirst, we had wholly negative results, By our routine methods of staining we were unahle to detect any bacteria within the cells, even when we employed scctioi.s that had been cut in paralVm. So ojjposed to all our |>rovious results and conclusions did these aj.pear that for a time we were on the point of relin(pushing this paper, it is possible that either the carbol-thionin used by us for the experiments was defective or our techni(iue motlified in some sli.'ht degree, for at the best the carbol-thionin method does at times show itself wanting. I'>ut our failure was so constant that we hardly believed that tliis explanation would suiUce. Now we have attempted to stain other sections from the same blocks bv other methods, and we eventually found that staining for half an hour with Loelller's methylene-blue, washing with te[iid water, and then passing thr<mgh alisolute alcohol and zylol, we obtained sections in which the tissue is relatively faintly stained and in which we are able to detect witliiii the cells peculiar small diplococci having the faintest brown tinge. These were obtaincil from the livers of animals whicli had been inoculated two and f-^ur hours before death. Our failure to recognize these bodies is in fact due to their minute size and their very faint stain. We have examined control livers also from young animals by the same methods with negative results. Tt would seem clear to us that the rate at which the colon bacilli are taken up and destroyed in the liver varies to some extent in difter- ent animals according to tlie condition of the tissues and the virulence of the microbe. It is to be noted that the culture employed in this latter series was from the same stock as that employed previously — a stock which had been grown outside the body for an additional six nKmths. And here we may notice that the most powerful staining diplococci, and also those having the deepest brown tinge, were in the livers of rabbits dying from three to four weeks after inoculation, as again in certain of our control animals. Our experimental animals, wiiich had been kept alive at the most for twenty-four hours, have DIPLOCOUCOI!) FOKM OK TIIK C()I,()N ItAiMI-M'S ; obtiiiii more 3 to six wcrka 'oun^ riihbits Tpnn iiiakiti}^ I, uTiil killing to HCttle the ilts. Hy our any bacteria iiul been cut (1 c'oiicliiHioiis reliiKHiishing II used by us ifieil ill sonio does at times lat we hardly e same blocks fj; for halt' an ater, and then ■d sections in rthich we are -i having the ers of animals death. Our linute size and ers also from ts. c colon bacilli ctciit in dift'er- l the virulence ployed in this previously — a additional six Aerful staining ye, were in the inoculation, as lental animals, ur hours, have yielded us only diplocoecus forms, showing but a (h'licute brown stain- ing within the cells. While we were in doubt with reganl to tiiis second series of livers, it seem"d to us well to study another excretory organ not in connec- tion with the portal circulation. Hxaminiiig the kiilneys of several control rabbits, we have in no ease been able to find the diplocoecus forms present within the organ in the great numbers in which we have come across them in the livers in the same animals. We have met with occasional diplococci within the cells of the convoluted tubules, but these have been rare. We have found that the examination of the kidneys for these modified colon bacilli has been a matter of consider- able difficulty. Undoubtedly they are taken up by the cells of the convoluted tubules. Of this we have abundant evidence, and occa sionally we have come across well-staining liiploeoccus-like forms in the outer portion of the kidney cells, but the diplocoecus forms appear to be desti''^ 1 with great rapidity, and in the process of destriictiim do not assuni the brownish tinge already referred to in coiineetion with the liver; thus it has been a matter of extreme dinieulty to trace them. We have, however, seen them in great numbers in the cells of the convoluted tubules of the rabbit two hours after inoculation (vide Fig. 1 1, Plate I.) and again at twenty-four hours, in this latter the number being greater, lleie, alsf), in the cells of the tubules in very thill sections we have come across numerous minute vacuoles of an elongated oval shape, often slightly dented in the middle; ami within these we have at times been able to distinguish two very minute dots, evidently the very final indication of the disappearing and destroyed bacillus. ( Vide b. and c. Fig. 11.) Independently, Dr. A. G. Nicholls' has studied the kidneys of the animals inoculated by us and has met with these diplocoecus forms, fully confirming what we here state. CoNcriUSioNS. Our observations, therefore, upon the whole would lead us to the following conclusions : 1. That the colon bacillus injected into the circulation is rapidly taken up both by the liver and the kidney. 2. That within fifteen minutes after inoculation some bacilli are already ingested by the endothelial cells in the liver, this process of ingestion continuing until these cells are full of bacilli. 3. That in this process of ingestion the bacilli are broken up into 18 ADAMI, ABHOTT, AND NICHOLSON, slii.rtcr IfUf^ths, and tliiit tlifso sliort stumpy bacillary forms may— already within ^he endothelial cells— present themselves as two deeply stainin<5 dots, and may thus resemble diplococci. 4. That already in two hours the modified bacilli may be discharged outwardly from the endothelial '^ells and be taken up by the under- lying liver cells. ,'). The exact stages of this discharge we have been unable to fol- low. In tlie liver cells the modified bacilli are to be recognized as small iliplococci of a size varying from that eijual to the diplococci seen in the endothelial cells down to points of extreme tenuity. Evi- dently these forms are undergoing destruction. In the first place, they lose their power of staining ; in the second, if the destruction is not too rapid, they assume a brownish tinge. What is the causation of this brownish tinge we have not yet dete' - mined, but it is to be made out in the unstained sections, and our studies upon the human liver indicate to us that not a little of the fine pigmentation common in liver cells is brought about by the existence in these cells of these minute elements of bacterial de- struction. During this process of destruction the modified bacilli lie in diges- tive vacuoles, and the fre(iuent appearance of the halo around these forms is in a great part due to the existence of the vacuole. We have occasionally been able to make out wIk.i ..ppoar to be these vacuoles in the liver cells without the evidence of the contained microbe, that having been apparently entirely digested. (We have seen the same appearance also in peritoneal leucocytes nine hours after intraperito- neal inoculation with modified colon bacilli.) ( Vule Fig. 10, Plate I.) 6. In the kidney the same process is at work. We have recognized the diplococcus form within the cells at tlie expiration of two hours after inoculation, and have also seen the vacuoles within the cells and convoluted tubules, and there have occasioiuilly met with two dots just visible, being final indications of the process of digestion of the bacillus. We sincerely hope that others will repeat and confirm these obser- vations, though to those repeating them we would point out that it is absolutely essential to employ higher powers than those ordinarily used for bacteriological investigations, while the fi.iest sections are recjuisite to give clear results. Very careful technique in the matter illiiry forms may — selves as two deeply i may be discharged 1 up by the under- been unable to fol- to be recognized as lal to the diplococci reme tenuity. Evi- ling ; in the second, e a brownish tinge, have not yet dete - jd sections, and our t not a little of the )ught about by the iits of bacterial dc- d bacilli lie in diges- e lialo around these e vacuole. We have to be these vacuoles itainod microbe, that have seen the same urs after intraperito- uh Fig. 10, Plate I.) We have recognized liration of two hours within the cells and r met with two dots js of digestion of the confirm these obser- d point out that it is ban those ordinarily e fiiiest sections are hnique in the matter UIPLOCOCCOIl) FORM OF T H K COLON HACILLUS. Ifl of intensive staining and of decolorizing of the ti.ssues is also an essen- tial. Unless these points are attended to our fVe(|Uent difficulties in forging the chain of evidence here biougiit forward will certainly present themselves, and, without great jiatience, we cannot expect ot'" s forthwith, to corroborate our results. We are prepared, that is, to find these results called in question ; but after many months, and on the part of one of us many years, puzzling over these peculiar pigmented bodies, seen more especially in the liver, we do not see what other conclusion to reach. Merc we may say that we are pre- pared to find bacteria other than the colon when taken up at the cells of the liver and kidney assume very similar forms. Indeed, we already have evidence of this in connection with the typhoid bacillus. If the above conclusions are correct, it is clear — ;)U(lj:ing from what we have said concerning the appearances seen in many normal livers of rabbits, and seen also, we may add, in the human livci' — that the liver as an organ possesses the most important function of taking up and destroying bacteria, more especially the colon bacilli, which have gaineil admission through the portal blood, while the kid- nev possesses a like power of rapidly destroying bacteria circulating in the general systemic blood. As our paper is more especially upon this diplococcoid form of the colon bacillus and its modifications within the body, we will not here dwell upon this subject, especially as one of us has alreaily called attention to this conclusion elsewhere.' We purpose, if possible, making a series of observations upon those conditions which lead to the taking up of bacteria from the intestines and upon their course through the blood, and again through the lym- phatic SL-ystem. Finally, a few words regarding the structure of the colon bacillus to be deduced from the above observations. It is difficult to arrive at any other conclusion than that the type colon bacillus is a stumpy bacterium with rounded ends. This bacillus consists of at least two parts — one which takes deeply the stain, the other relatively non- staining. Under ordinary conditions of free and rapid growth these are not to be distinguished from each other ; under other conditions, more especially those of difficult growth, the chromatin or sti.ining portion tends to be aggregated along the long ta.xis of the bacillus, most characteristically in the stumpy bacillary form, as two rounded bodies, 20 A DA MI, AUBOTT, AND NICHOLSON, aiul thus the iippejuiiiico is i^ivcii of a diplococcus, a capsulated diplo- coccnis, tlic apparent capsule beini; the non-staining botly-substance. Where the hacilius is of the large or filamentous type our observa- tions would seein to show us that the tilanient is capable of being broken up with a certain amount of ease — f. //., in the endothelial eells — into its component stumpy or l;aeterial forms, each of these being eitlier a single oval deeply staining body, or, as above men- tioned, two rounded staining bodies, so that it assumes the diplococ- cus form. The size of these chromatin bodies varies, as would be the case were the chromatin capable of varying degrees of concentration. Judirinii from what is oli.served within the hepatic and renal cells, these chromatin liodies consist, at least, of a basal non-staining ma- terial and a chromatin, for the power of staining (witii aniline dyes) may completely disappear, and, nevertheless, a substance is left behind still capaliieof recognition as a minute diplococcus, unstained by ordi- nary reagents, but. within the liver cell, capable of assuming a brown- ish tint. From tlie appearance within the Innly cells tliis central substance is oliviously more resistant than the remainde of the ba- cillus. It is the last part of the bacillus to be destroyed; indeed, these diplococcus like shadows of bacilli may accumulate more espe- cially within the liver cells, the n)esenteric and retroperitoneal glands. We will not liere discuss tlie recent work upon the existence of nuclei or nuclear material in the scliizomycetes. It is, however, impossible not to i)e struck by tlie analogy in structure between the colon bacillus as here described and nucleated cells in which nuclear division jM-ecedes cell division. For we have encountered the following forms : 1. z. 55*' '4 % S. % % « ■iuliited (liplo- (T -substance. ! our observa- iblc of being le L'ndotlielial }acb of these ^ above nien- tlie (liplococ- would be the .'onoentration. (I renal cells, i-staining ma- aniline ilyes) is left behind lined by ordi- iiinji; a brown- < tliis central le ■ of the ha- lved ; indei'd, te more espe- toneal glands. ,' existence of , is, however, .' between the which nuclear I the following DIl'LOCOCCOlI) KURM OK THE COLON BACILLUS. 21 This paper being already longer than we had intended to present, we have omitted any consideration or criticism of the observations by Giirtner,*' Klein," Thiercelin,"'and many others which show a recogni- tion or failure of recognition of the existence of this diplococcoid form of the colon bacillus, and very freciuently a tendency to mistake the diplococcoid and the encapsulated form of the colon bacillus for an entirely different species. This subject will be discussed at greater length by one of us (M. E. A.) in a separate article. EXI'LANATION OF PlATES. The figures of bacteria have all been (Iniwii (by J. (i. A.) under the same magnification— i.e., Heichert • i^ in. immersion, ocular 1— by means of a Zeiss camera Incida, latest imltern. AU were snbjeeteil lo the same process ol staining : Zlelil-Neelsen I'arliol-fnchsin dilnted witli iiO per cent, of water. They were relatively deeply stained and tlien decolorized in water con- taining a minute proportion of acetic acid— about one tlroji of Klncial acetic acid to the ounce of water. The drawings of sections were made under tlie same con<litions, with the e.xceplion of I'ig, II, I'late I., which is a tracing from a photograph. Pl.ATK i. Flii i._(irowth originating from liileof guinea-pig whicli died twenty-four days after intra- jieritoneal inoculation with Id c.cm. of ascitic lluid from case of liepatic cirrhosis ; forty-eight hours' growtli in alkaline peptone broth iuoculalcil from forty-eight hours' culture n|ion agar- agar, wliich in its turn had been seeded from a pijietle of the guinea-pig's l)ile removed a few minutes after death and kept for eigblcen hcairs at ;M°. Fl(i. 'J.— From film made from the ascitic lluid of the above case of hepatic cirrhosis left in sterilized Mask at 37'' for seventeen days. Plii, ;i_From lirst broth cullnre, forty-eight luairs old, made from the almve ascitic lluid. Note miniue ovoids as well as diploi'occi. Fill. 1.— From forty-eight-liour culture upon I.oelller's blood-serum made direct from the above a.scitic lluid : forms a shailc larger than those from broth, with slight (endency to be arranged in shorl chains. p,,;, ."i.— The same micro-organism after repeated transfer upon agar agar during si. k months: iniiividuals much larger, although slill relatively snnill, with shorl bacilhiry, di|ilo-bacillary, and di|ilococcoid forms. Fiii. (i.— The same after transfer through three guinea-pig.s, followed by three successive transfers through -•.."> iKjr cent, lactose broth : Ihe bacillus now is m. rpliologically indistinguish- able from the forms seen in Fig. 1 and (save in absence of llagella) from tlie ordinary colon l>acillU9. Flo. 7. -Microbe isolated from peri:oueal exu<iate in case of peritonitis witli iKU'lhepalitis after parallel passage through three gniuea-pigs and transfer through lactose broth. The microbe, npcui lirst isolation, was a minute diiil(«!(H'cns : it will be seen to be uudistinguishable from that shown in Fig. (1; cnlt\irally. it was identical. i.-|,j. n,— To sliow arrangement of diplococcoiils and ovoids in filamentous form <ir a colon bacillus isolated from the spleen In a case of p-ogre>sive hepaiic cirrhosis. From a colony on an agar iilate kept live days under unfavorable conditions (vide «r(7(.-7( Mnliral Joiinml, Octo- ber -ll. l.siW). Kt(i. '.(.—From film made from tlie bile ol a rabbit killed seven hoinsafter intravenous Inocu- lation with the colon bacillus; the l)ile was kept fourleeii days in pipette before examination. Note variety of forms : rare short l)acilli and di|ilo-biicilli with slight capsule, diploeocci witli well-marked caixnles, minute diploeocci devoiil of capsule (■.' destroyed). Fio. 10.— fells from peritoneal lliiid of guinca-|iig killed nine laairs ofter intraperitoneal inoculation with a forly-eight-lionr broth culture of form shown in Fig. ,'i—(. e, from agar cultures deriveil from the ascitic lUiid from a case of cirrliosis. Stained with carbol-tliicmin, oo DISCUSSION' llciclierl 1 ,s in. iminei>iiin, dculiir I, ilrawii under a Zeiss camera Incida. a. Deeply staining Ijncterial and diplcirceeoid Inrins. h. Atlenuatod dipNieoeeolds in large vacuoles, c. Still fur- tlier atlenuate<l diploeoecoids. (/. Vacuoles vciid cif contents. Kic n.- Kroni section ot convoluted tulailes of Icidney of youns rabliit killed two lio.irs after inl ravenous inoculation with pure culture of Imcillus coli. Traciiig from photograph under Zeiss i ,s in. immersion, compareil witli original section (tlie iiliotograpli not being pcr- lecti. 11. Iieoply staining diplococcoid Ibrm just witliin ceil of tubule. It. Attenuated diplo- co('coi.l form in vac'uole. r. Empty vacuoles of oval sliape. Kic. IJ.— To compare with Kig. 1. ■•"roni tilm of bile from guinea-pig kept in pipette and placed in incubator at :t7 • lor eigliteen hours. A drop of this same bile i)assed through broth and asar gave the form shown in Kig. 1. Note presence of ovolds, diplococcoids, and diplocoi'ci. I'l ATK II. Pui. K!.— Section of liver of rabbit killed liflecii minutes after intravenous inoculation with 0.."ic.c. of fiirly-ciHlit-liour brolli eullure of H. coil ; carbolic lliionin ; Zeiss camera lucida; Vi» oil inmiersion. a. Marked swelling and enlargement nf endollielial cell witl'. ingestion of bacilli, which have become short and stumpy. b. l'"ree bacilli, nniandug long. c. Leucocytes conlaiuing Ijacilli. l.'m. M. —Section .pf liver of rabbit killed lifteon minutes after intravenous inoculation with 0.5 e.c. of tbrty-eiglu-liour ciilttire ol li. coli ; carbolic thiouin ; Zeiss camera lucida; '/is oil immersion lens. a. bacillus Iree in caiiillaiy. b. Enddtheliai cei: niuch swollen atid contidniiig several bacilli, both stumpy and tending to asstuue diidococcoid form. ri..vTK III. Flo 15.— Section of liver of rabbit killed loin- hours after Intravenous inoculation with 0.5 3.C. of forty-eiglit-liour culture of P.. cod ; carbolic thionin ; Zeiss camera lucida ; ' i» oil immer- sion lens. Abundant very mintite diplococcoid forms in liver cells, part of which only are shown. liliil.liXillAPllY. 1. Adami. liriti^h Medical .Inurnal, Is'.is, ii. ),. rj.w. ■2. Schmidt, A. Wiener klin. Woehenschrift. Is'.rj, v. p. (143. :). Rodet. Archives de I'liysiologie, isiii'., .'ith series, viii. p. tUlS. 4. I.iviugood. Centralbl. f Uakteriologie, IS'.kS, xxiii. pp. 98!), 1002, and 1043. ."i, lilacbstein. .lolms Hopkins llosjatal linlletin, IS'.U, Xo. 11. (i. Uiiubar Zeitschr. f. llyi;iene, is'.fj. xii. p. 4S5. 7. N'icbolis. Philailclphia Monthly Medical .Iimrual, .lanuary, 18U9, No. 1, and .Montreal Meiiical Journal, isiw, xxviii. p. bil. s. (iiirtner. Centralbl. f. Uakteriologie, 1S91, xv. p. I. a. Klein. Ibid., p. 276. 10. Thierclein. Comptes rendus de la Soc. de liiologie, IS99, 10th series, vi, p. 209. . Doeply stiiining )les. c. Still fiir- killeil two hotirs rniiii photograph It not being pcr- .ttenUiUe<l diplo- It in pipette and ed 111 rough broth plococcoids, and inoculation with »niera liicida; Via leilli, which have inoculation with ra lucida ; '/is oil nipy and tending iculiUion with 0.5 ia ; 'is oil inuncr- ■ are shown. 113. 1, and Montreal p. 2m. TRANS. ASSOC. AM. PHYS. ft / • \ t » \ / I *\ 12 VOL. XIV. 1899 % "«Cr*ssMz» >"^v .'> ■I-^ -S?' «?a> IM.ATK I Adatni — Diplococcolil Form of tlie Colon Bacillus. TRANS. ASSOC. AM. PHYS, VOL. XIV, 1899 Ci JO \ 'lX^f:5-V\^^'^^^ J » a o o o MlilUWIIIl wm TRANS. ASSOC. AM. PHYS. VOL. XIV, 1899 FIG. 15. I'l.A'iK III I'l.A'iK III Aaiimi -Dil'locociMiid Kcrin <>f tin- Colon Uiirillus. DIPLOCOCOOII) FOKM oK T If K CoLoN HACII.M'rt. 2."{ DISCUSSION. I)K. WRi.f'll ; Dr. .Vilnmi's olwerviitioim iire of f^reiit iiitercHt mid mont «iigge«tive, nlthoiigli tlieir intiTprotiUioii Ih imt witliout ilillicullif!*. The colon bacilluH in undoubtedly fiicountereil very ofUii In bHeterioloniciil p.\- aminations* ul auto|mieH, but only in ii relatively wmall number of tb< .!• runes doeH itM presence seem to be of any pallioj;eiiii! si>;ni(icance. UbieliNtein's experiinentH Hhowed that colon bacilli injected into the circulation not infre- quently cau«e necrotic foci in the liver, where they may nurvive a long time, and that the bacilli are often elimiiuited by the bile. We found \veek«, and even monthH, after the injection the bacilli in the bile, in which they had induced certain chanjte« miggestive of the causation of gallstone by biliary infections. Dr. Adami and his coworkers have brought forward new observations in support of the opinion that bacteria are very frequently absorbed from the intestinal tract, and that certain organs, particularly the liver, dispose of these bacteria by intracellular digestion. Inder the designation of the "diplococcus form" of the colon bacillus, Dr. Adami describes conditions and appearances of this bacillus, which I judge not to be identical, and it is possible that some better designation miglit be used. I confess to a feeling of some uncertainty as to the exact interpretation of the small coccoid bodies demonstrated by Dr. Adami in the hepatic and other cells, but I ilo not <iues- tion that he lias brought forward to-day and in his previous papers strong arguments in favor of hi.*) conclusion that they are bacterial forms or are derived from such forms. Dr. Adami: This subject opens up to a large Held, and there is yet so much to be done, in order to establish the fundamental points in connection with the reaction of the tissue cells on the colon bacillus under normal and pathological conditions, that I fully acceile to Dr. Welch's statement, that this matter must at the present time be received tentatively. I cannot expect others who have not worked at the subject to accept freely and imme- diately the observations which I have just recorded; indeed, the observations upon this very minute form require very great care. Thus 1 recogiii/e that it may be some years yet before the statements here made receive general acceptance, and I am myself prepared to speml some years yet in working at the subject. I have, however, seen sufficient during my work this last year to feel assured that others must eventually see that my observations are fundamentally correct. R