;05 = 05 = U1 «i = 00 '>' = m = Q DC iO io = ;i 2^ 30 / her_ 3 ■¥ s s ~r a s /o // /2 /? /■f /5 /6 /7 106 /as' /of /03 /02 /of /OO 99' 98' 97 si M r M r Me .. /*f£- n E MF ^ E >>Tf ff£- M£ M £ /^ E M £ HE t^e fts ns »£ /I e ME f^e ME- ME ME ME ME ME ,.l \ M \ 4^ I' %'^ Is 1^ %i \% §^ ^i l| n ,s< 1^ ?l -1 <1^ f i ] 'A / \ A « A h f / \ \ / / 1 n / u I . / /^ f •A / V^ A / 1 / / A ft X r 7- \\ n f\ , A «^ / \ / / / / % /' A A /' / i u / / i W \ y * I V ** i T / / * y V V Chaet I. Portion of the temperature chart of Case No. 25 ; showing a slight but constant reaction after as small a dose aiS \ million iStreptococcus pyogenes vaccine. very considerable benefit in bringing the patient's temperature down to normal and keeping it at that level, in preventing complications such as cellulitis and erysipelas and in improving the patient's general condition. In cases where the wound was inefficiently drained vaccine therapy, as was expected, was quite ineffectual. As regards dosage the study of certain cases appeared to show that when the wound surface was extensive, thus giving a large area of absorption, and also during the period before the surface became covered with granulations, it was necessary to use very small doses. For instance, the examination of the temperature chart of case No. 25, a patient who was suffering from an extensive and deep wound of the thigh, shows that even as small a dose as half a miUion cocci repeatedly caused a distinct but slight reaction a few hours after the dose was given. This case was, however, exceptional and the usual procedure was to give a dose of about 1 or 2 million cocci at intervals of from five to seven days. The dose was slowly increased 13 to 5 and occasionally 10 million cocci ; larger doses than the latter were not often employed as no greater benefit was gained. Several other stock vaccines were employed on occasions, their use depending on the result of the bacteriological examinations ; for instance, B. proteus vaccine in doses of 5 to 10 millions, B. pyo- cyaneus vaccine in doses of 2 to 10 millions. Streptococcus salivarius vaccine in doses of 2 to 10 millions and Staphylococcus vaccine in doses of 50 to 500 millions ; all these vaccines were prepared from different strains of micro-organisms isolated from various patients. (9) The Advantage of using an Autogenous Vaccine in certain cases. On several occasions, in spite of vaccine treatment on routine lines, a patient's temperature or progress remained unsatisfactory Date 26 Z6X>26jO 37 £6' J^/02S/0 3Q 26/oj^jo 30 ^6/oj^a 3/ S c5 2^/a3ii/o F° 1 \ 1 1^ /OS- -^ ■1 ^1 11 5, ^1 /O-f ' '} U ft V 'J^ V /03 / \ % I /OZ V \ \A M t \ M /O/ V V ' is / A A /oa f ^' \ / 1/ ^ V i\ f\ r~\ J< S9 \ I ^ rA A 96 97 Chart II. A portion of the temperature chart of case No. 13 showing the effect of giving small doses of B. pyocyaneus vaccine. The patient had had fever of the same type for the previous three weeks and had been treated without any obvious benefit with two different streptococcus vaccines. until, as a result of careful bacteriological investigation, a different vaccine was employed, when the conditions changed for the better with almost dramatic suddenness. For instance, case No. 13, a patient suffering from an extensive Wound in the neighbourhood of the right scapula, complicated with bronchitis, continued to have a considerable amount of fever even after the lung complications had disappeared. Inoculations of siuall doses of vaccines made from Streptococcus pyogenes which was present in the wound and from a Streptococcus which had grown in one out of ten tubes planted with the patient's blood, had no effect either on the temperature or on the condition of the wound, which was at this time not at all satisfactory. Cultures havmg shown the presence of B. pyocijaneus in large numbers, a small dose of vaccine prepared from the strain isolated from the patient was given. The result was most striking, as the temperature, which had Chabt hi. A portion of the temperature chart of Case No. 54 ; showing the effect of B. pyocyaneus vaccine. The temperature had hecn of the same type for the previous 8 days. remained consistently high since the patient's admission three weeks previously, fell the same day and assumed a lower level. Three days later another small dose was given after which the temperature fell to almost normal, and throughout the remainder of the treatment fever was absent ; at the same time the appearance of the wound and the patient's general condition improved enormouslj^. A portion of this patient's chart is given (see Chart II) showing the effect of the pyocyaneus vaccine on the temperature. Later in the same patient's treatment a marked improvement in the condition of the wound surface was brought about by inocu- lating doses of a vaccine prepared from an unclassified Streptococcus 15 which cultures showed to be consistently present in very large numbers. The next case, No. 54, demonstrates the result of giving small doses of B. 'pyocyaneus vaccine in certain cases infected v/ith this bacillus. The patient was suffering from a fractured femur, which had been complicated with gas gangrene and was suppurating very freely. The temperature about ten days before the treatment had taken a very much higher level, and in spite of everything possible being done to improve the drainage, &c., it remained high. When Chart IV. A portion of the temperature chart of Case No. 8 during an attack of erysipelas, showing the rapid fall which followed the administration of Streptococcus pyogenes vaccine when doses of a vaccine made from another variety of Streptococcus had had no beneficial effect. small doses were given of B. pyocyaneus vaccine, this bacillus being present in large numbers in the pus taken from the deeper parts of the wound, improvement was very rapid and progressive after .the administration of each dose. Chart III shows the effect on the temperature, which very quickly reached the normal line. After this date fever did not return. The last instance that will be given is case No. 8, who shortly after his admission developed erysipelas. A blood culture having given a positive result in one tube, out of the ten planted, of an unclassified Streptococcus, it was thought that this coccus was pro- 16 bably the cause of his condition. A vaccine was, therefore prepared and small doses administered without the sUghtest benefit, it was, therefore, decided to give doses of Streptococcus pyogenes vaccme, this coccus being the common cause of erysipelas and bemg present in the patient's wounds. The result was most satisfactory, the temperature quickly faUing to normal and remammg at that level. (10) The operation of secondary suture. Our small experience served, so far as it went, to confirm that obtained on a much larger scale elsewhere. In particular we learned to lay great stress upon the choice of time at which secondary suture should be performed. The moment when sloughs disappeared and the surfaces of the wound took on a raw-meat appearance generally coincided with a reduction of the bacteria to a minimum. At this time also the exposed surfaces were only too ready to unite when brought into apposition, and the newly-formed granulation tissue was still soft and supple enough to permit of closure of the wound with but very little tension on the sutures. When the operation was performed under these optimum condi- tions our experience was that wounds could be successfully sutured which showed a much larger number of bacteria per field of the microscope than Carrel has laid down as permissible. We also found that the presence of streptococci did not preclude a successful result.^ Subsequent work published by Wright, Fleming, and Cole- brook throws light on these findings and the general experience of secondary suture operations. The leucocytes found in wounds are shown to have enormous power of killing all the bacteria which infect war wounds, not excepting the streptococci, but this destruc- tion cannot be counted upon except in the following circumstances : the leucocytes must be present in large numbers and must have recently emigrated ; they must be protected from the inimical influence of desiccation, and — most important of all — the bacteria must not be allowed access to pools of fluid, in which they can multiply out of reach of the leucocytes. In an open wound there is usually no lack of fresh leucocytes and it is not difficult to give them protection from drying— on the other hand, the surface of such a wound is almost never without crevices and pockets which serve for the collection of serous fluids, and for this reason probably it is quite exceptional for such a wound to completely sterilize itself. When, however, its surfaces arc brought into close apposition by sutures, not only are the leucocytes protected from desiccation and their concentration doubled at every point, but — if the operation is undertaken when the tissues are supple and very ready to unite — all the crevices and pockets, commonly referred to as dead spaces, are obliterated. The success of secondary closure operations, in our judgement, depends chiefly . upon the_ completeness with which the dead spaces are eliminated ; and this, in turn, upon the surgeon's skill ia effecting a close apposition of the tissues without undue tension upon the sutures, and — a factor too Httle emphasized— in the application of uniform pressure bv firm bandaging after the operation. If the closure is attempted ' Wright, Fleming, and Colebrook, Lancet, 1918, i. 831. 17 ■while sloughs are stilt adherent to the wound, they will not only constitute dead spaces in which serous fluid will collect, but also, since the sloughs are foci of bacterial growth, will ensure the pollution of the fluid and thus lead to certain failure of the operation ; and the same result is to be expected if the operation is deferred until cicatrization of the tissues compels the surgeon to put undue tension upon his sutures. One point remains to be made here : Fleming has shown by means of bio-cultures (vide Wright, Fleming, and Colebrook, Lancet, 1918, i. 831), made at intervals after the dressing of an open wound, that the conditions are most in favour of sterilization by the agency of leucocytes between four and eight hours after dressing. The secondary suture operation should, therefore, be undertaken during that period. (11) The persistence of living bacteria in scar tissue and seguestra. The question of bacteria remaining alive in scar tissue has also very important bearing on the performance of operations such as the resection of joints, the plating of fractures that have united in bad position, and the suture of nerves after suppurating wounds have healed ; and not only has it been impressed on the present authors during the present investigations, but also by facts brought to their notice from time to time in the past, that streptococci and other bacteria may remain embedded in scars without producing any symptoms for months and perhaps years. These bacteria, although they may lead to no symptoms for long periods, are capable of producing very active suppuration if the scar becomes injured in any way, such as being opened up during an operation, and it is not jmcommon to hear of a joint which has long before been the seat of a bacterial infection becoming intensely inflamed or even suppurating after violent movements made to break down adhesions. One of us can recall a case where a gunshot wound of the right fore- arm, which had been healed after much suppuration for over five months, was operated on in the hope of obtaining union in an ununited fracture of the ulna ; twelve hours after the operation the wound had become very septic and an abundant culture of Streptococcus was obtained from the discharge. Another point bearing on this subject brought to the notice of "the same individual is, that horses used for the preparation of anti- streptococcic serum by injecting cultures into the muscles of the neck, will, months after the injections have been stopped, develop abscesses due to streptococci at the site of the former inoculations if from any cause their condition becomes poor. During the present investigations it was found that, whenever sequestra were removed from a patient who had suffered from gas gangrene, cultures of B. welchii and other anaerobes and aerobes could be always obtained from the sequestrum even though the ' B. welchii and other bacteria had been absent from the discharges for many months. After the sequestra had been removed from the body and had become quite dry, the bacteria still remained alive. Thus from a sequestrum which had been removed from the body 1727 B 18 and kept quite dry for two years, not only were spore-bearing bacilli like B. welchii and B. sporogenes easily cultivated, but non-sporing micro-organisms such as B. pyocyaneus and Streptococcus were found to be living in considerable numbers. It is probable that sequestra lying in the body will be the resting place of bacteria for very long periods, and even though the wound may heal without the sequestrum being extruded,, the bacteria will remain alive and capable of pro- ducing very unwelcome effects if the surrounding tissues are in any way injured or even if the patient's general health deteriorates. From a study of these facts it is obvious that operative inter- ference should not be undertaken too lightly in the region of wounds which have healed after suppurating freely, and that as long a period as possible should b5 allowed to elapse between the healing of the wound and the carrying out of the operation. Further, as far as is possible the scar tissue should be left uninjured, the necessary incisions being made through the healthy tissue. How far the danger of suppuration following such operations could be lessened by such measures as massage of the scar, gentle at first and gradually becoming more vigorous, together with inoculation of vaccines made from Streptococcus pyogenes, has not been worked out, but both these procedures appear to be worthy of trial. B. GENERAL OBSERVATIONS ON THE BACTERIA FOUND IN THE WOUNDS (1) The faecal origin of wound infections. The results of the bacteriological investigations on those cases which arrived in this country shortly after the infliction of the wounds showed, as had been pointed out by one of us (A. P.),i that the primary infection is largely made up of faecal bacteria. Woirnds in this stage practically always yielded cultures of B. welchii and other Spore-bearing anaerobes ; associated with them were B. proteus,. Streptococci (generally of the faecalis or enterococcus types, but even, in this stage Streptococcus pyogenes was sometimes found), varieties of diphtheroids. Staphylococci, and tetrad cocci. As the wounds cleaned under treatment the bacteria became fewer and fewer in number and the anaerobes quickly disappeared, so that after a few days they were only to be found m sloughs and other dead tissues. When the wound had quite cleaned and had reached what has been frequently described as the ' raw meat stage " the bacteria present in the discharge were usually extremely few m number and, as has been mentioned above, this was a very favourable period for the performance of secondary operations. (2) The importance of Streptococcus pyogenes. As the wound surface became covered with granulations and any cavities or irregularities were filled up with granulation tissue the number of bacteria tended rather to increase, and day by dav » Fleming, Lancet, 1915, ii. 638. 19 it became more and more evident that Streptococcus pyogenes was present in practically every septic wound and was able to resist the bactericidal action of the body fluids and cells to a much greater degree than any of the other babteria present. It was this coccus that was responsible for nearly every complication that occurred in the cases during their stay in the wards of the Inoculation Depart- ment, the commonest of these being erysipelas, the formation and persistence of sinuses, and also such constitutional symptoms as fever. This coccus having been once found in a wound generally remained there, though frequently in very small numbers, until the wound was completely healed. It was during the later stages of the healing process that films made from the discharge from the wound showed practically no bacteria, but on making cultures, especially if. these were incubated under anaerobic conditions, one was often surprised at the number of Streptococcus pyogenes colonies that developed. Associated with these streptococci were usually other micro-organ- isms ; the commonest were Staphylococcus and diphtheroids, but B. proteus and B. pyocyaneus were also present at times. If during the healing process the surface of the wound showed portions where the granulations had an unhealthy aspect, it was usually found that the films made from the discharge taken from unhealthy looking patches showed that the bacteria were very much more numerous in thesS situations than in the discharge taken from the more healthy portions of the wound. (3) The recrudescence of bacterial infections after operations. After such operations as amputations by the flapless method or the removal of sequestra from the neighbourhood of fractures the number of the bacteria present on the wound surfaces increased greatly, so that twenty-four hours after the operation films made from the discharge showed enormous numbers of bacilli. It was also found, especially after the removal of sequestra, that bacteria which had long been abselit from the discharge again made their appearance ; thus a wound in connexion with a fractured femur which contained before operation only a very few bacteria, chiefly Staphylococcus and B. proteus, two days after the operation for the removal of a sequestrum showed enormous numbers of micro- organisms, amongst which were Streptococcus pyogenes, diphtheroids, and B. sporogenes. The latter had not been found before, although numerous examinations had been made since the patient was wounded six months previously. The explanation of this phenome- non is, as has been shown above, that the bacteria contained in the cavities of sequestra remain alive but quiescent for many months, but when the site is disturbed during the operation all the bacteria present tend to proliferate and infect the surrounding tissues. Under treatment, however, they rapidly disappear and the wound returns to the former condition, in which the discharge contains only a very few bacteria, the commonest and most important being the Streptococcus pyogenes. 20 PAET II A DETAILED ACCOUNT OF THE CHARACTEKIS- TICS OF THE VARIOUS BACTERIA ISOLATED FROM THE WOUNDS (1) Introduction— and routine procedure employed in studying the bacterial flora of the wounds. The routine technique employed for studying the bacteriology of the wounds was as follows : n j. j As soon as the case was admitted samples of pus were collected and from these (i) films, and (ii) cultures were made. (i) The films were stained by Gram's stain and counterstamed with either neutral red or weak watery fuchsin, and the results of the examinations were so recorded as to give the conditions of the pus cells, the amount of phagocytosis, and the types of the various bacteria in order of their prevalence. (ii) Cultures. Two series of cultures were made from each sample of pus, one from the unheated pus, the other from pus which had been heated to 65° C. for thirty minutes. The temperature of 65° C. was employed in preference to that of 80° C, which has been recom- mended in the past, as, while it was found to kill off satisfactorily all the non-sporing micro-organisms, with the exception of an occasional variety of streptococcus,'- it did not prevent the spores from rapidly growing out when placed under favourable conditions ; whereas, when the pus had been heated to 80° C, many of the spores appeared to be so damaged that the resulting growth even on the most favourable media was very scanty. The media and type of cultures used in each series were as follows : (a) Glucose agar plates. (b) Milk tube. (c) Trypsin-blood-broth tubes. The glucose agar was prepared from trypsin broth described by one of us (S. E. D.^), to which 0-25 per cent, of glucose had been added. This trypsin broth is a much better basis for solid media than broth prepared from meat extracts and dried peptones. The trypsin blood broth was made by adding 1 c.c. of fresh human blood and 0-25 c.c. of the Inject. Trypsin Co. (Allen & Hanbury) to 5 c.c. of the trypsin broth mentioned above. This medium has proved a very favourable one for nearly all micro-organisms, and its use has frequently enabled an abundant culture to be obtained when little or no growth developed in ordinary broth.^- * One glucose agar plate and one trypsin-blood-broth tube made ' It has been found that occasionally streptococci from wounds resist even a temperature of 80° C, and Simonds has shown that this also holds for some strepto- cocci in faeces, a result which has been since confirmed. ' Douglas, Lancet, 1914, ii. 891. ' Douglas and Colebrook, Lancet, 1916, ii. 180. • From observations made subsequently to those included in this paper we do not consider that this trypsin blood broth is a better medium for growing the spore bearing anaerobes than is a medium consisting of minced meat and water rendered 21 from the unheated pus were incubated under aerobic conditions at 37° C, the remaining glucose agar plate and trypsin blood broth tube, together with a milk tube made from the unheated pus, and the whole series of the cultures made from the heated pus were incubated under anaerobic conditions — the method used being the paladium-black fitted tins described by Mcintosh and Fildes.^ After a period of incubation the plates were examined and the various colonies picked off and planted on trypsin broth agar so as to obtain pure cultures. The milk tubes were of little use except to demonstrate the presence of B. welchii by the production of the typical clot in twenty-four hours. The trypsin blood broths were planted out on to glucose agar plates, after films had been examined to give some idea of the number and variety of organisms that had developed, and the resulting plates were incubated aerobically or anaerobically according as the original fluid cultures had been incubated. After this further incuba- tion the plates were examined and the various types of colonies were picked off and planted on to trypsin-broth-agar slopes. Further samples of pus were collected from time to time and the various micro-organisms isolated from them. By these methods a large number of pure cultures were collected and classified. They will be discussed in detail under the following headings : (1) The anaerobic bacteria. (2) Streptococci. (3) Diphtheroids. (4) B. proteus. (5) B. procyaneus. (6) Staphylococci. (7) Various coliform bacilli. (2) The anaerobic bacteria. (a) General Considerations. Prior to 1914 the literature on the spore bearing anaerobic bacilli was voluminous but very confusing, and it has become evident that much of this confusion was due to the fact that in many cases the cultural characteristics were described from impure cultures. Thus even such a well-defined bacillus as B. welchii was described with characteristics quite foreign to it. In describing the technique employed for the isolation of the various micro-organisms present- in the pus, attention is drawn to two points : (i) when the pus was heated to destroy the non-sporing bacilli a temperature of 65° C. was not surpassed ; (ii) the media used for making the plates — trypsin-broth-agar — is capable of giving a much more abundant growth than that made from meat extract and commercial peptone. It is believed that attention to these details proved an important factor in obtaining pure cultures of the slightly alkaline to litmus. Thia medium is very easily prepared, and it has the advantage that it reveals the colour changes produced by the action of the different microbes on the meat. The meat medium also is of the greatest use for storing the cultures. ' Mcintosh and Fildes, Lancet, 1916, i. 768. 22 various anaerobic bacilli, since owing to a very much smaller implantation of the pus being necessary to obtain a satisfactory number of colonies on the plate, the chances of other spores being included within the limits of the colony were greatly lessened. It was foimd to be much easier to obtain pure cultures direct from pus heated to 65° G. than from mixed cultures obtained by planting pus into fluid media, and the latter method was only used when the plates made directly from the heated or unheated pus failed to give growths of certain bacilh which had been observed in the examination of stained films. Before passiug on to describe the various anaerobic bacilli the role played by them and the period duriug which they were present in the wound will be discussed. As has been pointed out by one of us (A. F.), the primary infection of all the wounds is largely a faecal one, and the vast majority of the micro-organisms found in the pus during the early stages of the infection consist of various anaerobic bacOli, coliform bacilli, and streptococci. They are, during the earhest stages, confined to the 'damaged area, multiplying with extraordinary rapidity owing, partly at any rate, to the beneficial action of symbiosis. It has been shown by the present authors ^ that, when mixed with these other bacteria, an abundant culture of B. welchii will often develop, although the number of bacilli implanted was 10,000 or even 100,000 times smaller than that found necessary to produce a pure culture in the same serum. It is during the stage of very rapid proliferation of the micro-organisms in the damaged area that gas gangrene occurs by the invasion of the tissues by certain anaerobic bacilli. "When under efficient treatment the wound is cleaned up, or when in those cases where gas gangrene had developed that process is arrested, all the anaerobic bacilli rapidly disappear, so that even in a few days they may become comparatively very few in number and have usually completely disappeared from uncomplicated flesh wounds in ten or twelve days. IE, however, any bone has been injured or sloughs remain attached to the tissues, the various anaerobic bacilli can be almost invariably found, and in the cases where bone sequestra are present the common anaerobes can be isolated from the necrosed bone months or even years after the infliction of the wound. Sometimes these anaerobic bacilli cannot be recovered from the pus exuding from the wound, even when there is a sequestrum in situ in which they can be shown to be present. In the further study of the anaerobic microbes the pure cultures Tvere planted on trypsin broth agar, trypsin broth, and also on these media with the addition of glucose. In addition to the growth on these media the action of the bacilli on neutral red egg medium was noted ; the fermentation reactions were also investigated by growing the bacilli in broth prepared from casein, as suggested by Cole and Onslow^, to which the various sugars had been added. The appearance and power to liquefy gelatine cultures was also recorded. The neutral red egg medium was introduced by one of us (A. F.) in 1908, and is prepared exactly as is Dorset's egg medium with the ' Douglas, Heming, and Colebrook, Lance', 1917, i. 604. " Cole and Onslow, Lancet, 1916, ii. 9. 23 addition of 1 per cent, of a 1 per cent, freshly prepared watery solution of neutral red. It should have an orange colour and a firm dry surface. The formation of acid and the action of many of the bacteria on the neutral red produce a variety of colour effects which often prove helpful in identifying micro-organisms and ascertaining if the culture is pure. (b) Classification. . With one exception the anaerobic bacteria which were isolated from the various wounds can be grouped in the following three classes: I. The non-sporing bacteria, (i) Cocci. (ii) Diphtheroids. II. The non-putrefactive spore bearing bacteria, (i) B. welchii. (ii) B. tertius. (iii) B. cochlearis. (iv) B. butyricus. (v) B. sphenoides. (vi) An unnamed bacillus isolated from two cases, known as Sindrey I and Liddicoate I. (vii) An unnamed bacillus morphologically resembling B. tetani isolated on one occasion only, known as Tholby II. The general characters of this group are that their power to digest coagulated albumens is relatively slight, while in most cases they are able to split up many of the carbohydrates commonly used in testing the fermentation reactions of bacilli. The fact that several have considerable power of splitting up uncoagulated proteids must not be lost sight of ; for instance, it is widely recognized that B. welchii growing in serum splits up some of the proteid and produces large quantities of gas. III. The putrefactive group of spore bearing bacteria, (i) B. sporogenes. (ii) An unnamed bacillus isolated on one occasion only, known as Cullingford 3 C. (iii) An unnamed bacillus isolated on one occasion only, known as Cullingford 3 B. (iv) An unnamed bacillus isolated on one occasion only, known as Cullingford X. (v) An urmamed bacillus isolated on one occasion only, known as McFadzean I. (vi) An unnamed bacillus found on one occasion, known as Hancock II. The general characters of this group are that the bacilli have the power not only of breaking down and producing intense putrefaction in uncoagulated proteids, but also of rapidly liquefying and putrefying coagulated albumens. ^As a group they have little action on the carbohydrates. There remains but one bacillus which was isolated on one occasion only and was known by the laboratory name of Boone I ; this bacillus possesses characteristics which make it impossible to 24 place it in any of the above groups ; it appears to occupy an inter- mediate position. (c) The Non-spoeing Anaerobic Bacteria. This group has been very imperfectly worked out, and practically nothing is known of the role played by it in the infection of wounds. (i) Cocci. ' Forest's ' anaerobic tetrad. This microbe was isolated from a glucose agar plate which had been planted directly from the pus immediately after the patient's admission to hospital. Microscopic examination showed it to be a coccus arranged in regular groups of four, or masses of such groups ; in size it was similar to Staphylo- coccus ; Gram's stain was retained. At times very many of these cocci were seen ia films of the pus, each group of four being surrounded by a well-marked capsule. On agar and glucose agar slopes it grew hi very minute rounded flat transparent colonies which were difficult to see without the aid of a lens. These cultures died out in four or five days. No growth ever took place under aerobic conditions, and in glucose agar shake cultures no colonies developed in the upper centimetre of the medium even after the organism had been frequently subcultured during a period of several weeks. Tholhy's anaerobic tetrad. The morphology of this organism was similar to the above. The colonies on agar and glucose agar were, however, quite unlike those of the ' Forest ' organism, being round, heaped up, and very opaque in size, similar to a colony of Strepto- coccus pyogenes. This coccus retained its anaerobic properties for several generations, but later it was found to have acquired the property of growing aerobically. Harris's anaerobic coccus. This microbe was isolated from a glucose agar plate planted directly from the pus immediately after the patient's admission to hospital. Morphologically it was a coccus, in size and shape similar to a Staphylococcus. Gram's stain was however not retained. The colonies on agar and glucose agar resembled those of Streptococcus pyogenes. Stab cultures gave good growth in the depth of the tube but none within 1 -25 centimetres of the surface. Broth cultures showed a considerable granular deposit with shght clouding of the medium. On neutral red egg a fine growth was observed after 24 hours' incubation, but no reddening of the medium occurred. No growth took place on gelatine at 20° C. Milk was neither clotted nor rendered acid. Planted into broth containing various carbohydrates, acid was produced in that containing cane sugar, but glucose, maimite, glycerine, raf&nose, salicin, inulin, dextrin, starch, and dulcite M'ere unaffected. (ii) Diphtheroids. Sindrey's anaerobic diphtheroid. This was a long slender bacillus. Gram-positive, and non-motile. Its arrangement was of a diph- theroid character. Cultures rapidly died out and the strain was lost before it could be examined thoroughly. This was probably the same bacillus as Wright i and Fleming ^ described under the name of ' Wisp ' Bacillus. 1 Wright, Brit. 31. J., 1915, i. C29. = Fleming, Lancet, 1915, ii, 638. ((?) The Non-putrefactive Group of Spore-bearing Anaerobic Bacilli. (i) B. welcJiii, B. perfringens or B. aerogenes capsulatus. The ordinary cultural characteristics of this bacillus are so well known that they will be only briefly referred to. On agar the colonies are rounded, somewhat opaque with a granular texture, the edges smooth and regular. In broth an even turbidity is at first produced, but this condition is followed by a sedimentation of the bacilli which, after a few days, leaves the supernatant broth quite clear. Milk is rapidly clotted, the clot being of a peculiar spongy texture due to its being broken up by gas formation. No digestion FiC4. I. Forty-eight hours culture of B. ivelchii in serum. of the clot ever occurs. On neutral red egg bright yellow colonies are formed in 24 hours, the surrounding medium being crimson ; this is due to the bacillus destroying the dye in the immediate neighbourhood of the colony while the surrounding red colour is due to the formation of acid. Even after months no digestion of the egg can be demonstrated. Morphologically the bacillus is a short stout rod with slightly rounded ends, retaining Gram's stain. It is non-motile. Its length varies greatly according to the media on which it is grown ; thus films made from cultures grown on egg medium show a bacillus almost as broad as it is long, those from cultures on agar are considerably longer, while the blood agar cultures show still longer organisms. No growth takes place on gelatine at 20° C. Fig. I shows the appearance of a culture grown in serum for 48 hours. Sjwre formation. It has been commonly stated that B. tvelchii is incapable of forming spores when cultivated in a medium con- 26 tainmg a carbohydrate which is fermented with the production of acid dnvinff the srowth of the bacihus. The present Authors have been unable to demonstrate micro- scopically spores m young cultures grown either on broth or agar, H such cultures were not sterilized by exposure to ^ temperature of 60° C.-a temperature quite sufficient to kill the vegetative forms, so It must be presumed that spores are formed. In broh containing 0-5 per cent, glucose spore formation could never be detected, and such cultures were invariably rendered sterile by heatmg them to 60° C. for one hour. . ^ ,, . , -n ^;i „ When investigating the fermentation reaction of this bacihus, tilms were made from the various cultures and examined microscopically ; it was then found that the bacilli grown in broth containing 1 per cent starch showed ^■erv abundant spore formation although tlie Flu. II. Twenty-lour hours culture of B. nxkhii in starch broth. media gave a very strong acid reaction. This phenomenon was a constant one in the six strains that were examined, but the number of baciUi forming spores varied considerably. In three of the strains sporulation was extremety abundant, so that in 24 hours at least half the bacilli could be demonstrated by staining to have formed spores. See Fig. II. The remaining three strains showed a somewhat different picture, most of the bacilli having become swollen and staining in an irregular manner so that portions of the bacillus remained uncoloiired. At times these unstained portions were oval in shape and simulated a spore, but they did not retain the spore stain. Fig. Ill shows the appearance of these organisms. In every culture, however, some typical spores could be demonstrated by staining, and in the case of all the six strains starch broth cultures 5 months old when heated to 80° C. gtive abundant growth when planted into milk, showing conclusively that many spores had been formed. In broth con- Im<.. III. 4,S]i..ni-s- ciiltinv ,,[■ Jj .n-l^hn ill j.ntato l-'rijUi, ^liowiii^ i iT('L;ii)ar stain i ml;. fC ', "^ 1 %m ^ \ •t j Lc3 i 2^ Fic: IV. Aty])iral colcmiets "f /;. /it/, ///; ,111 :mar. 'I'lie \\vr,- iihtaiiiei.l from one sti-aiu ,,iiiv. Fig. V, At.\i,i,M! tnriii, ,,| /;. „,/,/,„ tin- ^aiii, strain a^ tliat ^li,.uii in I'lt;. [y. 27 taining 1 per cent, dextrin the bacilli assumed the same swollen appearance and irregular staining properties, but spore formation could not be demonstrated. One of us (S.E.D.) has shown that spore formation is very rapid and profuse when B. ivelchii is grown in serum "symbiotically with Staphylococcus, and Simonds observed spore formation even in media 'containing carbohydrates capable of fermentation, when the bacilli were grown in symbiosis with other faecal micro-organisms. Some spores can always be demonstrated in serum cultures several days old. Viability. Cultures in glucose broth, milk, or on glucose agar die out in about a week. Even in ordinary broth most of the cultures were found to be incapable of subculture after two or three weeks, but marked exceptions of this rule were noticed ; for instance a broth culture made in Boulogne in 1914 gave an abundant growth when planted on neutral red egg medium exactly two years later. Cultures on serum, broth containing starch, on solidified egg and any other medium containing a large proportion of albumen remain capable of being subcultured for very long periods owing to spores being formed. The spores appear to be very resistant. Thus a culture was obtained from a vaccine made three years previously by heating a 48-hour culture to 60° C. for one hour and then adding 0-5 per cent, carbolic acid. One strain of B. ivelchii which was isolated from one of the cases deserves special mention on account of peculiarities both as regards the morphology of the bacillus and the appearance of the colonies. These, instead of being evenly rounded and granular in appearance, showed a marked thickening at the edge, which usually commenced at a certain point of the periphery, and spread all round, leaving the centre thin and much more transparent. Fig. IV shows the appear- ance of such colonies. Morphologically the bacilli after 24 hours' growth appeared larger than normal and with the end more rounded. The length and thickness of individuals varied, but long forms were more common than usual, curved forms were frequent, and there was a tendency to form chains. There were also present many large crescent-shaped deeply staining forms, and bacilli could be seen in all stages from normally shaped organisms to these remarkable forms. The crescent-shaped organisms were probably involuted bacilli. They are shown in Fig. V together with stages of transition between this condition and the normal forms. When the culture was examined by dark ground illumination many of the bacilli were seen to have four or five bright refractile spots imbedded in them, which varied considerably in size, sometimes even distending the bacillary body. If such a specimen was stained by' the common method used to demonstrate spores the refractile spots retained a pale pink colour, but an emulsion of these bacilli exposed for an hour to a temperature of 60° C. became sterile, thus proving that the spots were not true spores. Apart froih the above peculiarities this organism gave all the fermentation reactions of B. ivelchii and also the typical clot in milk cultures. 28 Incidence. B. welchii was isolated from fourteen of the 61 cases investigated. The number of days after the infliction of the wound that the bacillus was isolated, a positive or negative history of gas gangrene, and the presence or absence of bone injury are in each case given below : ' Table I. Case. No. of days after infliction of wound that bacillus was isolated. History of gas gangrene. No. 10 „ 11 „ 13 6 6 and 55 5 Negative Negative Positive „ 29 „ 34 22 from sequestrum 8 Positive Positive „ 38 „ 39 8 8 Negative Positive „ 43 „ 45 150 from sequestrum only 13 Negative Positive „ 48 28 Positive „ 53 13 Positive „ 54 26 Positive „ 56 31 Positive „ 60 7 Positive Presence or absence of injury to bone. + + + It is to be noticed that in the case No. 29 who had a very extensive wound of the buttock involving the whole right sacro-iliac joint and who had had a gas infection, the pus from the wound obtained 15 days after the infliction showed neither by film nor culture any B. welchii. On the 22nd day a large sequestrum was removed and from this B. welchii was readily isolated. This sequestrum was allowed to dry at room temperature, and three years later cultures both of Streptococcus pyogenes and B. welchii were readily obtained. Fermentation Beactions of B. welchii. The following table gives the reactions of six strain's : Table II. Cabeohydkates. Name of Strain. 1 1 1 8 s C5 1 .g 1 .8 1 ! is s Laboratory stock. MacFadzean + + + + '0 + + + + + ?0 + + + + + Grout . . . . Cation + + + + + -r + + + + + gas only. gas only. + o Hancock . . + + + + - + + Lockhart . . + + + + — + + gas only. The sign + indicates the production of acid and gas ; o that the carbohydrate was unaffected ; — that the test was not carried out. 29 Two of the strains fermented inulin with the production of acid and gas, three other strains produced a quantity of gas in tlie presence of inuHn without the production of any acid being observed. Mannite, sahcin, dulcite, and raffinose were not fermented by any of the strains. (ii) B. tertius. This name was applied by Henry -"^ to the liacihus as it was the third most common in his first series of cases. (It has since, however, been shown that it is not in reahty the third commonest in these wounds, so the title is somewhat inapt, although it is more satis- factory than the various titles under which it has been described in the'past, viz. B. Hibler IX, B. Eodella III, and B.Y. (Fleming).) Hibler - believed that this organism was a cause of emphysematous gangrene, but although it is present in the lesions of a number of Ten days culture of B. tvrtiiis. cases suffering from gas gangrene there is very little evidence that it is the causative agent. In the past B. tertius has been not infrequently mistaken for B. tetani when attempts were made to diagnose the presence of the latter by film preparations. The principal points in which it differs from B. tetani is that the spore is very large and oval and has a much thinner ' capsule ' and that the bacillary bodies are more slender and frequently curved. Morphology. The bacillus is long and thin, sometimes curved and sometimes straight. It is motile and retains Gram's stain. It spores rapidly on all media after 48 hours' incubation, sporing forms being very abundant. The spores are situated terminally and are very large, being much broader than the body of the bacillus. Fig. VI shows the appearance of a ten-day old culture on egg medium. > Henry, ./. Path. & Bacteriol, 1916-17, 21, .344, "- Hibler, Jena, 1908. 30 CulMral characteristics. On agar after 48 hours' incubation the colonies are round, semi-transparent discs up to 2 mm. in diameter with slightly heaped-up centres and a marginal area slightly but distinctly crenated. Broth cultures frequently fail to develop, but when successful the medium is evenly clouded. On red egg small yellow colonies are developed on a brick-red field, the colour con- trasts being much less striking than in the case of B. welchii. There is no trace of digestion of the albumen even after 5 or 6 weeks' incubation. Stab cultures in gelatin after 10 days' incubation at 20°-22° C. give a beautiful arborescent growth simulating the traditional growth of B. tetani in this medium. No liquefaction and no gas formation were observed. Milk cultures are rendered acid in 24 hours, and this is later followed by a firm, uniform clot. Gas formation is not obvious and the clot remains undigested. Fermentation reactions. Table III gives the results obtained with three strains : Table 1 III. Name of S s a '8 J .g s .s 1 s 'S Strain. s S 1 S 1 Si 1 1 C3 s s *^ °a ^^ tt Cl CQ € q Forrest I + + + . , acid in 24 hrs. alk. in 48 hrs. + Cullingtord + -1- + + -1- -1- + + VIII Bloor + + o + oo-(--l--(-o acid in 24 hrs. alk. in 48 hrs. It is to be noted that the first strain, Forrest I, has rather less power of splitting up the carbohydrates than the other two. Further, that two strains, one in the case of glycerine and the other of rafiinose, produced an acid reaction at the end of 24 hours, which was replaced by an akaline one after 48 hours' incubation. Incidence. B. tertius was obtained from eleven cases out of the 61 investigated ; the period that had elapsed since the wounds had been received, the positive or negative history of gas gangrene, and the presence or absence of bone injury are given in the following table : 31 Table IV. No. of days after infiicUon of wound History of Presence or absence Case. that bacillus was isolated. gas gangrene. of injury to bone. STo. 1 17 Positive + „ 2 9 and 14 Positive + .. 5 19 Negative + „ 8 11 Positive „ 12 5 Positive — „ 20 77, 146, 151, and 302 ; the last 3 were from sequestrum. Positive + „ 29 15 and 22 from sequestrum Positive + „ 32 61 Positive + „ 34 8 Positive + „ 38 8 Negative — „ 39 8 Positive — In films of pus it appeared generally as long thin straight or curved bacilli, only an occasional spore-bearing form being seen;: in two cases, however, such spore-bearing forms were very numerous, and in films made from a small slough from one case the appearance of the film resembled a preparation made from a pure culture. (iii) B. cochlearis. This name has been applied to the bacillus because of the likeness in shape of the fully developed sporing bacillus to a spoon. It was isolated from one patient (case no. 32). Morphological characters. In appearance it is very similar tO' B. tertius, being a long, slender. Gram-positive, motile bacillus which forms large oval terminal spores. The spores are formed with less freedom than in the case of B. tertius, so that in a 48-hour agar culture very few are to be seen, although after ten days sporulation was abundant. The bacillus does not, like so many of the anaerobic spore-bearing bacilli, degenerate soon after the spore is formed ; thus for a considerable period large numbers of fully developed sporing forms are to be seen which resemble straight-handled spoons. It is actively motile and this is in quite sharp contrast to the sluggish motility of B. tertius. Cultural characters. The growth is relatively sparse on all media. On agar the colony is small, flat, and very transparent with slightly crenated edges. It shows no tendency to spread over the surface of the medium. In broth growth takes place slowly and produces an even clouding of the medium. On coagulated egg medium no digestion is visible and there is no putrefactive smell. In milk there is no clotting or acid formation. Fermentations. This organism fermented none of the test sugars. In this it differs markedly from B. tertius, which is a powerful carbohydrate fermenter. [In three cases a bacillus was isolated which, when grown anae- robically, gave a colony on agar and also film preparations exactly similar to B. tertius. (Fig. VII b). This bacillus, however, grew as well aerobically as anaerobically. In all three cases it was found associated with B. tertius. It fermented glucose, lactose, saccharose,. 32 dextrin, starch, salicin, mannite, and glycerin with the formation of acid and a small quantity of gas.] Fig. VII a. B. coddec, rii;. VII b. Bacilli resembling B. Ii if ins but growing aerobieally. (iv) B. hutijricus. This bacillus was isolated on one occasion only from a patient who had received a severe wound of the thigh complicated with 33 a compound fracture of the femur sixteen days previously. Gas gangrene developed shortly after the infliction of the wound but had been arrested by extensive incisions. Its general characters are very irregular, but it always has the power of fermenting many carbohydrates with abundant gas formation. Morphology. It is a medium sized Gram-positive bacillus, somewhat fusiform in shape, with blunted ends. It forms spores rapidly on all media ; the spore is large and oval, situated in the centre of the body or rather nearer one end. In most media the bacilli are non-motile, but a young culture in minced meat medium showed actively moving bacilli. In preparations made from cultures on egg medium the bacillus is constantly seen to take the form of threads, so long as to stretch across the whole field of the microscope. These threads show no signs of division into segments, but large irregular fusiform swellings can be noticed at intervals ; these do not retain the usual spore stains. Cultural characters. On agar round, semi-opaque colonies were produced in size up to 2 mm. in diameter. These colonies showed iDut little tendency to spread, but if planted thickly a confluent growth was formed. In broth and glucose broth a copious, even cloud was developed in 24 hours ; later multiplication ceased, and the bacilli settled to the bottom of the tube, leaving the supernatant fluid quite clear. On neutral red egg the growth resembled that of B. loelchii. In milk both acid and clot were produced, and the appearance of the culture was very similar to one of B. welchii ; microscopical examination, however, showed that abundant spore formation had taken place, which would at once differentiate the bacillus from B. welchii, which never forms spores in milk. Gelatin stab cultures incubated at 20° to 22° C. show at first a white line of growth along the track of the needle with some irregular prolongations into the surrounding medium. Later the gelatin is broken up by gas formation. No liquefaction was ever seen. The following fermentation reactions were recorded : glucose, lactose, saccharose, glycerin, starch, raffinose, and salicin were all fermented with the production of acid and gas. Mannite, dulcite, and inulin were unaffected. This bacillus appears to be quite unimportant, the only point which should be borne in mind is that the naked eye appearance of the milk culture is very similar to that of B.' ivelchii. (v) B. sphenoides. This name has been given to a bacillus, isolated from wounds, on account of the wedge-shaped appearance of the fully developed sporing bacillus. Morphology. This is a small motile Gram-negative bacillus, fusiform in shape and often arranged in pairs placed end to. end. The spore when first formed is subterminal but as it enlarges it takes & terminal position ; ' in shape it is perfectly round and of large size, 1727 • 34 being considerably broader than the body of the bacillus. When the spore reaches its maximum development the body of the bacillus is at first seen to be wedge-shaped, tapering from the spore to a point ; later the body contracts near the spore so that the appear- ance becomes more drum-stick-like, but the end opposite the spore always remains pointed. Cultural characteristics. On agar the colony is small and rounded or very slightly irregular, about 1 mm. in diameter. In broth growth is profuse, an even cloud being produced. On neutral red egg medium, at first yellow colonies surrounded by a brick red area are seen, at the end of a week the whole surface becomes brick-red. In milk, acid is produced in 24 hours, a day or two later clotting takes place. The clot is firm and contracts very little. One strain produced acid in 48 hours, but no clotting took place even after six weeks' incubation. In gelatine stab cultures incubated at 20° to 22° C. rather small woolly colonies are produced along the needle track. After further incubation, the medium around these colonies is blackened, producing a very characteristic appearance. Fig. X shows this condition. There is no liquefaction of the gelatine. The fermentation reactions of three strains are shown in the following table : Table V. Name of Strain. i 3 1 1^ 1 "w Q e 1 ^ § Ingram I + + + + + + 4- Acid. Clot. Tholby IV + -1- + + o + + + Acid. Clot. Kilby I + + — + Acid. No clot. It is to be noted that the third strain, Kilby I, had a markedly inferior power of splitting up the various sugars, &c., employed in these tests. Incidence. B. sphenoides was recorded as present in five cases, but was only isolated in pure culture from three. All these patients had had gas gangrene. The following table gives the number of days that elapsed between the receipt of the wound and the finding of the bacillus : Table VI. Case. No. 9 „ 21 „ 4 „ 20 „ 39 Period that elapsed from infliction of wound to finding of bacillus. . 25 days . 16 days . 24 days 5 months (from sequestrum) 8 days Pathogenicity. No experiments have been made to determine the pathogenic properties of these baciUi. Fio. VIII. Forty-eight hours culture of B. spJienoides in starch broth. Fig. IX. Forty-eight hours culture of B. sphenoides in potato broth. C 2 36 (vi) An unnamed bacillus isolated from two cases, hnown as Sindrey I, or Ldddicoate I. Morphology. The average size of this bacillus is similar ta that of B. sporogenes ; there are, however, some long forms always present in preparations made from cultures. The ends of the bacillus are square ; Gram's stain is positive ; no motility was ever observed. Spore formation is rapid, cultures 24 hours old showing large numbers. The individual spores are very large and oval in shape, and are either central or subterminal ui position, occasionally a large spore appears to be terminally situated. , Cultural characters. On agar the colonies are very flat and translucent and show a marked tendency to spread over the medium so as to form an almost imperceptible film which shows a wavy, ragged edge. If the surface of the medium is very dry the colonies remain separate and are then seen to have a very small opalescent centre from which several flat branching processes radiate ; these colonies resemble in some respects one of the forms taken by colonies of B. sporogenes, but they are much more transparent and the radiating processes are relatively much longer. In broth cultures growth is at first very rapid, leading to the medium becoming evenly clouded. Very soon, however, the bacilli and spores tend to settle to the bottom, leaving the supernatant broth quite clear. Milk cultures showed no change in 48 hours. On gelatin incubated at 20° C. no growth occurred. On red egg growth takes the form of a flat film which bleaches the medium underneath it, the surround- ing medium takes on a red colour. After a week's incubation the medium begins to fissure, and there is an increase of the water of condensation, indicating a slight digestion of the coagulated albumen. Digestion does not, however, advance beyond this stage, even after the cultures have been incubated for a month. In fluid cultures containing pieces of meat there was no apparent digestion, the meat was not blackened, and no smell of putrefaction could be detected. Fermentation reactions. Glucose and lactose were both fermented, acid and gas being formed ; in the case of the bacillus known as Sindrey I, acid was also produced in broth containing salicin. Neither bacillus had any action on the other carbohydrates which were tested. (vii) An unnamed bacillus isolated from one ca^e only, morphologically resembling B. tetani, known as Tholhy I. Morphology. The bacillus is a long one with square ends, it retains Gram's stain and in young cultures shows motility ; after forty-eight hours' growth many of the bacilli show a large round terminal spore and at this stage their appearance was identical with that of B. tetanus. Cultural characteristics. It was only with the greatest difliculty that a pure culture of this bacillus was obtauied, for, owing to its habit of spreading over the surface, and to other factors, the cultures were always found to be mixed with B. sporogenes. On agar, isolated i f f Fk;. X BlackfiiiiiL; cif gelatiije iiround ^tali ciiltuie of J:. spl'Cii'nilcf, 37 colonies were only obtained on a medium the surface of which had been allowed to dry very thoroughly ; these appeared as round, very transparent, structureless plaques. If the surface was even slightly moist the growth spread over the whole surface and appeared as a very transparent scum which was almost invisible, and unless the plate was very carefully examined, escaped notice. A gelatine stab culture incubated at 20° C. gave no growth. On red egg the growth appeared as a film. There was no evident digestion of the coagulated proteid, but the medium became fissured and the water of condensation became slightly increased. This condition remained unchanged even after the culture had been incubated for one month. Milk cultures showed production of neither acid nor clot. Fermentation reactions. These were tested, but the resultmg growths were very unsatisfactory, so that the results were not recorded. When grown in serum this bacillus produced large quantities of gas. Although morphologically identical with B. tetani many of its cultural characteristics were utterly unlike that bacillus ; further, neither the living cultures nor the broth in which it had been grown showed any pathogenic effect on guinea-pigs. (e) The Putrefactive Group of Spore-bearing Anaerobic Bacilli. The outstanding character of bacilli belonging to this group is their power to rapidly break down proteid material, evolving in the process gasses with an intensely putrid smell ; on the other hand, their power to ferment the various sugars, &c., is very limited. They were by far the commonest anaerobic bacilli found in the wounds. The classification of bacilli of this group has been rendered extremely difficult by reason of the very unsatisfactory nomenclature and confusing descriptions of the different bacteria in the literature, and when this research was conducted in 1916-17 there was little help to be obtained from the older work. The greatest confusion arose in connexion with the identification of B. sporogenes, B. oede- matis maligni, and Vibrion septique. Many authors seemed to regard these as different names for the common putrefactive organism of wounds, but it was clear that Vihrion septique and B. sporogenes were different organisms, and although it was probable that B. oedematis maligni of Koch was identical with one of these it was not very evident from the literature whether it was the common putre- factive organism of the wounds or whether it was the more pathogenic Vihrion septique. One source of trouble has been that Vibrion septique has been described as possessing putrefactive properties, whereas it is now abundantly clear that it is not a putrefactive organism. Doubtless these putrefactive qualities have been ascribed to it owing to the use of mixed cultures of Vibrion septique and B. sporogenes. 38 (i) B. sporogenes. Morphology. The bacillus is usually straight, in length similar to an average sized B. loelchii, but very distinctly thinner than that organism ; occasionally curved forms are seen (Fig. XII). It is motile and retains Gram's stain when the preparations are made from young cultures, but even after 48 hours many of the bacilli have already lost their Gram-positive properties. Spores are formed rapidly in all cultures : they are large, bulging the bacillary body and oval in outline. As regards the appearance of the fully developed spore-bearing bacillus the different strains seemed to divide themselves up into two more or less distinct types. In the first and common type the spore is definitely sub- FlG. Xr. B. sporogenes. terminal and occasional central spores are to be seen (see Fig .XI), while in the second and less common type the spore, which is pro- portionally rather smaller, appears at first sight to be quite terminal (Fig. XII). On closer examination, however, there is seen to be a very slight thickening of the spore envelope on the end distal to the bacillus, so that this is in reality a subterminal spore. The bacilli of this second type also are usually longer and many curved forms are to be seen. When stained for spores (with carbol fuchsin and methylene blue) the blue stain in the bacillary body was always seen to be more intense close to the spore and at the other end of the bacillus, leaving the central portion only faintly stained. In cultures 18 hours old spores are comparatively few in number, but after 48 hours they are very numerous ; soon after the spores are formed the bacillary bodies degenerate, leaving only free spores. The motihty is best seen in young cultures, but even sporing forms are frequently still motile, progression taking place with the spore either in front or behind. Fig. XIII II. Medusa-head type of colonies of B. s[utinij<' Fic. XIII t. B. sponirjriiis, flower-like colonies. 39 Cultural characteristics. The traditional form of a colony grown on agar, or glucose agar, is the so-called ' medusahead ' type, which consists of a thickened opaque whitish central spot from which radiate a number of fine wavy tangled hairy processes (Fig. XIII a) . This type of surface colony can only be produced if the surface of the agar is thoroughly dried. If the agar remains moist, as it commonly does in the case of freshly poured plates, the colonies show quite different characters and have the appearance shown in Pig. XIII b. The centre portion still remains rather heaped up and opaque, and from it thin flat opalescent processes spread over the agar in various directions, giving the colony an appearance which has been likened to a flower. Eventually, if the plate remains sufficiently moist, the whole surface will become covered with a fine opalescent film of growth due to these processes coalescing with one another, the thickened central portions of the original colony being still apparent as mere opaque Fie. XII. B. sporogenes. spots. That the very marked differences in the appearance of the colonies are entirely due to the moistness of the agar surface is proved by the fact that with a little manipulation both types of colony can be produced from the same culture. Another peculiarity of certain strains was a tendency to send prolongations into the depth of the agar, and from the pus of one case two types of colonies were constantly produced, one of which sent prolongations into the depth of the agar, while the other did not. Subcultures from these types of colony preserved their characters, but no other differences could be detected in the bacilH. In broth B. sporogenes gives an evenly turbid growth, in gelatine stab cultures incubated at 20° C. growth rapidly takes place along the track of the needle ; very soon, however, Lquefaction commences and quantities' of gas are formed ; eventually the whole mass becomes liquid In milk the most noticeable feature is the digestion of the niedium but if it is carefully examined after 4 or 5 days' incubation, when the upper portion of the milk has become clear, it is seen that the milk has been clotted, so that a mould of the tube has been 40 made, and this clot is, in time, completely digested. Occasionally this clotting was not observed, but the vast majority of the strains examined constantly produced it. The milk is at first alkaline, but after 3 or 4 weeks it becomes distinctly acid. On neutral red egg medium the colony first appears as a bright yellow spot on an orange-red background ; in 48 hours the medium shows distinct cupping in the immediate neighbourhood of the colony and around this depression is a blackened ring. Later the digestion of the albumen becomes more marked, so that the colonies are represented by pits containing a semi-fluid, foul-smelling material. The medium immediately around these pits is iDlackened. Eventually in 8 or 10 days the whole medium becomes liquefied, forming a multi- coloured, stinking mass. At the end of a month all trace of blackening has disappeared and the medium has become distinctly acid. Fermentation reactions. Glucose was constantly fermented by all the strains with the production of acid and gas. Some strains produced acid in glycerin broth in 24 hours, but after 48 hours the medium was again found to be alkaline ; all the strains produced some gas from glycerine. Many strains produced a trace of gas in broths containing dextrin and starch, and a few from salicin broth, but no acid production was ever observed. Lactose, mannite, saccharose, inulin, dulcite, and rafEinose were quite unaffected. Incidence. Bacilli of this type were isolated from 23 cases out of the 61 investigated, and in 7 it was the only anaerobic bacillus found. Of 20 strains investigated fourteen were of the type pro- ducing subterminal spores, the remaining 6 producing end spores. The number of days after the infliction of the wound that the bacillus was isolated, a positive or negative history of gas gangrene, and the presence or absence of bone injury in each case are given in the following table : Table VII. No. of days after injiiction of wound History of Presence or absence ('use. that bacillus was isolated. gas gangrene. of 'njury to bones. vfo. 1 17, 18, 32, and 46 Positive + „ 2 9 and 14 Positive + „ 4 24 Positive „ 5 19 Negative + „ n 10 and 37 Positive + ,, H 11 Positive ,, 1- 5 Positive „ 13 5 and 22 Positive + ,. 14- 16 Positive + „ 20 49, 77, 146, 151, and 302 ; the last 4 from sequestrum. Positive + „ 21 17 Positive _ 22 28 and 198 from sequestrum Negative -1- " 23 9 Positive „ 24 20 and 25 Negative + „ 25 7 Positive „ 29 15 and 22 from sequestrum Positive + „ 30 16 Positive „ 32 61 Positive + „ 33 11 Positive • -f „ 35 10 Positive + „ 38 8 Negative „ 39 8 Positive „ 50 19 Positive + 41 Serum tests. It was found that by inoculating rabbits with vaccines and Hving cultures of B. sporogenes and some of the other members of this group good agglutinating sera could easily be obtained. The doses used for this purpose were, for the first dose, 500 million bacilli which had been heated to 60° C. for 1 hour, given subcutaneously ; after an interval of 10 days 1,000 million living bacilli were given intravenously. By such means the serum gained the power of agglutinating emulsions of this bacillus up to a dilution of 1 in 2,000 to 1 in 3,000. In order to find out if B. sporogenes could be spht up into a number of strains by agglutination tests, a number of preliminary experiments were carried out. Two rabbits were inoculated, each with a different strain of B. sporogenes, the one being the smaller type with a subterminal spore — the most common form — the other being the longer type with an apparently terminal spore. The rabbits were inoculated with doses as described above, and after a suitable interval they were bled and the agglutination tests carried out. It was then found that the serum from each rabbit agglutinated the homologous bacilli. Further it was found that the serum of the rabbit immunized with the longer and pseudo-terminal sporing type of bacilli was able to agglutinate to a comparative titre some of the other strains having these characteristics, but had no effect on strains of the shorter subterminal spore type. The serum prepared by inoculations of the shorter subterminal spore type agglutinated in high dilutions most of the strains having the same characteristics, and at the same time were able to agglutinate — but only in dilutions of 1 in 100 and 1 in 200 — some of the strains belonging to the longer end-sporing type. Several strains, however, were not agglutinated by either of these sera, and many more experiments would have to be carried out before it would be possible to decide into how many types this group of bacilli should be divided. The preliminary experiments, however, show definitely that there are at least three varieties. Unfortunately, these researches could not be proceeded with. It may here be mentioned that attempts to produce immune bodies in the rabbit by inoculations of various cultures of B. welchii were, as has been noted by other authors, quite unsuccessful. Neither agglutinins nor amboceptor, as sought for by complement deviation tests, were found after repeated inoculations of doses covering a considerable range of both living and dead cultures. On the other hand, good agglutinating sera were easily prepared against B. butyricus, B. tertius, B. cochlearis, and B. sphenoides ; but again circumstances intervened which prevented this portion of the research being proceeded with. The results obtained in some of these experiments are given in the following tables : 42 Agglutination isms with serum terminal sparing B. sporogenes. Table VIII. of various strains of B. sporogenes and other organ- of rabbits immunized with the more comiJion sub- type and the less common terminal sparing type aj Result of agglutinating with serum of rabbit immunized iy : Strain. 8'poring type. Subtermiiial sparing End-sporing type. type. B. sporogems. Forrest I Subterminal + o Boone II jj Johnson J3 + Pretty + Cullingford I jj + McFadzean II )J + o IV Terminal + Harris IV )» + Dawson j» + + Ingram II » + o Parker A Sub terminal ■; B »5 + „ c »J + MoArdle I >J + „ III SJ Other organisms : McFadzean I B. tertius Cullingford Til b III c X Table IX. o Showing that from the same patient there loere isolated several different members of the putrefactive group of bacteria which seemed quite distinct serologically. Strain Strain name. no. Typt. Cullingford I B. sporogenes subterminal spore III 6 ? IIIc ? X ? McFadzean I II IV B. sporogenes subterminal spore „ terminal „ Parker A B. sporogenes subterminal spore (colonies grew down into agar). B B. sporogenes subterminal spore McArdle I B. sporogenes subterminal spore III Sporogenes serum. Subtermi- nal sporing type. End- sporing type. Cullingford X sertim. o o + + + + o + o 43 (ii) An unnamed bacillus isolated from one case only, knoivn as Cullingford III c. This bacillus was isolated from a glucose agar plate made from pus which had been heated to 66° C. taken on the patient's admission to hospital. Morphology. The bacillus is of medium size, straight or slightly curved, with square ends. After being incubated for 24 hours there is marked spore formation, the spores being oval and resembling those of -B. sporogenes in size and shape. A very marked character- istic was the rapidity with which the body of the bacillus disinte- grated after the spore had formed, even after 24 hours' incubation practically all the spores were lying free. In specimens made from very young cultures Gram's stain was positive, but this property was very soon lost, even a 24-hour-old culture showing but few Gram-positive elements. No motility could be made out even in the youngest cultures. Cultural characters. The colonies on agar were irregular with a wavy edge from which projected a few filamentous processes. They were less opaque than those of B. sporogenes, and the processes were less numerous and less marked. Cultures in broth showed in 24 hours an even clouding of the medium. In older cultures the bacteria had settled to the bottom, leaving the supernatant broth clear. On gelatine incubated at 20° G. no growth could be made out. Milk cultures remained unchanged for the first 12 days. After 14 days' incubation, clotting had occurred. The clot was very slowly digested, the digestion not being complete after 6 weeks' incubation. Throughout this whole period the reaction remained alkaline. On red egg, after 24 hours' incubation the colonies appeared as round depressions on a pink field, after 48 hours the depressions had increased in size and the surrounding medium had become bright red. After 14 days there was distinct liquefaction of the coagulated egg but even at the end of 6 weeks much of the medium remained solid. In this stage the culture gave off a very putrid smell. In broth containing pieces of meat, growth was rapid and abundant, much gas being formed. No blackening of the meat was observed nor was any digestion noticeable to the naked eye. The smell of these cultures was intensely putrid. Fermentation reactions. When grown on broths containing various sugars, &c., it was found that glucose and glycerine were fermented, both acid and gas being produced, while it was without action on all the other carbohydrates which were employed. « (iii) An unnamed bacillus isolated from one case only, hnown as Cullingford HI b. Morphology. This bacillus was rather small with rounded ends and frequently occurred in pairs. It stained readily and very sharply with the usual basic stains and retained Gram's stain. Examination of young cultures showed very distinct motihty, the progressive movements having a somewhat sinuous character. Spore formation was not very abundant, the individual spores being 44 rather smaller than those of B. sporogenes ; as regards their position in the hody of the bacillus they were either central or subtermmal. The body of the bacillus rapidly degenerated after the spore had formed so that most of the spores were seen lying free. Cultural characters. The colonies on agar were small, semi- transparent, round or slightly irregular. No filamentous processes were to be seen unless the plate was very wet, when there was formed a sheet of growth advancing with an irregular edge. Cultures in broth showed an even turbidity. Gelatine stab cultures incubated at 20° C. were rapidly liquefied and considerable amounts of gas were produced so that the medium was broken up. With milk cultures which had been incubated at 37° C. for 7 days the milk showed no change. At the end of 12 days it appeared to be some- what digested, but no clotting could be made out. When, however. Fu4. XIV. Anaerobic bacillus known as Cullingford III b. the cultures were examined after 14 days' incubation a definite clot was found to have formed. The digestive process continued very slowly so that after 6 weeks' incubation some of the clot still remained undigested. Cultures on red egg showed, after 48 hours' incubation, colonies of the ' medusa head ' type on a red background. After 8 days' incubation these colonies had formed yellow depressions. Immediateljr around them the medium was intensely blackened, the blackening being much more pronounced than in cultures of B. s-porogeyies. The remainder of the surface of the medium was a deep red. After 14 days the coagulated egg was markedly digested and in 6 weeks the liquefaction was complete and all the blackening had disappeared. The cultures gave off a strong smell of sulphuretted hydrogen in addition to the more usual smell of putrefaction. In broth cultures containing pieces of meat the blackening of the meat was very marked, and here again the smell of sulphuretted hydrogen could be recognized in addition to the smell of putrefaction. Spore 45 formation was marked, most of the spores lying free, but many were made out to be subterminal in position in respect to the body of the bacillus. Fermentation reactions. In broths containing the various sugars, &c., it was found that glucose was fermented, both acid and gas being formed. In the case of glycerine, gas was produced, but the media apparently remained alkaline ; there was no action on any of the other carbohydrates that were tested. The main points of difference between this bacillus and B. sporo- genes were as follows : (1) In size this bacillus was smaller and spore formation was much less abundant. The individual spores even in young cultures had usually lost all signs of the body of the bacillus. (2) Cultures on coagulated egg differed from those of B. sporogenes in that the digestion of the medium was very much slower, the blackening around the colonies was very much more intense, and the general appearance of young cultures was quite different. (3) Emulsions of this bacillus were not agglutinated by any of the sera which agglutinated emulsions of B. sporogenes. It may here be noted that from the same patient a strain of B. sporogenes was isolated which gave all the typical characteristics of that bacillus and which was agglutinated by standard anti-sera. (iv) An unnamed hacillus isolated from one case only, Icnown as Cullingford X. Morphology. This was a large square-ended bacillus, it was not motile, Gram's stain was positive. Spore formation was very profuse and even in very young cultures fully formed spores were seen. The individual spores were oval, the long diameter being greatly in excess of the short ; the body of the bacillus was not distended at the site of the formation of the spore. After the spore had been formed the body of the bacillus rapidly disintegrated, so that even in cultures only 24 hours old many free spores were seen. Films made from cultures which had been incubated for 48 hours or more showed practically nothing but free spores, the bodies of the bacillus being represented by some ill-staining debris. Cultural characters. The colonies on agar when growing on the surface were rather large, granular in structure, with somewhat irregular edges. When growing in the depths they appeared as opaque white spots fBom the surface of which passed fine tangled processes giving the colony a very woolly appearance. In broth the growth is very profuse and gives rise to an even clouding of the medium. On gelatine incubated at 20° C. no growth occurred. In milk after 7 days' incubation a clot had formed and much digestion had taken place. The reaction remained alkaline. The digestive process continued slowly and was almost complete after being incubated for 6 weeks. On red egg after 24 hours' incubation the colonies appeared as dirty yellow spots on a red background; after 48 hours' growth some liquefaction of the coagulated egg had com- menced. Later the medium was noticed to be slightly blackened in the neighbourhood of the colonies. The digestion and lique- 46 faction of the medium continued very slowly and was not complete even at the end of 6 weeks' growth ; the blackening of the medium persisted. In fluid cultures containing pieces of meat, blackening of the upper layers occurred after 24 hours' growth. The putrid smell was much less marked than in the case of B. sporogenes. Digestion of the meat proceeded very slowly. Fermentation reactions. Glucose was fermented with the pro- duction of acid and gas, none of the other carbohydrates that were tested being affected. A serum capable of agglutinating emulsions of this bacillus was obtained by injecting a rabbit with one dose of 500 million dead bacilli subcutaneously and three of 1,000 million intravenously. Fig. XV. Anaerobic bacillus known as Cullingford X. (v) An umiamed bacillus isolated from one case only, known as McFadzean I. Morphology. This bacillus was rather short and stout, straight with rounded ends; young cultures showed it to be slightly motile. Gram's stain was positive. Spore formation was not very profuse. The spores were formed about the centre of tl^e body of the bacillus, which disintegrated so rapidly after the spore was fully developed that it was rare to find a fully developed spore with the body of the bacillus capable of being stained. Cultural characteristics. On agar plates the colonies were flat, irregular, and very transparent ; there was a great tendency for the colonies to spread and to cover the whole surface of the medium with a thin film of growth. In broth culture growth appeared as an even clouding of the media. On red egg after 48 hours' incubation the colonies appeared as yellow depressions on a slightly reddish background. After a week's incubation there was considerable lique- faction of the egg but no blackening of the medium was ever noticed 47 After one month's incubation some of the egg still remained solid as if the liquefying process had ceased. A strong odour of putre- faction was given off. In gelatine cultures incubated at 20° C. no growth occurred. Fermentation reactions. Practically the only indication that any of the carbohydrates tested were acted on was a slight change in the reaction of the medium. Thus cultures in broths containing sac- charose, mannite, glucose, and lactose were found after 48 hours' incubation to have lost the alkalinity that was present and to have become neutral ; in the case of salicin, however, the alkalinity was increased, while inulin, glycerine, dextrin, and starch remained unchanged Milk cultures were clotted after six days' incubation, the clot being digested later ; the reaction remained alkaline. (vi) An unnamed bacillus isolated from one case only known as Hancock II. Morphology. This was a small bacillus about half the size of jB. welchii ; it was straight or very slightly curved with square ends. Gram's stain was positive and young cultures showed motility. Spore formation was not very profuse ; the individual spores were oval and somewhat smaller than usual, the bodies of the bacilli not being distended at the point where they were formed. Their situation was usually subterminal but occasionally terminal. Cultural characteristics. On agar or glucose agar surface colonies grew as flat very transparent structureless sheets varying in size from 0-5 mm. to 10 mm. in diameter. The margins were quite smooth and sharp-cut. In milk cultures, after 14 days' incubation the milk was clotted, later considerable digestion of the clot slowly occurred. In gelatine stab cultures at 20° C. there occurred^ lique- faction with slight gas formation. On red egg the growth after 48 hours' incubation appeared as a greasy looking slightly depressed scum on an orange background. The surrounding medium never took on a red tint which most of the other anaerobic bacilli produced. No blackening of the medium was ever noticed. Later liquefaction of the coagulated albumen slowly progressed until the whole mass was liquefied, but this occurred very much more slowly than in the case of B. sporogenes. Fermentation reactions. Glucose was fermented with the pro- duction of acid and a small quantity of gas, none of the other carbo- hydrates which were tested being affected. (/) Bacteria that cannot be placed in any of these Groups. (i) A bacillus isolated from one case only, known as Boone I. This bacillus was obtained from pus which had been heated to 65° C. for half an hour on the day that the patient was admitted. Morphology. A 'specimen made from an agar culture 24 hours old showed a large, stout, square-ended bacillus identical in appear- ance to B. welchii. No spores were observed. In specimens made 48 from cultures grown on red egg the bacilli were seen to be much longer and more slender than those taken from agar cultures — this is exactly the opposite to what is seen in similar, cultures of B. welchii. Specim.ens made from a ten-day-old culture on red egg showed a long and comparatively thin bacillus with many spores which were oval in shape and situated terminally. Cultural characteristics. This bacillus was not a very strict anaerobe, in this character it was similar to B. welchii. On agar the colonies were round, semi-opaque, flat and showed a number of concentric rings on their surfaces. In- size they were rather small, after 48 hours' incubation varying from the size of a pin point to 2 mm. in diameter. The colonies were well separated. In broth growth was rapid and profuse so that in 24 hours a thick, even Fig. XVI. Anaerobic bacillus known as Boone I. cloudiness of the medium was produced. In agar stabs the growth along the track of the needle was rather irregular ; it occurred to within 2 mm. of the surface. In gelatine stab cultures incubated at 20° C. irregular growth occurred along the needle track, later the gelatine was liquefied and some gas was formed. On red egg after 36 hours' incubation the colonies showed as yellow spots on a bright red background. They formed -slio-ht depressions in the surface of the medium which at first were quite sharply defined. After one month's incubation the colonies had spread considerably and tended to become confluent. There was no further sign of digestion of the coagulated albumen other than the shght but constant depression which formed in the immediate neighbourhood of the colony within the first 48 hours _ Milk cultures showed no change in 48 hours. After 8 days' lacubation a definite solid clot was formed which then showed no tendency to contract. After one month's incubation the clot had 49 contracted considerably and there appeared to be slight digestion. The milk remained alkaline throughout the whole period. Fermentation reactions. Glucose was fermented, acid and gas being formed. In glycerine broth slight acid formation was observed after 15 hours' incubation, but in 48 hours the reaction had again become definitely alkaline ; some gas had also been formed in 48 hours. The remaining carbohydrates which were tested remained unaffected. Serum. An agglutinating serum with a titre of 1 in 3,200 was obtained by inoculating a rabbit with one dose of 500 million dead bacilli subcutaneously and two doses of 1,000 million intravenously. This bacillus, although, when grown on agar, it closely resembled B. welchii in morphology, was more nearly related to B. sporogenes in its cultural characters ; further it had the power of slightly digesting coagulated egg, it liquefied gelatine and had very limited power of splitting up the various carbohydrates. (3) The Streptococci. Very shortly after commencing to investigate the bacterial flora of the wounds it became evident that streptococci of some type were present in practically every septic wound and that these cocci were responsible for most of the complications that might occur during the healing of these wounds. It was therefore decided to investigate this group of cocci with rather more than usual care. In order to carry this out a large number of strains of streptococci were isolated from the samples of pus which were examined from day to daj. (ffl) Methods of Diffbebntiation. Each strain was put through a series of tests and the following characteristics were noted : 1. The naked eye characters of a 24-hour broth culture were noted to ascertain whether the growth consisted of an even turbidity throughout the medium or a granular deposit at the bottom of the tube, leaving the supernatant broth clear. 2. Microscopical preparations were made from a 24-hour-old broth culture, one of which was examined unstained. According to the length of the chains the cocci were classed as either ' longus ' or ' brevis '. Another preparation was stained by Gram's method and counter-stained with neutral red to ascertain the shape, &c., of the individual cocci and the degree with which they retained the Gram's stain. 3. Fermentation tests. Cultures were made in broths containing lactose, saccharose, raffinose, mannite, and salicin, and the formation of acid was noted. (The medium used in these tests was made from casein as described by Cole and Onslow ^, the indicator being an alkaline solution of acid fuchsin.) 4. Cultures were made in litmus milk to ascertain if acid and clot were produced. ' Cole and Onslow, Lancet, 1916, ii. 9. 1727 D 50 5. A culture was made in a sugar-free broth tinted with neutral red and incubated anaerobically for 48 hours ; it was then examined for the presence or absence of fluorescence'. 6. A stab culture was made in gelatine and incubated at 20° C. ; after an interval of a week it was examined to ascertain if growth had occurred, and whether it had been accompanied by liquefaction of the gelatine. When the data given by tests referred to above had been collected the various strains were arranged according to Gordon's modification of Andrewes's and Horder's classification. Before the results thus obtained are described, attention may be drawn to the following points which were noticed during the isolation and testing of the various strains. (&) Some General Considerations with regard to Streptococci. 1. Practically all the Streptococci found in wounds grow much more readily under anaerobic than aerobic conditions, and it was by no means uncommon to find a Streptococcus which later proved to be Streptococcus pyogenes, that would, when first isolated, only grow under anaerobic conditions ; it was only after they had been subcultured on several occasions that growth of such cocci under aerobic conditions could be obtained. The importance of this observation cannot be over-estimated. In a case of rat-bite fever, for instance, the present authors ^ obtained the growth of a Streptococcus in 3 out of 5 cultures, incubated under anaerobic conditions, made from a lymphatic gland removed from the patient's axilla, whereas 5 other cultures which were incubated aerobic- ally all failed to give any growth. This Streptococcus when first isolated gave no surface growth either in agar stab cultures or on agar slopes under aerobic conditions, but after bfeing repeatedly subcultured it grew well under these conditions ; if none of the cultures had been incubated anaerobically an important piece of evidence pointing to Streptococcus pyogenes being the cause of the disease in this case would have been lost. Again, Streptococcus pyogenes was isolated on three different occasions from one of the patients (Case No. 26) of the present series, but none of the primary cultures nor even the first few sub- cultures would grow under aerobic conditions. It was only after several stab cultures in agar had been made that surface growth could be obtained. 2. Cultures of most of the varieties of Streptococci, made on Dorset's egg medium or coagulated serum, remain alive for a much longer period than those on agar and gelatine.^ The viability, however, of the different types was very variable. Some cultures died out unless subcultured every few days, but usually those made on coagulated egg or serum were alive and could be subcultured > Douglas, Fleming, and Colebrook, Lancet, 1918, i. 253. ' More recently we have found that minced meat medium such as is used for the cultivation of anaerobic spore-bearing baciUi has the same function of preservins the vitality of Streplococcus cultures and indeed in this medium almost aU the common microbes can live for long periods without subculture. 51 without any difficulty three months after being planted. They were kept at room temperature and the media prevented from drying up by a rubber cap fitted over the wool plug. (c) Classification of the Steains of Streptococci isolated FEOM 30 Cases taken at eandom. In all 160 strains were isolated from these 30 cases and, using the classification mentioned above, the following results were obtained : per cent. Streptococcus pyogenes . 118 =72-75 ii. Streptococcus salivarius 10 = 6-25 hi. Streptococcus faecalis . 8 = = 5 iv. Streptococcus equinus . 5 = = 3-125 V. Streptococcus anginosus 2 = 1-25 vi. Unclassified Streptococci 17 = = 10-625 Total 160 Prom 28 out of the 30 cases Streptococcus pyogenes was isolated on one occasion at least and in 15 out of the 30 cases it was the only Streptococcus found. The 10 strains of Streptococcus salivarius were isolated from 3 different cases and were always associated with other streptococci, these being Streptococcus pyogenes in all 3 cases and Streptococcus equinus in 2 cases. Streptococcus faecalis was isolated from 5 cases ; in all of these they were associated with other streptococci, in 4 instances with Streptococcus pyogenes, and in 1 with Streptococcus equinus and unclassified Streptococci. The 5 strains of Streptococcus equinus were isolated from 4 cases. In 2 cases they were associated with Streptococcus pyogenes, in 1 case with Streptococcus faecalis and unclassified Streptococci, and in 1 with unclassified Streptococci only. The 2 strains of Streptococcus anginosus were both isolated from the same case, they were associated with Streptococcus pyogenes. The 17 unclassified Streptococci were isolated from 7 cases, in 5 cases they were associated with Streptococcus pyogenes, in 1 case with Streptococcus faecalis and Streptococcus equinus and in another case with Streptococcus equinus only. (d) The Charactees of the Various Types of Steeptooocci. (i) Streptococcus pyogenes. Morphology. Microscopical preparations made from a 24-hour- old broth culture show the microbes to be arranged in long chains and the individual cocci to be round and sharply cut, very few irregular or involution forms being seen. Gram's stain is well retained. Broth cultures after 24 hours' incubation show a well- marked granular deposit and often some granules sticking to the sides of the culture tube, but the main mass of the broth remains quite clear. Fermentation reactions. The majority of strains ferment lactose, D2 52 saccharose, and salicin, with the produation of acid, leaving raffinose and mannite unaffected. Occasionally, however, mannite is also acted on. Strains which ferment mannite appear to always retain this p,roperty. Some strains produce acid and clot in milk, others appear to have no action, no clot being produced, and the reaction remains alkaline ; others again produce only a slight amount of acid, but no clot.^ Cultures in broth containing neutral red when incubated anaerobically did not as a rule show fluorescence. Gelatine cultures incubated at 20° C. showed good growth ; there was no liquefaction of the medium. As was mentioned above this coccus always grows better anae- robically, and some cultures when first isolated will only grow under anaerobic conditions. The pathogenicity of the various strains was not tested. Taking the whole series of the 118 strains of Streptococcus pyogenes that were isolated and tested, the impression acquired was that this group of micro-organism forms a constant and compact group with very constant distinguishing characteristics, the only important variable factor being the power of some strains to ferment marmite. In order to confirm this impression it was decided to carry out a series of agglutination tests, and to ascertain if a serum prepared by inoculating a rabbit with a series of doses of a vaccine made from one strain would agglutinate emulsions of this strain, and also the other strains, to approximately the same titre. A vaccine was therefore made from a Streptococcus pyogenes isolated from Case No. 20 by heating a serum broth culture to 60° C. for 30 minutes. This vaccine contained 8,000 million cocci per c.c. and as a primary dose 1,000 million cocci were given sub- cutaneously ; four or five days later 1,500 million cocci were inocu- lated intravenously and three further doses of 8,000 million cocci were administered at intervals of one week. The rabbit was bled nine days after the last dose. The serum thus obtained was found to agglutinate an emulsion of the Streptococcus from which the vaccine had been made in dilutions up to 1 in 500. The technique employed in carrying out these agglutination tests was to mix 1 c.c. of the dilution of the serum with 1 c.c. of a faintly opalescent suspension of the strain of the Streptococcus to be tested in small test tubes which were then placed in a water bath at 45° C, and the result was noted four hours later. The dilutions of the serum that were used were 1 in 50, 100, 150, 200, 800, 400, 500, 600, 800. The emulsion was prepared as follows : the strain of Streptococcus to be tested was planted in a tube con- taining about 5 c.c. of a mixture of trypsinized meat broth and serum or hydrocele fluid, the latter having been heated to 60° C. for 30 minutes. The proportion of serum to broth was about one-half to one-third. The tubes were then incubated at 37° C. for from 24 to 36 hours, preferably with the tube in a sloped position, 1 Fleming and Porteous have since shown (Lancet, 1919, ii. 49) that the clotting of milk by Streptococcus pyogenes is largely dependent on the size of the tuhe in which the test IS carried out. In very narrow tubes clotting is more rapid than in wide tubes and in narrow tubes clotting may be observed when even after prolonged incubation no change can be seen m the wide tubes. 53 as a greater quantity of growth occurred when this was done. In this naixture all the strains of Streptococcus pyogenes were found to grow in such short chains that in shaking up the culture an almost even turbidity was produced throughout the medium. After being thoroughly shaken, the cultures were diluted to a faint opacity with salt solution. Twenty-four strains of Streptococcus pyogenes which had been isolated from different patients were tested in this way. The results are given in the following table : Table X. Dilutions of Serum. No. of case C5 cj ^ s ^ ^ Q5 ^' from whom the § 1^ 1^ § § § § streptococcus was .g •i .s ■5 :s ,=^ _s .s ,S isolated. »»S '-I 1^ f-H f-H ^ >H '..i ^-H No. 2 • + + + + + + + — „ 5 . + -r + + -|- _]_ ~r — — „ 7 ■ + -r + + + + + + — , 8 • + + + + + + + + - — ., 9 • + + + + + + + -f — „ 10 ■ + + + + + + _i_ + _ „ 11 . + + ~r J_ + + + + + „ 12 . + + + -i- + + + + — „ U • + + + + + + + + -;- „ 15 ~T + ~r + + + + + — „ 16 ■ + + + + + + — — — „ 18 ■ + + + + + + + ~r — „ 19 ■ + + -r "T + + + -1- _ „ 20 ■ + + -;- "r + + + _ — 21 ~r + -r + + 4- + — — „ 23 ■ ' ~f + + + + + + -f" — „ 24 . + + + + + + + — — „ 26 -f + + + _!_ + + — _ „ 27 H" + + + + + + — — „ 29 + + + + + + + — — „ 30 . + + + + + + + -}- + „ 31 . + + + + + -i_ + + „ 33 . + + + + + 4- + + — — A.B. . . + + . + + + + + + + o The strain from the vacome used in inoculating the rabbit was isolated from Case No. 20. The sign + indicates that agglutination was present with that dilution of serum. The sign — indicates that no agglutination took place. The following experiment was then made to ascertain if the various strains of Streptococcus pyogenes isolated from the same case were agglutinated by the immune serum to the same titre. Emul- sions were prepared by the method described above from 4 strains isolated from Case No. 5, 6 strains from Case No. 7, 2 strains from Case No. 10, and 5 strains from Case No. 11. These emulsions were then mixed with dilutions of the immune serum similar to those employed in the former experiment. An emulsion was also prepared from strain 1, Case No. 20, which furnished the vaccine with which the rabbit yielding the immune serum was inoculated, and the agglutination given by the serum with this emulsion gave the standard titre. 04 The results are shown in the following table : 1 Iable XI. Dilwtioi^s of Serum. Cs ^ (^ ^ ^ ■a s ■Strain from which § X5 S5 § § § 50 emulsion was made. •i s •i ■f •S •S •S T*H ^H >-H l-H i-H >*H I^ ^ Case No. 20 (1) + + + + + + + — Case No. 5 (1) + + + + + + + — (2) + + + + + + + — (3) + + + + + + + — (4) .. + + + + + + + — Case No. 7 (1) + + + + + + + — (2) .. + + + + + + + — (3) .. + + + + + + + — (4) .. + + + + + + + — (5) .. + + + + + + + — (6) .. + + + + + + + — Case No. 10 (1) + + + + + + + — (2) .. + + + + + + + — Case No. 11 (1) + + + + + + + — (2) .. + + + + + + + — (3) .. + + + + + + + — (4) .. + + + + + + + — .(5) . . + + + + + + + — s These experiments indicate that an immune serum prepared by inoculating a rabbit with a vaccine prepared from a single strain of Streptococcus pyogenes is capable of agglutinating not only emulsions of the strain from which the vaccine was prepared but all the strains that were classified by the characters given above under the heading of Streptococcus pyogenes. Two points, however, remained to be investigated — one being whether these agglutination tests were truly specific or whether they were due to co-agglutination ; the other was .whether the small group of Streptococci which fermented mannite, but were otherwise similar in every way to those which failed to do so, were agglutinated to an equal titre by sera prepared by inoculating animals with vaccine made from strains which did not ferment mannite. The following experiments were made to ascertain if the serum prepared by inoculating a rabbit with a vaccine made from a typical non-mannite fermenting strain of Streptococcus pyogenes agglutinated to a corresponding titre emulsions of the mannite fermenting strains. Emulsions were prepared from the non-mannite fermenting strain used in the preparation of the serum. Case No. 20, Strain 1, and from three different mannite fermenting strains isolated from Case No. 13. These emulsions were then mixed with dilutions of the serum — 1 c.c. of the emulsion to 1 c.c. of the dilution of serum. The results are given in the following table : Table XII. 8 Dilutii ■)ns of serum. No. of Case and Strain from which emulsion was s § s § § % § made. 4 8 1^ c<3 ■S ■S Case No. 20. Strain 1 — + + + 4- + + + „ „ 13. „ 2 + + + + -1- + + trace 99 99 99 99 3 + + + + + + + trace 99 99 99 )> O + + + + + + + -1- 55 Former experiments showed that a 1 in 500 dilution of the serum was the highest giving any agglutination with the emulsion of Strain I, Case No. 20. In the next experiment the emulsions were prepared from five other strains of the mannite fermenting type isolated from three different cases and the same serum was used in the tests ; here again a similar result was obtained, the serum agglutinating these emulsions in dilutions up to but not beyond 1 in 500. In these experiments the tubes containing the dilutions of serum and emulsion had been placed in a water bath at 45° C. for at least 4 hours before the readings were made. In the following experiment emulsions from a non-mannite fermenting strain, Case No. 16, Strain 1, and a mannite fermenting strain, Case No. 8, Strain 5, were employed and the readings were made after 2 hours in the water bath at 45° C. The result is given in the following table : No. of Strain and Case Table XIII. s Dilutions of serum. ■S S • ^- ^ ■=> ^ i~i 3s "^ f^ ^ '^ "^ ^ ^7,.- gS ^ *^ "^ '-H f>i 9 tne emulsion. gg ^ ^ g a g "g ^=^1^ ^^ ^M ^ >^ O Case No. 16. Strain 1 — + + + + trace — „ „ 8. „ 5 + + + + + trace — These experiments show conclusively that a serum prepared by inoculating an animal with a vaccine prepared from a typical non- mannite fermenting strain of Streptococcus pyogenes has the power of agglutinating emulsions of the mannite fermenting strains to an exactly similar titre as is the case with emulsions of the non-mannite fermenting strains. The next experiment shows that a serum prepared from a strain of Streptococcus pyogenes has no power to agglutinate emulsions of Streptococci belonging to the faecalis group. In carrying out this experiment emulsions were made from three different strains of Streptococcus faecalis, and also to act as a control from the strain of Streptococcus pyogenes used in the preparation of serum. These were then put up in small test tubes with the varying dilutions of serum and the results were recorded after incubation at 45° C. for at least 4 hours. The following table shows that the serum had no power to agglutinate emulsion of Streptococcus Jaecalis even in dilutions of 1 in 60, while the emulsion of Streptococcus pyogenes was agglutinated in dilutions up to 1 in 500 : Table XIV. Dilutions of serum. ■a QJ <» cj ^ ^ .^■ No. of Strain and Case .'o'-i'-hc^i'^'^'o £ furnishing §. .g .g .g .g .g .g .g § the e7nulsion. ^r-('~i'-i'--i'~('-H'~(C Case No. 1. Strain 6 faecalis — — — — — — — — „ „ 20. „ 4 faecalis — — — — — — — — „ „ 33. „ 4 faecalis — — — — — — — — Control 1 Case No. 20. Strain 1 pyogenes + + + + + + + — 56 Lastly an experiment was made to ascertain if, after the serum had had its agglutinating properties removed by adding to it an emulsion of a typical Streptococcus pyogenes, it would be incapable of agglutinating emulsions of other typical strains of Streptococcus pyogenes and also emulsions of mannite fermenting strains. The experiment was carried out in the following manner : an emulsion of Strain 1, Case No. 16, a typical non-mannite fermenting Streptococcus pyogenes, was obtained by centrifuging three 24-hour- old broth cultures of the coccus, pipetting off the supernatant broth, and emulsifying the resulting deposit in 1 c.c. of normal saline solution. This gave a very thick emulsion, to which was then added 1 c.c. of the serum which had been prepared from Strain 1, Case No. 20, and was that used in the various agglutination experiments described above. The mixture was then incubated at 45° C. for 4 hours to allow the emulsified cocci to absorb the agglutinating property present in the serum. Dilutions of the serum were then made and mixed with emulsions prepared (i) from the strain used in absorbing the serum, (ii) and (iii) from two other typical non-mannite fermenting strains of Streptococcus pyogenes, (iv) from a mannite fermenting strain of Streptococcus pyogenes. The small test tubes containing these mixtures were then placed in a water bath at 45° C. and the results which are given in the following table were recorded after an interval of 4 hours. Table XV. No. of Strain and Case Dilutions of the absorbed serum, furnishing the emulsion, 1 in 20. 1 in 50. 1 in 100. Cotitrol Strain I. Case Xo. 16 (typical pyogenes used to absorb serum) . . . . + _ Strain VIII. Case No. 7 (typical pyogenes) + _- _ _ Strain I. Case No. .32 (typical pyo- genes) + — _ _ Strain V. Case No. 8 (mannite ferment- ing type of pyogenes) . . . . + — _ _ The serum had been shown by previous experiments to have the power of agglutinating emulsions of all these strains in dilutions up to 1 ■ in 500. The results given above, therefore, show that the serum after being absorbed by an emulsion of a typical Streptococcus pyogenes lost the power of agglutinating not only emulsions made from that particular strain but those made from other typical strains of Streptococcus pyogenes and also mannite fermenting types. This indicates that all the Streptococci described under the heading of Streptococcus pyogenes belong to one serological group and that'^the agglutmation results obtained in the former experiments were specific and not due to the in'esence of group agglutination. These experiments indicate that the various groups of Strevtococci can be_ identified by the use of a specific agglutinating serum. A few prehmmary experiments, however, showed that the precautions 57 found necessary in using this method for other bacteria are equally important in the case of Streptococci. For instance, it was found that the specific serum for Streptococcus pyogenes agglutinated certain strains more nearly related to the pyogenes group in comparatively low dilutions of the serum. It is, therefore, important that the exact titre of the serum should be estimated and only those cocci which are agglutinated to about full titre should be placed in the group from which the serum was prepared. (ii) Streptococcus salivarius. Ten strains of this Streptococcus were isolated out of the 160 strains especially studied, that is 6-25 per cent. These 10 strains were isolated from 3 different cases out of the 30 from which the 160 strains of Streptococci were isolated. The characters of the cocci were as follows : Morphology. A microscopical preparation made from a 24-hour- old broth culture showed the individual cocci to be rather small and round in shape. Involution forms were rather more common than in the case of Streptococcus pyogenes. The chains of cocci were distinctly of the type ' brevis '. Gram's stain was retained much less strongly than in the case of Streptococcus pyogenes. To the naked eye 24-hour-old broth cultures showed an even turbidity without any marked deposit at the bottom of the tube. Fermentation reactions. Broths containing lactose, saccharose, and raffinose were fermented, acid being produced ; those containing mannite and salicia were unaffected. Litmus milk was rapidly clotted with the production of acid. No fluorescence was produced by growiag the coccus ia broth containing neutral red under anaerobic conditions for 48 hours. No growth took place on gelatine slopes or stabs incubated at 20° C. Cultures on agar and even on coagulated albumen died out very rapidly in comparison with most of the other varieties. All the strains isolated were exactly similar to one another, but it was considered that as only 10 strains had been isolated from 3 cases agglutination tests would fail to give reliable results as to the unity of the group. No experiments were made to ascertain its pathogenicity for animals, but from the persistence and the abundance of the cocci in two of the cases from which they were isolated it was thought that they probably played an important role in these two wounds. Both of these cases were treated with apparent benefit with a vaccine made from the coccus isolated from the wound. (iii) Streptococcus Jaecalis. Cocci having the characters of this group were isolated on eight occasions. The general characters of this group were as follows : Morphology. Microscopical preparations made from a 24-hour- old broth culture showed oval cocci which were very regular in size and shape and frequently occurred as diplococci. Gram's stain was retained to a very marked degree. To the naked eye 24-hour-old 58 broth cultures gave a perfectly even turbidity with no tendency to form a deposit at the bottom of the tubes. Fermentation reactions. Broths containing lactose, mannite, and salicin were in every case fermented with the formation of acid ; in certain strains which will be referred to later in detail saccharose was similarly acted on. Litmus milk was rapidly clotted with the production of acid. When grown in broth containing neutral red under anaerobic conditions for 48 hours the media became markedly fluorescent. In gelatine stabs or slopes incubated at 20° C. growth was rapid and abundant. . Two strains, which rapidly fermented saccharose, also had the power of slowly liquefying not only gelatine, but also coagulated albumen. This action was, compared to that exerted by many bacteria, very slow, as is indicated by the appearance shown in Fig. XVII, which is reproduced from a photograph of a culture grown for 8 days at 20° C. It was, however, progressive, and if the cultures were kept for several weeks or months, gradually the whole medium was rendered liquid. This was also true of cultures on coagulated albumen, at first the only sign of liquefaction being the formation of a slight trough under the line of growth and some cracks in the surrounding medium ; after some months, however, for instance, in a culture on coagulated egg kept for 6 months at room temperature, practically the whole mass of coagulated proteid was liquefied into a grumous mass, brownish in colour. (iv) Streptococcus equinus. Five strains were isolated out of the 160 strains=5-125 per cent., and these were found in the wounds of 4 different patients. The characters of this group of Streptococci were as follows : Morphology. In microscopical preparations made from a 24-hour- old broth culture the individual cocci were seen to be round and regular in shape. The chains were always of the type ' brevis '. Gram's stain was retained less intensely than was the case with Streptococcus pyogenes. The naked eye appearance of a 24-hour- old broth culture was similar to that of Streptococcus faecalis, there being even turbidity throughout the medium and little or no tendency to form a deposit at the bottom of the tube. Fermentation reactions. Broths containing saccharose and salicin were fermented with the production of acid ; lactose, raf&nose, and mannite were unaffected. Litmus milk usually remained unchanged. Broth cultures containing neutral red showed no fluorescence after being incubated under anaerobic conditions for 48 hours. Gelatine cultures incubated at 20° C. showed good growth. No experiments were made to test the'pathogenicity of this coccus, and from the fact that it was always isolated in the early stages of the wound and that it was never found on a number of different occasions from the same wound, its presence was thought to be most probably without pathogenic significance. Fig. XVII Staliculturrof Slrfjitocuce lis farrall i, ahmviuis liqucfartiim (jf the gi-latiiir. 59 (v) Streptococcus anginosus. Only two strains were isolated — both from the same case. The characters of these Streptococci were as follows : Morphology. In microscopical preparations made from a 24-hour- okl. broth culture the individual cocci were round and regular in shape. The chains of cocci were of the type ' longus '. Gram's stain was only moderately well retained. Broth cultures 24 hours old, examined with' the naked eye, showed a marked granular deposit at the bottom of the tube, the upper portions of the medium usually remained clear but also occasionally showed some cloudiness. Fermentation reactions. Broths containing lactose and saccharose were fermented, acid being formed. Eaf&nose, mannite, and salicin were unaffected. In litmus milk acid and clot were produced. Cultures in broth containing neutral red were rendered fluorescent after being incubated for 48 hours under anaerobic conditions. Gelatine cultures incubated at 20° C. showed little or no growth. (vi) Unclassified Streptococci. Streptococci giving characters which made it impossible to place them under any of the above headings were 17 in number, that is 10-625 per cent, of the 160 strains investigated. The 17 strains were isolated from 7 different patients. (a) Six of these strains which were isolated from four different patients formed a group with the characteristics given below. Three of them were obtained from blood cultures, on each occasion only one tube giving growth out of ten that were planted with the blood, it was, therefore, probable that the cocci were present only in very small numbers. In one of the above cases it was also isolated from the wound. The three cases were all treated with a vaccine made from the strain isolated from the individual case, but improvement as a result of this procedure was not marked. Morphology. In microscopical preparations made from a broth culture 24 hours old the individual cocci were very small, regular and round in shape. Gram's stain was not retained very intensely. The chains were usually of the type ' brevis ', but this was not so marked as in the case of Streptococcus faecalis or salivarius. To the naked eye a 24-hour-old broth culture generally showed some turbidity of the whole bulk of the medium and some deposit at the bottom of the tube. Fermentation reactions. Cultures made in broth containing saccharose were fermented, acid being formed ; lactose, raf&nose, mannite, and salicin were quite unaffected. Grown in milk, neither acid nor clot were formed. Gelatine cultures incubated at 20° C. gave good growth. Cultivated under anaerobic conditions for 48 hours in broth containing neutral red fluorescence was produced. When planted on coagulated egg cultures, if kept at room tempera- ture and drying up of the medium is prevented, the cultures remain alive for as long as seven months. No experiments were performed to ascertain its virulence for animals ; as regards its pathogenic 60 action in the wound the impression gained was that its presence was not of great importance. (b) The next group consisted of four strains which were isolated from two patients, and which had the following characteristics : Morphology. Microscopical preparations made from 24-hour-old broth cultures showed the individual cocci to be usually round, but even in young cultures a certain number of irregularly shaped involution forms were found. The chains formed by the cocci were rather intermediate in length but more nearly approached the type ' brevis ' than ' longus '. Gram's stain was retained less strongly than in the case of Streptococcus pyogenes or Jaecalis. Examined with the naked eye the 24-hour-old broth culture showed that growth occurred as a granular deposit at the bottom of the tube, the super- natant broth remaining clear. Fermentation reactions. Broth cultures containing lactose and salicin were fermented, acid being formed ; saccharose, raf&nose, and mannite were quite unaffected. In milk cultures acid and clot were rapidly produced. Cultures made in broth containing neutral red and incubated under anaerobic conditions for 48 hours showed no fluorescence. Gelatine cultures incubated at 20° C. gave good growth. No experiments were made on animals to ascertain if this Strepto- coccus was pathogenic, but in one of the cases, at least, it appeared to play an important part as regards the condition of the wound during a period in the healing process. This was Case No. 13 who had an extensive wound in the region of the right scapula. The wound after an initial period of severe sepsis had been progressing favourably, and during this period Streptococcus pyogenes had been found in moderate numbers. The wound, however, again became ■unhealthy looking and cultures made on several occasions showed the presence of very large numbers of the Streptococcus under discussion. The aspect of the wound was quite striking, the granula- tions being very flat, glazed, and rather dusky in colour. The skin round it was reddened and sore. A vaccine having been made from the Streptococcus isolated, small doses were administered ; the improvement was very striking and the wound rapidly became healthy, so that a short time afterwards a very successful skin- grafting operation was carried out. (c) The remaining 10 strains all showed individual differences which prevented them being allotted to definite groups. A consider- able proportion were isolated from pus which had been heated to 65° G. for 30 minutes. These heat-resisting strains differed among themselves with respect to their fermentation reactions. Their presence in recently inflicted wounds — from which they were usually recovered — is readily explained by the fact that they are commonly to be found in normal faeces. The impression was gained that the heat-resisting types had no pathogenic significance in connexion with the wounds. 61 (4) The Diphtheroid BacilU. (a) Definition. All Gram-positive bacilli which showed the characteristic so-called ' Chinese letter ' or palisade arrangement have been classed as ' Diphtheroid BacilH '. (6) Number of Wounds Infected. These bacilli were found either in cultures or in films of the pus from 54 patients at some period of their stay in hospital. From the specimens taken at the time of the patients' admission into hospital diphtheroid bacilli were found in 32 cases. Generally speaking it may be said that these baciUi are less commonly found in recently inflicted wounds than in the later stages, when granulation and healing of the wound is taking place. In many of the pus films examined, diphtheroid bacilli were to be seen in enormous numbers both inside and outside the pus cells. It was a common thing to see here and there a pus cell packed with them, just as in gonorrhoeal pus occasional leucocytes are seen to be filled with Gonococci while the majority contain none. When such a pus was cultivated in the ordinary way there was often a surprisingly small yield of diphtheroid bacilh ; indeed frequently none grew although they had been seen in large numbers in microscopical specimens of the pus. It is possible that some of them were obligate anaerobes, and from one case (No. 24) such an anaerobic diphtheroid bacillus was recovered. Wright i, and Fleming', have described under the name of the ' wisp ' bacillus an anaerobic diphtheroid bacillus occurring frequently in the early stages of wound infections. (c) Possible Sources op Infection. Diphtheroid bacilli are commonly found on the human skin, and since many of them are notably serophytic organisms (i. e. are able to multiply in the unaltered serum), their presence in the wounds may well have been due to direct infection from the surrounding skin. Doubtless also they were at times passed on from case to ease, but in the present series of cases it is unlikely that this was so to any marked extent ; for, as will be shown later, diphtheroid bacilli differing markedly from the more ordinary types and showing the characters of true diphtheria bacilli were found in four cases. These bacilli were found in patients widely separated from each other in the wards, and there was no evidence of spread of this type of bacillus from any of these patients to others in the neighbourhood. It is possible that in some cases the bacilli seen in pus films were in reality acne bacilli. This bacillus, which in pus presents a very definite diphtheroid arrangement, is one of the commonest inhabi- tants of the skin and, if it gained access to the wounds, would be missed in the routi'ue examination by cultures examined after 24 or 48 hours as its colonies do not become visible, even on a specially 1 Wright, Brit. M. J., 1915, i. 629. = Fleming, Lancet, 1915, ii. 638. 62 suitable medium, for three or four days. The possibility of some of the diphtheroids being acne bacilli did not occur to the authors until after the investigation was completed, so there was no opportunity of testing it. (d) Methods op Isolation and Differentiation. The bacilh were isolated by picking off single colonies from plate cultures of the pus grown either anaerobically and aerobically for 24 or 48 hours at 37° C. The cultures were maintained on agar, but at some period cultures were made on coagulated serum and egg medium so that the morphology could be studied and the presence or absence of Ernst Babes granules could be noted. Tor the fermen- tation tests Cole and Onslow's digested casein broth was used as a basis, with the addition of acid fuchsin as an indicator and 1 per cent, of the appropriate fermentable substance. La a number of cases it was noticed that in- cultures which were believed to be pure, there were colonies of very different sizes, some being very large like those of Staphylococcus, while others more resembled those of Stre'ptococcus pyogenes. On a number of occasions these were picked off and thoroughly tested, but it was found that there was no difference in the appearance of the organisms composing the large and the small colonies ; and that subcultures made from each type gave rise to a mixture of large and small colonies. (e) Characters of 61 Strains of Diphtheroid Bacilli. The characters of 61 strains, isolated from 21 patients, are set forth in Table XVI, and it can be seen from this that the bacilli arrange themselves into fairly definite groups. By means of the fermentation reactions the bacilli were split into four groups, and by other reactions two subgroups were separated from them. The main characters of these groups and subgroups are set forth in Table XVII. There were certain minor differences within the groups. Thus in Group I, one strain (Case No. 1, Strain 2) showed bacilli which were larger than usual and which took the strain very irregularly, in this way resembling the bacilli of Group II. Three strains also of Group I grew on agar in colonies resembling streptococcal colonies and not in the large opaque colonies which were usual. In Group II also one strain (Case No. 4, Strain 1) gave Streptococcus-like colonies. One strain of Group IV fermented lactose. It is possible that Groups I and II are in reality the same and that the mannite fermentation is of little importance. The authors have shown that in the case of Streptococcus pyogenes some strains ferment mannite although serologically they are apparently identical with the non-mannite fermenting strains, and possibly the same holds true for the diphtheroid bacilli. Mannite is much less easily broken up by these bacilli than either glucose or saccharose, and the acid formation, in almost all cases, did not appear before the second or third day of incubation. 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S jg dgd'^S'd ^ g g'^ 3 o d § d^ £ o*^ g'^^ g d f S oS3g5 So o°S SSBgo &" o gS &3 >i o L/^ Lj o o F-^ o lj f^ bO tiD tioTs QJ bJO le taO bO bD m^ ^ n-, '"' hn fan '-^ --I '^ i-it-i t-i'^ uir-( ji i^ F-iti ce ^^A H^iZ i-1h1 oQi^ h^Hl iA >A tA a % 3 ~ 00 " -~ 00-00 05 . -00 . Cl § (M t— < lO C-l to e rH M CO riJ lO O --I --H C^ CO "* lO CO I> ^ Cq CO ri4 rt Cvl fe; ^^5 ■mimij OOO OO ocooo c o o o o ooooo o 'uioiing coo CO ooooc o o o o o ooooo o ■3soniffv}j OOO CO ooccc o o o o o ooooo o ■uufx-}(j <;oo ou ooooo o o < o o oooo<; o ■uuxi/i20 2 1^ y^ h^ .t3 o rt "3 " S 02 ■8-2 ? Tjl id rH t^i fO Tj^ id ^ r-H ^ 00 o '^ '^ ^ "-^ -t -^ « -o O * " 'O "O o o 1727 nHM>— I r-* NCO"^ F—^i— iCqCO <-H(Mi— iC^'fO-^ld 66 + M "-a .2 , ^ 3 art 5P® jP p^ DO q o c3 Hi n ■< EH fH l>>2 -.T3 ^ x bo aj n-i *! T) n p! rrt ^ rh q > l-t t-H 3 s § .2 ■s g ^ S fi 5 >: (-( Qo © o-^ Er« fJ2 rJ2 o -*3 4:1 09 <1 El ' ^ to ia 3 w°. f^ op S3 -1* -a Q m 6 a 9, 67 Group la seemed a very definite entity because of its marked coloration of the culture medium, but in all other respects it resembled Group I. It is to be noted that in several cases it occurred in wounds together with bacilli of Group I. Group III appears to be identical with Hoffman's bacillus both in its morphology and in its inability to ferment any of the test substances. Considerable interest attaches to Group IV, as the bacilli included in it appear to be true Klebs-Loeffier bacilli. Their extraordinary resemblance to true diphtheria bacilli was noted at once by an examination of a film of the original cultures isolated. In specimens made from young cultures on serum, Neisser's stain showed polar granules in almost all the bacilli, and when guinea-pigs were injected with 1 c.c. of a 24-hour broth culture they died with lesions identical with those which follow the injection of true diphtheria bacilli. One of the cultures resembling B. diphtheriae was non-virulent for guinea-pigs, as were all the members of Groups I, la, and II which were tested. The patients from whom these cultures of true diphtheria bacilli were isolated showed no clinical signs of such an infection. There was nothing at all resembling a diphtheritic membrane, and there were no special constitutional disturbances. (/) Incidence of the Different Types OF Diphtheroid Bacilli. Table XVIII shows the arrangement of all the strains tested into their different groups, and it will be seen from this that of the 21 cases from whom diphtheroid bacilli were isolated, bacilli of Group I were found in 16, Group la in 4, Group II in 7, Group III in 3, Group IV in 4, and Group IV a in 1 . In a very large number of cases the diphtheroid infection was a mixed one. In four of the cases no less than three types were found, and it is worthy of note that in each of the wounds harbouring virulent diphtheria bacilli there were found representatives of one or more of the other types. Note. In September 1917 Fitzgerald and Eobertson^ reported that true diphtheria bacilli were found in the wounds of 40 out of 67 patients arriving in Toronto between May 20 and June 7, 1917. In some cases, but not in all, the wounds showed a characteristic diphtheritic membrane. The extraordinary prevalence of this mfection was explained by the fact that one of the nurses in charge of these wounded men had a slight wound on the finger, from which B. diphtheriae was isolated. Fitzgerald and Eobertson's report led to an investigation of the bacteria of the diphtheroid group in wounds in Canadian Hospitals in England, and the results of this investigation have been pubhshed by Adami and others.^ Out of 306 cases investi- gated, bacilli were found in four which had the morphological and cultural characters of B. diphtheriae and of these two were found to be pathogenic to animals. 1 Fitzgerald and Robertson, J. Am. M. 'Ass., 1917, 69, 791. " Adami and others. Bull. Can. Army Med. Corps, June 1918. 68 Table XVIII. No. of strains tested which fell into Group s. Case No. / la II. III. IV. IVa 1 4 2 2 1 3 3 3 1 4 3 1 G 1 1 . 3 8 6 9 1 10 2 11 1 12 1 13 1 14 2 18 2 2 1 21 1 1 24 1 2S 1 3 26 1 27 1 28 2 1 29 1 1 31 4.«1 AT^ ^£ 3 1 1 Total No. of cases in which the different types were found . 16 4 7 3 4 1 The Canadian workers investigated the characters of the various diphtheroid organisms in the wounds and classed them into five groups by means of their fermentation reactions. The results are shown in the following table : Table XIX. No. Dextrose. Lactose. Saccharose. Dextrin. 6 A A A A 6 A A A 23 A A ^1 ^ A A 2 ■ Type. Wound diphtheroid No. 1 . „ 2 . »» >» »> "^ * (Xerosis type) \ B. diphtheriae : Virulent Non-virulent . Hoffmann's bacillus . These tests were carried out in Hiss's serum water medium. It is to be noted that the Canadian observers found that a con- siderable number of the strains had the power of fermenting lactose, whereas the present authors in their series found only one strain (a true diphtheria bacillus) to have this property. Eeference to the older literature on the subject indicates that the fermentation characters of B. diphtheriae are not quite constant, and possibly the difference in the nature of the test media employed accounted for the difference in the results obtained. There seems no doubt, how- ever, that the common diphtheroid bacillus found in war wounds in England is a bacillus of the xerosis type which ferments only glucose and saccharose and is non-pathogenic for laboratory animals. {g) Significance of Infection with Diphtheroid Bacilli. Diphtheroid bacilli in wounds have almost always been regarded lightly. They are practically never found alone, and as their direct 69 pathogenicity is slight they are overshadowed by the Streptococci which are almost invariably present. It has been shown, however, that they exercise a powerful symbiotic influence on some of the pathogenic bacteria (vide Douglas, Fleming, and Colebrook ^), and in this connexion their presence in an infected wound may be of considerable importance. (5) B. pioteus. (a) Number of Wounds Infected and Pebsistbncb OF THE Infection. B. proteus was isolated from the wounds of 40 out of the 61 cases investigated, on some occasion during their stay in hospital. Unlike the Streptococci, however, it was often only found after the patient had been in England for a varying period ; thus 17 out of the 40 cases failed to give cultures when examined immediately after their admission, but did so at varying intervals of from 1 to 59 days later. It is probable, therefore, that this bacillus isusuallyahospital infection. As regards the length of time that the infection persisted in the wounds, there was considerable variation. In those oases where the wound was well drained, involved only soft parts and was in a healthy state, the infection frequently disappeared in a few days, but when sloughs, or especially sequestra, were present, the infection lasted for months, the bacillus apparently finding a nidus quite inaccessible to the antibacterial properties of blood fluids and cells. Wounds infected with B. proteus showed no very distinctive characters ; frequently, however, the surface had a somewhat unhealthy appearance and any newly formed epithelium tended to break down. (b) The Isolation of B. Peotetjs and its General Characters. B. proteus, as its name implies, is very pleomorphic in its mor- phology. Specimens made from young broth or agar cultures showed the predominating form to be a short bacillus Avith rounded ends, but long thread-like forms are not uncommon. The bacillus is easily stained with any of the basic dyes but doesnot retain Gram's stain. It is actively motile and, when suitably stained, shows peritrichous flagellae. Owing to the characteristic growth on agar being the formation of a spreading film, which usually, in 16 to 20 hours, covers the whole agar surface of the plate or tube, the isolation otB. proteus is peculiarly easy, and for the same reason it is very seldom that it escapes recogni- tion when present in a sample of pus. All that is necessary for the isolation of pure cultures is to make a subculture from the film as far as possible from the track of the needle with which the plate or tube was inoculated, so as to avoid the chance of picking up other bacteria that may be growing below the film. The appearance of the growth on agar is fairly characteristic, the most marked feature, as already mentioned, being the formation of a film which rapidly spreads over the whole surface of the medium. This film is usually quite uniform, very little if any thickening being present to show where the colonies commenced. Its edge is usually ' Douglas, Fleming, and Colebrook, Lancet, 1917, i. 604. 70 smooth but somewhat irregular, and slightly thinner than the central portions. In appearance it is iridescent, especially when an agar slope is examined with tra.nsmitted light, but so colourless that in plate cultures after 16 to 20 hours' incubation, when the whole agar surface is covered with a thin layer of growth, it is almost invisible, and it is only when a portion of the film is removed with a platinum needle that the growth becomes evident. In older cultures the film becomes much thicker and assumes a dirty whitish colour, and often the medium throughout its whole thickness becomes stained a dirty brownish tint. The consistence of the agar is also altered in old cultures, the jelly becoming less coherent so that the surface breaks easily under the platinum needle. Broth Cultures. In ordinary broth growth is abundant, an even turbidity being rapidly formed. With age the density increases and a collar of growth collects round the tube at the surface of the broth, but usually no true veil of growth is formed over the surface. At the same time there is a considerable deposit of growth at the bottom of the tube, but the intervening broth remains cloudy. The medium may assume a dirty brownish tint. These cultures have a very disagreeable smell. Gelatine Cultures. In stab cultures growth at 20° C. is rapid, a line following the track of the needle being visible in 24 to 30 hours. Liquefaction of the gelatine rapidly takes place, commencing almost as soon as the growth is visible at the surface of the medium immedi- ately around the needle track. The liquefaction spreads rapidly over the surface and downwards along the track of the needle, and a large proportion of the gelatine is usually completely liquefied in seven to nine days. On gelatine plates, prepared with 3 to 5 per cent, gelatine, growth at 20° C. is rapid and a characteristic appearance is shown soon after liquefaction has commenced. The centres of the colonies appear like dewdrops lying in a depression of liquefied gelatine, while very fine branching threads of growth can be seen radiating into the still solid medium. Milk. Growth on milk is rapid and abundant ; the casein is precipitated and digested, and if litmus has been added the colour is usually rapidly bleached. The culture at this stage usually shows a faint dirty brownish colour with some partially digested casein at the bottom of the tube and turbid dirty whey above. The reaction is often slightly acid in the very early stages, but the medium rapidly becomes increasingly alkaline. Fermentation of sugars, dc. Fermentation tests for the identifica- tion of B. proteus are very unsatisfactory, as the bacillus has the power of bleaching almost all the indicators ordinarily used in such tests. Further, although the bacillus has the power of breaking down certain sugars, for instance glucose, with the production of acid and gas, it has a very marked property of attacking proteid and its derivatives and during the process liberating a considerable amount of alkali. In consequence a culture may, after showing an initial acidity with accompanying gas production due to the action of the bacillus on the sugar or alcohol, again become alkaline as the result of the breaking down of peptone, &c., with the production of an excess of alkaline products ; under these circumstances cultures may show gas production and, at the time of examination, a strongly alkaline 71 broth. It may be noted, however, that mannitol is unaffected by all strains of B. proteus. One biochemical test useful in recognizing B. proteus consists of planting the organism into slightly acid or neutral urine and incubating the culture at 87° C. Growth takes place rapidly, and after 4 to 6 hours' incubation the urine is found to be strongly alkaline and free ammonia is given off. Indol was not produced by the strains of B. proteus obtained from wounds (Wolf).^ When grown under anaerobic conditions in a mixture of serum and trypsin, large quantities of gas are produced. Wolf working with certain selected strains isolated from these wounds found that B. proteus had but comparatively feeble proteolytic properties and should not be placed among the putrefactive organisms. (c) Serological Eacbs of B. proteus. A series of experiments were carried out to ascertain if B. proteus isolated from wounds consisted of a single or a number of serological races. Two agglutinating sera were prepared by inoculating two rabbits, each with a vaccine prepared from a single strain of B. proteus. The agglutinating titre of these two sera to emulsions of the homo- logous organism was found to be 1 in 64,000 in the one, and 1 in 8,000 in the other. Thirteen strains were then taken, and, emulsions having been prepared by diluting down broth cultures to a suitable opacity with salt solution containing 1 in 1,000 formalin, agglutination tests were carried out, each emulsion being tested with both the above sera. The results showed that the more powerful serum agglutinated all the thirteen strains, which included the homologous bacillus, to practically the same titre, namely a dilution of 1 in 64,000, but the other serum only agglutinated two -or three of the same strains tO' approximately the same titre (1 in 8,000) as an emulsion of its- homologous strain. The conclusion deduced from the above tests and a large number of absorption experiments was that certain strains of B. proteus contained in their structure a large number of antigenic components, while other strains only contained a very limited number, with the result that a serum prepared from a strain containing the large number of antigenic components would tend to agglutinate a large number of strains, while that made from a strain possessing but a limited number of components would have the power of agglu- tinating but a small number of strains. (6) B. pyocyaneus. (a) Number of Wounds Infected — and Variation of the Strains as Eegards Chromogenic Power. B. pyocyaneus was isolated from 31 of the 61 cases investigated, i. e. 50-8 per cent. The cultural, morphological, and most other characteristics are so well known that it is not proposed to discuss them. One point, however, was especially noted — namely, the variations in the chromogenic power of the different strains. This appears a constant property of an individual strain and showed ' Wolf, J. Path, and Bacteriol, 1919, 22, 289, 72 very wide differences, the colour produced in agar cultures varying from a very pale green to a deep reddish black. Occasionally strains isolated from the same patient gave different depths of colour. As regards the period of infection, this pursued a very similar course to that described in the section dealing with B. proteus, and the conclusion arrived at was that B. pyocyaneus showed a great tendency to be spread from patient to patient during their stay in hospital. In some cases the length of time the infection persisted was very short, in others, however, as with B. proteus, probably owing to the presence of sloughs or sequestra, it was much more prolonged. (&) Pathogenicity. At times the presence of the organism in the wound seemed to cause no change in the condition of the wound or the patient ; at others the infection coincided with an increase in general symptoms, such as pyrexia, and a cessation of the healing process together with an unhealthy appearance in the wounds. In such cases the treatment with vaccines gave very satisfactory results (vide Part I, p. 14). Owing to the difficulty of producing satisfactory agglutinating sera for B. pyocyaneus the question of the existence of a single or a variety of serological races was not investigated. (7) Staphylococci. These cocci were very commonly found in the wounds, but as they appear to differ in no way from the Staphylococci which flourish in the ordinary open wound or superficial ulcer, no detailed description of their characters is necessary. Staphylococci were found in 41 wounds at the first examination after the patients' admission to hospital. The patients had been wounded at intervals varying from 4 to 65 days before their arrival in London. In 14 of the wounds Staphylococci were not discovered at the first examination, but were found subsequently during their stay in hospital. The interval between the admission of these patients and the discovery of Staphylococcus varied from 2 to 58 days. In 9 of the wounds no Staphylococci were found at any time. It is clear therefore that infection with Staphylococci was extremely common in these wounds. The type of coccus varied, and although the ' aureus ' variety was the most common, cocci of the ' alhus ' type were frequently found. Staphylococci giving the pale greyish colonies characteristic of S. epidermidis were only rarely found. (8) Conform bacilli. In this group are classed all the bacilli, except B. pyocyaneus and B. proteus, which in their morphological characters and in their profuse growth on ordinary media, resembled B. coli. They did not liquefy gelatine. Such bacilh were found in most of the wounds at some period, but they did not usually persist. They were so varied in their characters that their classification has not been attempted, but a brief description of each of them will be found in the bacteriological notes appended to the case report of the individual patient from whom it was isolated (Part I). PART III EXPERIMENTAL WORK BEARING UPON THE ACTION OF CERTAIN ANTISEPTICS IN IN- FECTED WOUNDS (1) Introductory. In determining by laboratory tests the usefulness of this or that antiseptic in the treatment of septic wounds many methods have been employed. The old method of determining the lethal action of the antiseptic on bacteria in a watery medium, while it is useful in other spheres, is valueless when it is desired to estimate the value of a bactericidal agent in a wound. For the treatment of an infected wound an ideal antiseptic ought to possess, amongst others, the following properties : 1. It must be capable of killing bacteria in the discharges of a wound, i. e. in serous fluid containing many leucocytes. 2. It must be capable of penetrating into small diverticula such as exist in all septic wounds, and of destroying the microbes in such recesses. 3. It must have the power of penetration into the walls of the wound so as to kill the bacteria which may flourish there. 4. It must not exercise any local destructive action on the tissues or interfere with the physiological properties of the tissue fluids or cells. 5. It must not, in the concentration employed, exercise any general toxic effect on the individual. Up to the present no antiseptic has been produced which ap- proaches the ideal. All the antiseptics in common use have a much greater destructive effect on animal cells than they have on bacteria ; none of them are capable of penetrating into the walls of a wound and exercising any destructive action on the bacteria without at the same time killing the tissue cells ; and none of them can penetrate into small diverticula of the wound and kill the microbes in such situations. It was with such a scheme of the properties for an ' ideal ' anti- septic constantly in mind that the following experiments were carried out. (2) Experimental methods. A large part of the v^ork consisted of in vivo experiments con- ducted upon two cup-shaped wounds, which lent themselves par- ticularly well to investigation since a measured quantity of fluid could be readily introduced into them, retained for a while, and then removed for examination. These two wounds will hereafter be designated ' P ' and ' C '. Both were cup-shaped depressions leading down into the cancellous tissue of the tibia, and both resulted from a compound fracture of this bone. They were each capable of retaining about 2 cubic 74 centimetres of fluid, and, being old wounds of many weeks' standing, were lined with granulation tissue. In many respects, however, they differed from one another ; the one ' P ' had walls of granulation tissue, but the base was occupied by a large sequestrum of spongy bone. From this wound there was a copious discharge of pus containing about 1,000,000 leucocytes per c.mm., which was heavily infected with anaerobic spore-bearing bacilli, Streptococci, coliform bacilli (mostly B. proteus), and diphtheroid bacilli. The volume of the discharge varied considerably from day to day and sometimes was as copious as one c.c. per hour. The other wound ' C ' was wholly lined with granulation tissue, and, except under the action of certain solutions which will be described later, the quantity of discharge was fairly constant, amounting on the average to 0-2 c.c. per hour. This wound was infected only by pyogenic cocci. The following procedure — or some slight modification of it — was employed in working with these wounds : the wound cavity, having been completely emptied, was washed out with repeated changes of normal saline solution until the washings were quite clear. All fluid was then carefully removed. After an hour the total exudate into the cavity was measured by sucking it up into a graduated pipette — and was retained for examination. The quantity and character of this exudate served as a control observation. After again washing out the cavity with normal saline and emptying it, exactly 1 c.c. of the antiseptic under investigation was introduced. This was allowed to remain for 5 or 10 minutes and then removed for examination after carefully mixing the whole contents of the cavity by sucking into and blowing out of the pipette. The quantity of fluid so removed .was noted as before. The wound was now filled in a second time with 1 c.c. of the antiseptic solution which was left there for a longer period, say half an hour, and then again removed for examination. This pro- cedure was repeated as often as was required. A number of samples of the antiseptic solution after different periods of contact with the wound having in this way been collected, each was examined in order to ascertain the following points : 1. The number of living bacteria present. 2. WTiether the antiseptic solution had diminished in strength while it was in the wound. 3. The number of leucocytes present, and their condition. 4. Wliether there had been exudation of serous fluid into the cavity — or,_on the other hand, absorption from it — while the anti- septic was in the wound. 5. What was the anti-tryptic value of the serous exudate ? ■V^Tiere other experimental methods were employed, they wfll be described separately in connexion with the results obtained from each. (3) Experiments bearing upon the ability of antiseptic solutions to kill bacteria in the discharges from an infected wound. An answer was first sought to the question— Does a marked reduction in the number of living bacteria actually occur when antiseptics are introduced into an infected wound ? 75 The antiseptics chosen for this inquiry were Dakin's solution, eusol (hypochlorite solutions) and chloramine T, as being bactericidal agents of rapid action — and flavine, as representative of the more slowly acting agents. Each of these was instilled on different occasions into one of the wounds ' P ' or ' C ' as described above, and the bactericidal effect determined by enumerating the living organisms per c.c. of fluid present after varying periods of treatment with the antiseptic. The enumeration of the bacteria was made as follows : imme- diately a sample had been pipetted out of the wound cavity 10 c.mm. were taken up into a capillary pipette and thoroughly mixed with 50 c.c. of agar which had been melted and allowed to cool to 45° C. This mixture was poured into a Petri dish and allowed to solidify. To avoid surface growths which were very apt to spread over the medium, a second layer of melted agar at 45° C. was now poured into the dish. The plates were then incubated for 48 hours, when the colonies were counted. Experiments with Dakin's solution^ (1) The wound ' C ' was washed out, a sample of the normal exudation collected after one hour, and the viable microbes in it enumerated. Dakin's solution was then placed in the wound, replaced by fresh solution 5 times within 75 minutes and then withdrawn. One hour and 2 hours later, samples of the exudate were taken from the wound and the living bacteria in these enumerated. The 3 samples gave practically the same number of colonies. (2) The Dakin's solution was applied to the wound 4 times during four and a half hours and then withdrawn. A sample of the exudation occurring before the application gave 91 colonies — a similar sample of the exudate taken 1 hour after the antiseptic had been withdrawn gave 95 colonies. Experiment with Eusol. This was conducted in a slightly different manner. The wound ' P ' was filled in with eusol ; 10 c.mm. samples were withdrawn at intervals of 5, 20, 30, and 60 minutes, diluted 1,000 fold in normal salt solution to avoid carrying over any appre- ciable amount of antiseptic, and the bacteria then enumerated as before. The result is shown in Table XX. Table XX. Time the Eusol remained No. of colonies Calculated no. of colonies in the wound. developing. per c.c. of fluid in the wound. 5 mina. ... 2 200,000 20 „ ... 50 5,000,000 30 „ . . . 40 4,000,000 60 „ . . . 25 2,500,000 With regard to this result it will be seen in the section immediately following, that the hypochlorite concentration of eusol was reduced more than 90 per cent, after being in the wound 10 minutes. There- fore, the lower bacterial count obtained with the 80 and 60 minute samples is probably not attributable to any bactericidal effect of the antiseptic ; it is much more likely that it was due to the destruction of bacteria by the leucocytes, which emigrate into, the wound in ' Daufresne's modification. 76 great numbers as soon as the hypochlorite concentration is sufficiently lowered (vide p. 78, Expt. 1, and also p. 108). Exveriments with Chloramine T. (1) A 1 per cent, solution was placed in the wound ' P ' and samples taken for enumeration ot the bacteria at intervals, as shown in the following Table XXI. Table XXI. Time the solution had remained Number of colonies developing in wound. on plates. 10 minutes . . . .No growth visible after 24 hours. Innumerahle colonies after 4 clays' incubation. 20 .... Ditto. 30 "... . Innumerable colonies after 48 hours' incubation. 60 "... . Ditto. ' This experiment shows that the appUcation of a 1 per cent, solution of chloramine T to a heavily infected wound caused no appreciable diminution in the number of living organisms present, whether that application was short or prolonged. The delayed growth of the agar cultures was presumably due to a certain amount of uncombined antiseptic carried over in the sample of fluid used for the enumeration. It will be noted that, in the case of the samples taken from the wound after 30 and 60 minutes appli- cation of the drug, growth occurred much more rapidly than in the earlier samples ; and reference to Section 4 will show that the reason for this, viz. that the active principle of the antiseptic had, within 30 minutes, been exhausted by combination with the tissues and exudate of the wound. It will be instructive to refer here to Fig. XVIII, which depicts a film preparation made from the same wound on another occasion after a 1 per cent, solution of chloramine T had been applied to it in four changes during one hour. When so applied the antiseptic may be said to have had its maximum opportunity of exciting a bacteri- cidal effect and yet the preparation shows a great proportion of microbes — not appreciably less, in fact — than were present in the wound when untreated. And the result given in Table XXI makes it clear that the microbes seen in the film were chiefly living organisms. It may be stated that film preparations made when testing the bactericidal effect of Dakin's solution and eusol (vide p. 75) showed precisely similar appearances. (2) With a 4 per cent, solution of chloramine T the following result was obtained : Table XXII. Source of sample tested. Mo. of colonies developing from 10 c.mm. samples. Preliminary wash with normal saline . . 872 (Surface film of B. proteus). Chloramine solution after 5 minutes in the wound 4 7j >j )j ^0 „ „ „ 5 )> ' >> » 20 „ „ „ 30 (Surface film of B. proteus). " " " 3*J » >> » 79 „ „ „ » '. " 50 .. ,. „ 764 „ „ Wash with normal saline solution immediately after the withdrawal of chloramine solution . . 800 Ki(_; XVHI. A tilm preparation of the exudate from the same wound pasHing; into 1 per cent, ehloramine T after that solution had been applied four times within 00 minutes. 77 Experiments with Flavine. These yielded unsatisfactory results, and as the figures obtained are quite unreliable they will not be given. It was necessary when investigating the antibacterial effect of such a slowly acting antiseptic to estimate the number of living bacteria after the fluid had remained in the wound for several hours. After such an interval a sputum-like membrane had formed on the walls of the wound, and during its formation most of the bacteria were found to have been caught up in its meshes, and in consequence it was quite impossible to emulsify them for the pur- poses of the test. The result of this was that the plate cultures made for the enumeration of the bacteria gave dense clumps of mixed colonies which it was impossible to count. Completely sterile cultures were never obtained ; and the number of living microbes in the fluid appeared to increase the longer the sample of flavine solution remained in the wound, up to 15 J hours. Conclusion. The above experiments show that, in the cases of Dakin's solution, eusol, and chloramine T, at any rate, no appreciable bactericidal effect was manifested upon the bacteria present in the wounds ; in the case of flavine, the results were inconclusive, but showed, at any rate, nothing like a sterilization of the wound. This last finding was confirmed by bacteriological observations on many other infected wounds treated with this dye. (4) Experiments carried out with a view to showing why the antiseptics in common use fail to sterilize infected womids. The fact that antiseptics fail, as has been shown above, to exert an appreciable bactericidal effect in the wound, suggests that their potency is in some way dissipated by contact with the walls of the wound, or its discharges, or by both these factors. The following experiments were undertaken to show whether, in the case of Dakin's solution, eusol, chloramine T, and flavine, such deterioration of strength could be demonstrated when these solu- tions were placed in a wound, and, if so, to indicate the rapidity and degree of the change. The destruction of antiseptic potency in loounds. Experiment with Dakin's solutio7i. The wound ' C ' having been carefully washed out, it was filled with Dakin's solution and samples removed at intervals for titration of the hypochlorite against sodium thiosulphite and potassium iodide solutions as described in Carrell's book 1 ; the technique being modified in order to make the method applicable to small volumes of fluid. The accuracy of using small volumes was tested by titrating repeatedly 10 c.c. and 0-1 c.c. volumes of Dakin's solutions, when it was found that the results corresponded closely. All estimations were carried out immediately the fluid was removed from the wound. By reference to Fig. XIX it will be seen that after the Dakin's fluid had been in the wound for 5 minutes the concentration of hypo- chlorite had fallen to one-fourth of its original titre, and in 40 minutes it was only one-seventh of its original strength. When it is remem- bered that the wounds employed in carrying out these experiments 1 Carrell et Dehelly, Le traitement des plaies infectees, Paris, 1917. 78 were cup-shaped depressions and in consequence the fluid in contact with the walls was only a small proportion of that contained in the wound, it will be seen that rapid as was this loss of strength of hypochlorite it would most probably have been even more rapid Tftre of Hypochlorfte 0-5%r 0-4% 0-3% 1 V, --— - Minutes 5 10 20 60 Time fluid remained in wound. Fio. XIX. Rate of destruction of Dakin's solution in a wound. 0-3% X 0-27o _ ^ o-^% O 30 "fO s /o so Time fluid remained in wound. Fig. XX. Rate of destruction of eusol in a wound. Unbroken line shows result of Experiment 1. Broken line shows result of Experiment 2. if the wound had been a comparatively flat surface, and the fluid had been syringed in under a dressing. This point will be raised again when dealing with eusol. Experiments with Eusol. In these the hypochlorite was esti- mated by titration as above. (1) The result obtained in an experi- ment on wound ' C ' is shown in Pig. XX. 79 This fig. shows the extraordinary rapidity with which the concen- tration of hypochlorites is diminished almost to a vanishing point. In 5 minutes the concentration of hypochlorites fell to about one- fifth, and in 10 minutes to one-tenth of the original strength. When these figures are compared with those giving the concentration of hypochlorite which inhibits the growth of micro-organisms (see p. 82) in a serous fluid, it will be seen that the antiseptic action of eusol applied to a wound is only momentary. (2) Another experiment was carried out on a clean shallow flesh wound of about one week's standing. This wound had a capacity of about 2 c.c. of fluid. Two c.c. of eusol were now introduced and kept constantly stirred, the technique in this way differing from that used in the first experiment when the eusol, after introduction, was left undis- turbed. After an interval 1 c.c. of this fluid was withdrawn and immediately placed in 2 c.c. of a 10 per cent, potassium iodide solution. The cavity was now emptied and dried and 2 c.c. of fresh eusol introduced. After an interval 1 c.c. of this second application of eusol was withdrawn and immediately placed in potassium iodide solution. By such procedures specimens of eusol which had been in a perfectly clean granulating wound for ^, 1 , and 5 minutes respec- tively were obtained. On titrating these against thiosulphite of soda it was found that in 5 minutes the eusol had retained only one-tenth of its original concentration of hypochlorites. This result is shown by the broken line in Fig. XX. Experiment iviih Chloramine T. The wound ' P ' was washed and then filled with a 4 per cent, solution of the antiseptic. At intervals 0-2 c.c. samples were withdrawn for estimation of their strength by the addition of 1 c.c. of 4 per cent, acetic acid solution, which caused precipitation of the chloramine. By the amount of precipitate the destruction of chloramine could be roughly gauged as shown in the following table : Table XXIII. Result of adding acetic acid. Preliminary wash "with chloramine T solution. Heavy white precipitate. Chloramine T solution after being in wound 5 min. Ditto. „ „ „ „ „ 10 „ White flooulent precipitate (much less). „ „ „ „ „ 20 „ No precipitate. 30 „ Ditto. „ „ „ „ „ 50 „ Ditto. These results indicate that chloramine T is very rapidly destroyed when brought into contact with. wound surfaces. Experiment with Flavine. A 1 in 1,000 solution was introduced into the wound ' C ' after it had been washed out, and was replaced by fresh solution after periods of contact with the wound varying from a few seconds up to 15^ hours. The strength of flavine in each sample was estimated by matching the colour on filter paper of drops of definite volume against similar sized drops of solutions of known strength. The results are shown in Table XXIV. 80 Table XXIV. Time during which flavine Concentration of flavine solution, solution had remained in the a similar volume of which placed wound. on filter paper gave the same colour. A few seconds 1 in 1,500 15 minutes . . .... 1 in 1,500 30 „ . . . 1 in 2,000 60 „ . . . . 1 in 3,000 2 hours . 1 in 3,500 4 „ ... . . 1 in 3,500 15J ,, 1 in 16,000 With regard to these results it must be remembered that the flavine solution was placed in a cup-shaped wound and in no way disturbed until the sample was withdrawn. Further, that before the last sample had been introduced the solution had already been applied to the wound (in several changes) for 7| hours, during which time the tissues had become heavily impregnated with the dye, and also coated with the sputum-like material which results from the action of the dye upon the discharges. These circumstances would tend markedly to delay the disappearance of the flavine from the solution, and this would have most probably been much more rapid . if the solution had been applied in a thin layer to the wound walls, a condition that would obtain in wounds dressed in the usual manner. Again, the conditions of the experiment differed from those of ordinary surgical practice in that no gauze or other dressings were applied to the wound. It will be shown later (p. 90) that cotton and other fabrics have the property of fixing very large quantities of flavine, so that when the solution is applied on gauze in the con- ventional manner of dressing a wound, the loss of strength of the dye must be very much greater and more rapid than was indicated in this experiment. (5) Experiments in vitro bearing upon the bactericidal efQcacy of antiseptics in the presence of serum. The foregoing experiments have shown that Dakin's solution, eusol, chloramine T, and flavine quickly lose a large part of their bactericidal potency when introduced into a carefully cleansed and empty wound. Infected wounds do not remain long, however, in that condition. Exudation of serum is usually abundant, and the leucocytes emigrate — as will be shown — in great numbers, as soon as the strength of the antiseptic has been somewhat reduced. It therefore becomes necessary to determine what bactericidal effect any given antiseptic is capable of exerting in a naedium consisting largely of serum, or serum mixed with leucocytes. In the experiments which follow, the quenching effect of leuco- cytes, -per se, upon antiseptic agents, has been left out of account, because it was impossible to obtain samples of pus which were in any way comparable. Serum alone has therefore been employed, or, rather— in order to approximate more closely to the conditions obtaining in infected wounds— serum to which has been added sufficient trypsin to just neutralize its anti-tryptic power. 81 Procedure and technique. It is, of course, necessary when testing antiseptics in serum to follow the routine of implanting the serum with microbes and then adding the antiseptic. This is especially important in connexion with the hypochlorites, which are very rapidly quenched by serum, since it would be manifestly unfair to such antiseptics to add them to the serum first and then at some later period to add the microbes. Trypsinized serum was therefore inoculated in bulk with the particular microbe chosen for the test and was then distributed in equal volumes into a series of small tubes, into each of which had previously been introduced the requisite quantity of progressively diluted antiseptic solution. Usually 9 volumes of serum were added to 1 volume of antiseptic ; in a few cases equal quantities were employed. In all cases control tubes were put up, in which the antiseptic was replaced by water or normal salt solution, according as the antiseptic had been diluted in one or other of these fluids. In a few experiments, where Staphylococcus was the organism used, the effect of the antiseptic was gauged simply by the complete prevention of growth in the serum, as judged by naked eye examina- tion or subcultivations ; more frequently gas-producing organisms were employed, and the amount of their growth in each of the mix- tures was roughly indicated by measurement of the gas formed. The technique employed for this latter purpose was practically that described by Beattie, in which melted vaseline is poured upon the top of the inoculated medium, thus making an air-tight plug which rises in the tube as the gaseous products of bacterial growth accumu- late. Small test tubes made from glass tubing of a calibre of 4 to 6 millimetres were employed. For each experiment these tubes were made from the same length of selected glass tubing, so that the calibre of all the tubes used in any one experiment would be the same. Thus the measurement of the height of the column of gas in the different tubes was directly comparable and served as a com- parative measure of the growth of the organism. Although there are certain objections to this method of estimating the amount of growth of a microbe by the amount of gas formation, the method was shown to be sufficiently accurate by two series of tests. 1. The number of microbes were counted in tubes showing, respectively, little and much, gas formation ; it was found that where there was more gas there were more microbes present. 2. On several occasions duplicate series of tubes were put up ; after an interval of 24 hours the amount of gas produced was the same in each tube within very small limits. In a few experiments conducted in this way, where the serum was inoculated with coliform bacilli (which do not make gas except in the presence of sugar) 1 per cent, of glucose was added to the trypsinized serum. Results of the experiments. Two sets of data have been furnished by these experiments. In the first place figures were obtained giving, for each of the common antiseptics, the concentration which was necessary to inhibit all growth of the microbe tested. And secondly, data showing the reverse effect, viz. a stimulation of the growth of the microbe in serum by smaller concentrations of some of these antiseptics. These two effects may be considered separately. 1727 F tel 8 8 ,3 =0 S •§■§1 i-H rH tH g a rt c "tils §s||£ og St , eq CD oo .a.s .s.s r— 1 r— 1 ^H 1— 1 1— ( T— 1 F-H o o ss (SO lOOO N eqoo -HOC .S.S .a .9 .a 1— 1 r-H .s.a 1— 1 r— 1 .S.a o'-ioX .a .a .a .a .a s s >< ^ -"S ■« .S.S I II ill c5 *0 O O CO o « .a .3 .a.s .S.S .S.S Kl sl^ w ^ "g o _ o oo -. O 00 (N lO (M 00 O O t-, O O So o_ I s|| .s.s.s.a.s.s l.s 11 X H n !? ■w O o « ^ .s to . iil'Ss Vj -5i iij ca ei S o 900 " CD o O |-<(MIN — I Til S5 .S .S.S .S.S I .s o§ 00 IM Highes concentr tion showint growth. (MtM S '-io -I TX Ttl 0^0 .3.3 i'Sf.ll o'.s s' 2 o a o ■=. 9 „ oo°.oco 'O -^H . cc CM (M iM rt .S.S I. a .3 .3 .3 .3 Izi < g.S § " fe : > c3 § -s a g " tt fe.2 0-3 wo 83 (a) Inhibitory Effect Exerted by Antiseptics on the Growth of Bacteria in a Serous Medium. Table XXV sets forth the inhibitory power of some of the common antiseptics when allowed to act in a serous medium. If it is examined in relation to the experiments quoted in Sections (4), (6), and (9), which show that the effective strength of antiseptic solutions is largely dissipated by contact with the walls of the wound, the cellular exudates and the dressings, it will be realized that these solutions can seldom exercise in the wound, even for a few minutes, any destructive or in- hibitory influence on the bacteria. In the case of Dakin's solution and eusol, for example, it has been shown on p. 78 that after 10 minutes in the wound these antiseptics have been reduced to at least one-tenth of their original strength, a concentration which is found incapable of even inhibiting the growth of microbes in serum. Similarly in the case of flavine, when allowance is made for fixation of the dye by the walls of the wound, the leucocytes and the dressings, it is clear that the -1 per cent, solution ordinarily employed will be incapable of destroying or inhibiting B. coli or B. fwteus, and it will not for long retain a strength of 1 in 16,000 which the table shows to be necessary in order to restrain the growth of the other bacteria. It will also be seen from this table that there is, with the commonly used antiseptics, very little variation in respect to the different organ- isms, whether they be spore-bearing or non-sporing bacteria. In connexion with the dye-stuffs it has long been known that members of the triphenyl-methane group, such as gentian violet, crystal violet, malachite green, &c., have a powerful bactericidal action in respect to the Gram-positive microbes, while they are very weak with regard to the Gram-negative organisms, such as B. coli or B. proteus. It was claimed for flavine that it had almost as powerful an action on B. coli as it had on the Gram-positive organisms, but this has not been confirmed by these experiments, for whereas it will in serum inhibit the growth of Staphylococci and B. welchii in a 1 in 16,000 dilution in serum, yet B. coli v/ill grow and produce gas in a con- centration of 1 in 1,000 and B. proteus in one of 1 in 2,000. With the dye-stuffs it is impossible to give a definite figure for the inhibitory power, just as it. is for their bactericidal power, as this varies so much with the number of microbes present. This difference of lethal concentration with the number of microbes acted on, manifests itself with all the common antiseptics, but whereas with a substance like carbolic acid it is very slight, with the dye-stuffs it is very marked. The extent of this difference is brought out in Table XXVI, which gives the results of adding different quantities of Staphylococcus to serial dilutions of carbolic acid, flavine, and crystal violet in nutrient broth. Two suspensions of microbes were used, one being 5 million times stronger than the other. Using carbolic acid the concentration necessary to inhibit growth with the stronger suspension was only twice that which inhibited growth with the weaker suspension, whereas with crystal violet it was sixteen times and with flavine at least sixty-four times as strong. F 2 84 Table XXVI. Antiseptic. Carbolic acid Crystal violet Flavine Strong suspension of Staphylococci. Greatest Least concentration concentration in which growth which inhibited occurred. growth. 1 in 400 1 in 400 1 in 320,000 1 in i:,o00,000 1 in 8,000 1 in 256,000 Least concentration which inhihiti^d growth. 1 in 200 1 in 100,000 1 in 4,000 Weak suspension of Staphylococci. Greatest concentration n which growth occurred. 1 in 800 1 in 5,000,000 (6) The Stimulant Effect of Antiseptics on the Growth of Bacteria in Serum. I I I I I I M I 'o O'S 0-25 0125 0-06 0-03 0015 Contrc-i Concentration of carbolic acid. Fig. XXI. Influence of carbolic acid on the growth of B. welchii in serum. ^_ % 0-5 0-25 0-125 0-0(i 0-03 0-015 0-007 Control, Concentration of carbolic acid. Fig. XXII. Effect of carbolic acid on the growth of B. sporogenes in semm. . In Fig. XXI is seen a set of tubules in which the stimulant action exerted by weak carbolic acid solutions, e. g. 0-03 to 0-25 per cent, (as well as the inhibitory effect of a stronger solution) , is well shown. The black columns represent the vaseline plugs. B. irelchii was the organism employed. Fig. XXII illustrates a similar experiment in which B. sjjorocjencs was used. All the results which follow were obtained in a similar manner, but for simplicity of representation the gas formation has Ijeen plotted out in columns or curves. Further experiments with carbolic acid are shown in Figs. XXIII and XXIV. Exjyeriments with iodine solutioti. Iodine, as can be seen by reference to Fig. XXV, showed only an inhibitory action when tested with B. sporogenes. With B. welclvU and B. tetanoides. however, there was evidence of stimulation in concentrations of 1 in 3'2,000 to 1 in 64,000, as is shown by Figs. XXVI and XXVII. UU "' cju/rj/o^ <^79{^ -/o :!i/^/Pf^ 86 Experiments icitli Eusol and Dakin's solutions. Eusol and Dakin's solution showed no stimulant action of B. icelchii, Aviiereas in the case of B. sporogenes the stimulant action was marked. Eusol also stimulated the growth of the B. tetanoides. These results are brought out graphically in Figs. XXVIII, XXIX, and XXX. In the experiment illustrated hj Fig. XXIX the implantation of microbes was small, so that the growth was very much delayed. In this case equal parts of serum and eusol proved a much better cultrrre medium than did equal parts of physiological salt solution and serum. Growth started first in the tube containing equal parts of serum and eusol and, as can be seen by referring to the Fig., subsequently developed in the other tubes of the series, last of all in the control. In a case such as tins, when a small number of microbes are implanted into unaltered serum the normal inhibitory action of the serum is sufficient to prevent growth uirtil some change takes place, either due to the products of the microbes or to some other cause. It would appear that eusol mai'kedly hastens this change. ■■ill i Jll O O o o o o c O O ^ O O O C ^trengzn -. r,, g ^j ;o ^ ^ o J o - ^ ^ o o - 22 /. „.— • / .^^ X --• SO if / /'' 5^ nV ,r-n •5 /6 - 7 1" - _ s / <§ / / • t ^ 'O " / r Control - / / / / ^.^■0-°"°'°" •^ / / .*-* ^ 6 - / / f / • •^ ' / y^-- 2 n 'l ^"T' 1111 1 1 1 1 1 1 1 1 1 i S /O ■, /S BO 2S SO ^5 -fo -fS so 56 SO eS TO 7£ BO Number of hours incubation. FiCf. XXVI. Effect of iodine on tlie growth of B. welchn in serum. so - // .-' ■40 - I-'/ .'' % \ V ^^f \ 30 > Ci / / .'- »-, ^ SO _ / / \ ■K> so so 70 SO 90 lOO HO /so Number of hotu-B incubation. Fig. XXVII. Efiect'of iodine on the growth of B. tetanoides in serum. Strength , ~ 7 T ? * 7 i Incubation 24 hours. Fig. XXVIII. Effect of Dakins solution on the growth of B. wekhii in serum Concentration of- 1 J J. 1 J. I Antisepdfi^ 2 f- 8 'i> 32. \ ll _/ J, s J. J. \ 2. *r 8 /6 32."^ IncvJaiioi: 27 lioiirs. 42 hours. Fic. XX\^[II a. Effect of Dakin's solution on the growth of B. xporogenes in serum. St-sr,gth Incxibaiion .3 days. 5 days. ^ n -o N i^ *- 6 dav8. ll_ IllU. C^ . L. ^ "J 'O j'- S^^^"9th -^ ^ 3 .3 Incufialiov 7 days. 8 days. Fig. XXIX. Effect of eusol on the growth of B. s-porogenes m serum. 81) X ■^'^H^tb J lui ^S:** -•^s C Q ft Ii ciibatiuii 24 hours. 48 hours. 72 houi.^. Fig. XXIX a. Effect o{ eusol on the growth of B. v:e\chii in serum. J l^ Li_ 1 1 , (\i »j- to lo M O -V"- -*. ^ ^ c; Incubation 3 days. 4 days. 5 days. Fig XXX Effect of eusol on the- growth of B. tetanoidcs in serum. X Strength o o "^ o o o o Tj CO to t\i *> OD "O - I-) '.6 <-M JO QOOQOOO Q O O O O O O D "}■ qj 'o '^ ^ 1) ''^ Incxihatxon 24 hours, 48 houra. 72 hours, Fig. XXXI. Effect of chloramine T on the growth of B. sforogevcs in serum, O O O O o o o 'J3 f»j T- IB lO ^J '3- Screnqt.'j - n ^ a oj « (a) Incuhaiton 24 hours. LiL Q o O O Q O i:i ♦ to lO C\J "* 13 iQ Strength - fro r>j m J o o o o Q o o *t m (O tM ^ ) 48 hours. Fig. XXXTT. Effect of chloramine T {a) on the growth of h. ivekhii in serum : {h) on the growth of B. profetis in serum. 90 (6) Experiments which serve to demonstrate the fixation of certain antiseptic dye-stuffs by the fabrics commonly employed in the dressing of womids. In Sections (4) and (5) attention has been directed to the quenching of antiseptic solutions by contact with (a) the fixed tissues of the wound and (fc) its serous discharges.. It will now be shown that a further considerable loss of bactericidal potency is traceable — in the case ■ of the antiseptic dyes — to the ready combination of the dye with whatever cotton or other fabric it encounters in the wound. In the first series of experiments a small pad of gauze was allowed to soak up a few c.c. of the dye solution. After contact with the gauze for varying periods the solution was squeezed out of the pad and its dye-content ascertained by ..matching the colour of drops against that of similar drops of known solutions of the_,'dye. The following results were -obtained ; (o) With brilliant green. 1 in 1,000. After contact with the gauze for 1 minute the strength of the solution was 1 in 25,000. After contact with the gauze for 3 hours the strength of the solution was 1 in 50,000. {b) With flavine. 1 in 1,000. Experiment 1. After contact for a few seconds the strength of the solution was 1 in 5,000. After contact for 2 hours the strength of the solution Avas 1 in 10,000. Experiment 2. With flavine solution. 1 in 4,000. After contact for 5 minutes the strength of the solution was reduced to 1 in 128,000. In a second series of experiments pieces of cotton bandage material of equal weight were packed into several lengths of glass tubing to form wads 2-25 cm. in depth. Equal quantities of various dye solutions were then poured into the tubes on one side of each wad and allowed to percolate through the bandage by gravity. Fixation of the dye by the cotton was indicated by the amount of fluid which passed through the wad uncoloured. The results are shown in the following table : Table XXVIII. Amount of filtrate passing through the bandage plug without any trace of colour. 1-7 cubic centimetres 0-75 „ 1-3 „ 1-5 ., 8 ,. (First drop had a faint pink tinge. ) (Tested by allowing to drop into alkali. ) FlaYiae ....,„„, , . Brilliant green. - - . '. " . Crystal violet ' ' . . Methylene blue Neutral red .... Acid Fuchsin (acid solution) „ „ (alkaline solution) Eosin . . . . . Phenol sulphonephthalejn . 91 (7) Experiments made to ascertain whether certain antiseptic solutions were capable oJ penetrating into — and kilhng the microbes contained within — small diverticula such as exist in all septic wounds. The ■sYGunds of the war were all, in the early sta^-c, ii-ivLjTilar in shape and had many small pockets and pi-ocesses passing off in different directions bet^Yeen the muscles or under the skin. In the hite stages of superficial -wounds the surface may Ijp flat but the granulation tissue can lie shown to contain many organisms below the surface. An attempt was made in the experiments descriljed below to reproduce these conditions, as far as possible, in vitro. Test tubes were converted into ' artificial wounds ' by draAving out from their lower ends several spike-like processes about a centimetre in length. The resemblance to the actual wound C(jndition was, of course, only rough ; actually in the wound the pro- cesses are much longer and freijuently they are more or less shut off from the general cavity by a narrow neck, whereas in the test tuljp they are widely open to the main cavity. This dissimilarity ought to have acted in favour of the antiseptic solution in our experiments. By the aid of such ' artificial wounds ' we were able to investigate the antiseptic action of fluids on bacteria in the cavity of a wound without the experiment being complicated by tire presence of organisms in the tissues or by pieces of slough in the wound. Fi<;. XXXIII. Test tube coiiA'erted into an ' artticial wound ' Experiment 1. Two similar ' artificial wounds ' were taken and into them was poured about 2 c.c. of human seruirr into which faeces had Ijeen planted. They were then incubated anaerobically for 24 hours. At the end of that time they coutaineil a mixture of bacteria very similar to those seen in a recently inflicted wound, i. e. B. loelchii, Streptococci, StapJujlococci, coliform bacilli, and other micro-organisms. One of the tubes was now ' dressed ' with eusol, by the simple method of pouring off the contents, and replacing them with eusol. After repeating this performance several times, the eusol was allowed to stand in the tube for 20 minutes, when it was poured off and replaced with sterile human serum. The other tube was treated in exactly the same way, using physiological saline solution instead of eusol. The tubes Avere then replaced in the inoul:>ator under anaerobic conditions tor 24 hours. This procedure was repeated every day for 12 days. Each day films were made from Ijoth tubes, and up to the end" of 12 days 'there was practicaUy no difference between the flora in the two tubes except that perhaps there was not such a copious growth of B. icekhii in the tube which had been ' dressed ' with salt solution. 92 Both tubes after 12 days showed B. welchii, Streptococci, Staphylo- cocci, coUform bacilli, and many long thin Gram-negative bacilli. This experiment would seem to show that eusol used as a single daily dressing is without antiseptic value in a wound, it is apparently incapable of penetrating into diverticula. Experiment 2. Tubes with processes similar to those used in Experiment 1 were filled in with serum planted with a faecal emulsion and incubated anaerobically for 24 hours. Films were made from each tube and examined microscopically and all showed the same flora. The tubes were now ' dressed ' with various fluids as under. Tube I. ' Dressed ' with normal saline solution. „ II. „ „ 5 per cent, saline solution. „ III. „ „ eusol. „ IV. „ „ Dakin's solution. „ V. „ „ carbolic acid, 1 in 40. On the first day each of the tubes was washed with three changes of its corresponding fluid ; this was allowed to act for 20 minutes and was then poured off, replaced with serum, and the tubes incubated anaerobically for 24 hours. The results then were as follows : Baotbriologioal Floba. Tube I. Much gas formation. B. welchii, B. coli. Streptococcus, diphtheroids, B. tetani. ** -^^* »j " »» j> ,» /, -7 » lii. „ ,j „ „ „ „ „ IV „ V. No gas formation. Almost pure culture of Streptococcus. Few B. coli. On the second day the ' dressing ' was carried out in a similar manner except -that the fluids were allowed to act continuously for 1 hour after the preliminary washings. After 24 hours' incubation in serum the flora was unchanged. The next day the fluid was allowed to act for 3 hours, but the tubes after 24 hours' incubation showed the same flora. On the fourth day the fluid was allowed to act for 6 hours. After 24 hours' incubation the flora was the same, the Tube IV (Dakin's solution) showed the most copious growth. The next day the tubes were again washed with three changes of fluid, after which the fluids were allowed to act continuously for 3 days. The fluid was then poured off, replaced with serum and incubated for 24 hours. In Tubes I and II (normal and 5 per cent, salt solutions) all the microbes grew. Tubes III and IV grew apparently pure Streptococcus.^ Tube V grew Streptococcus and Gram-negative bacilli. All these final cultures were diluted 3,000 times and 10 cmm. of the dilution was plated out and incubated aerobically. In all cases the amount of growth was approximately the same. It was thought that possibly some of the bacteria, which had apparently been destroyed in Tubes III and IV, had not had time to grow out into the general mass of the culture fluid. All the tubes were, therefore, ' dressed ' again and the fluid was allowed to act ' Other organisms were also present as shown in the next stage of the experiment. 93 for two days. The fluid was then poured off, replaced with equal parts of broth and serum, and the tubes incubated anaerobically for 2 days. The cultures were then examined with the followng results : Tube I. Gas formation. B. welchii. Streptococci, coliform bacUli & Staphylococci. " rr±: >> " !> !> » „ V. No gas formation. Streptococcus only. These experiments show that it is impossible to sterilize the cavity of a wound by washing it out with an antiseptic or by leaving that antiseptic in the wound for a long period. The reason of this is (apart from any question of bacteria in the tissues where the anti- septic does not reach them) that in the minute processes which are usually present ia the recent wound the antiseptic does not penetrate, and in the intervals between the dressings the bacteria grow out again from these processes, so that in 24 hours the result is very much the same, whether an antiseptic has been used or not. With the hypochlorite solutions (eusol and Dakin's solution) anae- robic and aerobic bacteria persisted after the tube had been left filled with the antiseptic for 3 days, and the fluid was then poured off and replaced by a fresh sample which remained in the tube for a further period of 2 days. The only difference between the tubes ' dressed ' with these antiseptics and salt solution is that Staphylococcus per- sisted in the ' salt ' tubes whereas they were not evident in the ' antiseptic ' tubes. Of course in such an experiment it was not to be expected that there would be any change in the ' salt ' tubes as the salt is not an antiseptic. It is interesting to note that 1 in 40 carbolic at the very first ' dressing ' seemed to destroy all the microbes except a few B. coli and Streptococcus. This latter organism, however, is the most dangerous one found in wounds, and it persisted in the ' carbolic ' tube up to the end. Experiment 8. Tubes were next made ^o as to simulate a wound with infected walls in addition to the pockets. To imitate the tissues a small tightly -rolled ball of asbestos wool was employed ; this was placed in a tube similar to those used in the first series of experiments. The preliminary infection of this ' artificial wound ' was made as before with faeces grown in serum for 24 hours ; the ' dressing ' was carried out in the same way as in the last experiments and the fluids were left in contact for the same times. The only difference was that no anaerobic precautions were taken. Two tubes were used, one of which was ' dressed ' with normal salt solution and the other with Dakin's solution. They were examined .at the same times as in the previous experiment, and on every occasion there was much gas production and the flora remained as it was at the commencement of the experiment — even after 8 days' soaking in the antiseptic. • Gas formation and the presence of B. welchii and B, coli were not evident in 24 hours, but were marked after 48 hours' incubation. 94 Experiment 4. An imitation of an absolutely flat wound with infected tissue was next made. The tissues were represented by a ball of asbestos wool the size of a small pea and the wound by a small test tube without processes. Four such tubes were taken and the infection was made by incubating for 3 days aerobically in serum a mixed culture of B. ivelchii, B. sporogenes, Streptococci, and B. coU. In the presence of the ball of asbestos wool all these organisms flourished and there was much gas formation. The culture fluid was poured off and the tube and ball of asbestos wool was washed with four changes of the antiseptic fluid under investigation. The fluid was then left in the tubes for 3 days and the tubes were shaken at intervals. At the end of this interval the fluid was replaced by a mixture of serum and broth and the tubes were again incubated. The results were as follows : Table XXIX. Dressing fluid used. Normal salt. Copious growth & gas formation. All the microbes have grown. Eusol. „ „ „ „ „ Dakia's solution. „ ,, „ „ „ Alcohol 90 per cent. No growth in 24 hours, but in 3 days' growth of anaerobes and Streptococci. One would have supposed that a small ball of asbestos wool would have been less difficult to penetrate than the infected tissues round a wound, but here it is shown that none of the antiseptic fluids used were capable of sterilizing even a small asbestos ball saturated with a serous fluid. The following exp^eriments were made to ascertain if certain dyes would show better results than the antiseptic used in the above experiments. Experiment 5. ' Artificial wounds ' made out of small test tubes were filled with serum implanted with faeces. After being grown anaerobically for 24 hours, there was much gas formation and films showed the ordinary faecal flora : Streptococci, diplococci, coliforms, large and small Gram-positive organisms. The tubes were then emptied, filled in with antiseptic and kept for 24 hours at 37° C. The antiseptic was removed and the tubes, having been washed out with normal salt solution several times, were filled with glucose broth and replaced in the incubator for 24 hours. The results were as follows : Tube I. Ma vine 1 per cent, solution was added to make 1 in 1,000 in the original serum culture of faeces. Eesult after final incubation. Growth with gas formation ; film showed B. coli. Streptococci, and Gram-positive baciUi. Tube II. Faecal culture emptied out. Tube washed with flavine 1 in 1,000 several times, then filled with 1 in 1,000 in water. Eesult after final incubation: Growth with gas formation ; film showed B. coli. 95 Tube III. Tube emptied of faecal culture, washed with flavine 1 in 1,000 several times, then filled with a gauze plug soaked in flavine 1 in 1,000 in serum. Eesult after final incubation. Growth with gas formation ; film showed B. coli, Streftococci, and Gram-positive bacilli. Tube IV. Brilliant green added to faecal culture to make 1 in 1,000. Eesult after final incubation. Growth with gas formation ; film showed B. coli, Streptococci,, Skud Gram-positive bacilli. Tube V. Faecal culture emptied out. Tube washed with brihiant green 1 in 1,000 in water several times, then filled with brilliant green 1 in 1,000 in water. Eesult after final incubation. Growth with gas formation ; film showed B. coli. Streptococci, and Gram-positive bacilli. Summary. 1. In a test tube with small processes, which simulate the pockets present in a wound cavity, a daily ' dressing ' with eusol has no effect on the bacterial flora. 2. In such ' artificial wounds ' eusol and Dakin's solution are incapable of sterilizing the tubes even after acting continually for three days. 3. Carbolic acid, 1 in 40, is incapable of killing the Streptococci in such ' artificial wounds ' even after acting continually for three days. 4. Antiseptic dye-stuffs, such as flavine, 0-1 per cent., and brilliant green, 0-1 per cent., acting for 24 hours, similarly failed to sterilize such ' artificial wounds '. 5. Eusol, Dakin's solution, and alcohol (90 per cent.) are incapable of penetrating a small ball of asbestos wool impregnated with a serous fluid. 6. In view of these experiments it is unreasonable to expect Dakin's solution, eusol, carbolic acid, flavine, or brilliant green to sterilize the cavity of a wound if there are any small processes present, or if the tissues around are infected. (8) Experiments dealing with the ability of certain antiseptics to penetrate the walls o£ the wound. No experiments bearing directly upon this problem have been carried out. In the first days of the war, however, when considerable discussion was taking place on the possibility of obtaining a rapid sterilization of wounds by the early application of a strong antiseptic, a series of observations was made upon the ability of antiseptics to penetrate into blood clot. Inasmuch as this process may be held to resemble, in some measure, the process of penetration into organ- ized tissues, our findings may be here recorded. In planning the experiments we proceeded upon the reflection that a chemical sterilization, such as was contemplated, could only be achieved if the antiseptic employed was able to kill bacteria and spores embedded in blood clot, for in every wound there occurs a certain amount of oozing of blood, if not definite haemorrhage, and most of the microbes are presumably caught up in the resulting blood clot. It was, therefore, necessary to test the feasibihty of 96 killing bacteria and spores embedded in blood clot. The antiseptic to be tested was used in a proportion (arbitrarily chosen) of one to four of blood (clot in serum) — a proportion which, it was thought, might fairly well represent the conditions obtaining in a wound when the latter had been ' opened up ' under an anaesthetic. Techiique. Into a number of small test tubes of approximately similar bore (-8 cm.) were introduced (1) a 10 cmm. volume of a broth culture of Staphylococcus, Streptococcus or other organism, or a dilu- tion of such a culture ; (2) one cubic centimetre of blood freshly drawn from a human vein. The tubes, after the contents had been thoroughly mixed, were then put aside, usually at 37° C, for an hour or two, to allow a firm clot to form, exude its serum, and shrink away from the sides of the tube. Such clots were of the shape and size shown in Fig. XXXIV and usually were about ^ to f cubic centimetre in volume. To each of the tubes with its one cubic centimetre of serum and clot was then added 0-2.5 cubic centimetre of the antiseptic to be tested, which was duly mixed with the serum by shaking. In some cases this mixing was far from perfect owing to the very dense viscid precipitate immediately thrown down. After exposure of the clots to the antiseptic in Serum for a period of several hours at 37° C, each was in turn picked out and washed in several changes of sterile salt solution with a view to diluting any antiseptic carried over. Each clot was then broken up with a stout platinum wire bent at an angle and several fragments were planted into tubes of sterile nutrient broth in order to ascertain if the organisms in the clot had all been killed. The approximate number of organisms originally present in each clot was usually ascertained by making appropriate dilutions of the broth culture employed and plating sample volumes of these. Fig. XXXIV. Table XXX. Phbnol : Staphylo- coccus. 'Sirejjtn coccus. B. sporo- genes. 90 per cent. B.P. Kille.l. Not killed. 90 Killed. Killed (15 hrs 90 90 .'jO per cent, with glycerine and almond oil. exposure to antiseptics). Not killed. Not killed. Not killed. .30 per cent, with almond oil and liquid paraffin. 20 per cent, with almond oil. Killed. Not killed. B. lelani. Approximate uumher of organisms in the clot. Slaphylococcns .5.5,000 ; B. sporogenes 6,000. Streptococcus 12,000; B. sporogeius lO.QOO. B. sporogenes 1,000. B. sporogenfs l.dOO. B. sporogenes 1 .000. B. sp.jrngcnes 50.000. Not killed. B. tttani oO,Om: Strep- tococcus not deter- mined. 97 Phenol : Stafhylo coccus Table XXX {continued). 20 per cent, suspended with Tragacanth. 10 per cent, in spirit 10 per cent, in spirit. 10 per cent, with formalin Not killed. 5 per cent., with alcohol and glycerine. 10 per cent, with formalin 5 per cent. 5 per cent, aq^ueous. Inhibited. 5 per cent, aqueous. 2-5 per cent. Lysol : 10 per cent, with Tragacanth Not killed, suspension. Ceesol : 30 per cent, with almond oil and liquid paraffin. 20 per cent, with almond oil and liquid paraffin. 20 per cent, with Tragacanth. 10 per cent, with Tragacanth. Killed. 10 per cent, with liquid Killed. paraffin. 10 per cent, with liquid paraffin. 10 per cent, with liquid paraffin and almond oil. 10 per cent, with spirit. 5 per cent. Meecuey Salts : Perchloride, 1 in 1,000 aq. 1 in 1,000 aq. Not killed. 1 in 500 aq. Killed. ,, 1 in 500 aq. Biniodide, 1 in 500 aq. Not killed. ,, 1 in 500 aq. „ 1 in 500 aq. with spirit in equal parts. Iodine : Tincture iodine, undiluted. Not killed. Strepto coccus. Killed. Killed. Killed. Killed. Not kiUed. Not killed. B. sporo- genes. Not killed. Not killed. Not killed. Killed. Killed. Not killed. Not killed. Not killed. Not killed. Inhibited. Killed. Not killed. Not kihed. Not killed. Killed. Not killed. Not killed. Killed. Not killed. Not killed. Not kUled. Killed. KiUed.1 Killed.' B. tetani. Approximate numher oj organisms in the clot. Not killed. B. tetani mfiW ; Strep- tococcus not deter- mined. Streptococcus not deter- mined. Streptococcus 3,000,000. Staphylococcus 166,000. Not determined. Staphylococcus 50,000. Streptococcus 3,000. Streptococcus 1,000. Staphylococcus 55,000 ; B. sporogenes 6,000. B. siMrogenes 50,000. Not killed. Streptococcus not deter- mined ; B. tetani 50,000. Not killed. Streptococcus not deter, mined ; B. tetani 50,000. Staphylococcus 55,000 B. sporogenes 6,000. Staphylococcus 55,000 B. sporogenes 6,000. Streptococcus 12,000 B. sporogenes 10,000. Streptococcus 3,000,000. B. sporogenes 1,000. Staphylococcus 50,000. Staphylococcus 48,000 ; StreptococcusliyOfiW. Streptococcus 15,000. Staphylococcus 166,000. Streptococcus 15,000. Streptococcus 3,000,000 Staphylococcus 55, COO B. sporogenes 6,000. Streptococcus 12,000 B, sporogenes 10,000. Streptococcus 3,000,000. Streptococcus 15,000. FOKMALDEHYDB : 20 per cent. (50 per cent, of formalin). Killed. . . KUled. Streptococcus not deter- mined ; B. tetani 50,000. ' When a ten-fold stronger emulsion of Streptococcus was added to the blood the result was ' not killed '. 1727 G 98 Table XXX (continued). FOEMALDBHYDE : 20 per cent. (50 per cent, of formalin). 15 per cent. 12-5 „ 10 10 10 10 5 5 Peem.\nganate of Potash : 5 per cent, aqueous. 5 1 Methylated Spikit : Undiluted. Staphylo- coccus. KiUed. Not killed. Inhibited. Strepto- coccus. Killed. Killed. Killed. KiUed. Killed. Killed. Not kiUed. B. sporo- B. tetani. Approximate nurnber of genes. organisms in the clot. Not killed. . . Streptococcus 40,000 B. sporogeneslOQ,OOQ Not killed. . . Streptococcus 40,000 B. sporogenesl(iO,QO^. Not killed. . . . Streptococcus 40,000 B. sporogenes 100,000, Not killed. .. Streptococcus 40,000 B. sporogeneslOO,000. Staphylococcus 48,000 Streptococcusl80,000. B. sporogenes 50,000. B. sporogenes 1,000. Staphylococcus 220,000; B. sporogenes 200,000. Streptococcus 40,000 ; B. sporogenes 100,000. Not kiUed. Not kiUed. Not killed. Not kUled. Not kUled. Not killed. Not lulled. Inhibited. Not killed.! KiUed. KiUed. Streptococcus 3,000. B. sporogenes 1,000. B. sporogenes 1,000. Not determined. Streptococcus 15,000 Streptococcus 1,000,000. (9) Investigation of certain changes brought about in the tissue and blood cells as the result of the introduction of antiseptics into the wound. It was laid down in Section 1 that the ideal antiseptic ' must not exercise any local destructive action on the tissues, or interfere with the physiological properties of the tissue fluids or cells '. (a) The Action of Antiseptics upon the Fixed Tissues. The onlj observations which appear worthy of record in this connexion are the following : (a) Solutions of malachite green and of brilliant green were not found to be inimical to the growth of epithelium in the wounds, but rather stimulated it (vide clinical section, Case No. 2). The strength in which these solutions actually came into operation upon the tissues cannot be stated, as a large part of their strength (0-1 per cent, solutions were employed) must have been expended upon combination with the cotton dressings (vide section 6). (h) The only manifest destruction of fixed tissues was seen in the case of flavine. A 0-1 per cent, solution of this dye was applied on muslin to a superficial wound upon which epithelium was growing rapidly around several recent skin grafts. After 2 days nearly aU. the new-formed epithelium had been destroyed. 1 With 1,000-fold weaker planting, result was ' killed ' 99 In connexion with another wound — the cup-shaped wound ' C ' — it was observed that the normal rate of exudation from the walls was reduced by 50 per cent, after the tissues had been exposed to flavine for some hours. (h) The Destructive Effect of Antiseptics on Leucocytes. The leucocidal action of antiseptics has been tested in several ways : (1) by introducing a volume of the antiseptic solution as a fourth volume in an ' opsonic mixture ', incubating for 15 or 20 minutes and then estimating the amount of phagocytosis ; (2) by estimating the power of the leucocytes to emigrate in the presence of the antiseptic ; (3) by mixing blood with the antiseptic and then testing, at intervals, the capacity of the leucocytes to ingest microbes. The first method has been frequently employed, but it is of little use as a^uide to the toxicity of an antiseptic for practical purposes. Antiseptics vary very much in the rapidity of their action on leuco- cytes, just as they vary in the speed with which they kill bacteria. If 15 or 20 minutes is allowed for the action of the antiseptic on the leucocytes, only those fluids which have a very rapid action on, the white corpuscles (and probably also on bacteria) will show their full effect. Again it must be remembered that in such an experiment ■ phagocytosis is going on all the time, so that the resultant number of ingested bacteria is in no way an indication of the capacity of the leucocytes to ingest bacteria after 20 minutes'' contact with the anti- septic. The greater portion of the phagocytosis occurs during the first 5 or 10 minutes of incubation. By using this test an antiseptic which has a very slow toxic action on the leucocytes, such as flavine, compares very favourably with quick-acting antiseptics such as the hypochlorites. The same objection applies, to a less extent, to the method of measuring the amount of emigration in Wright's emigration tubes. ■'• In this method emigration commences rapidly and reaches its maximum (where there is no special attractive force) in something like two hours. Thus the very slow acting antiseptics, such as flavine, do not show, even by this method, their full effect. (When the tubes are examined under the microscope, however, after, say, three hours' incubation, the emigrated leucocytes in tubes containing flavine show up as rounded yellow bodies in sharp contrast to the actively moving forms in the control tubes.) It will be seen, however, from Table XXXI that the antiseptics which act with moderate rapidity, such as carbolic acid and perchloride of mercury, show by this method practically their full toxic effect on the leucocytes. The be^t method of examining the effect of antiseptics on leuco- cytes is undoubtedly to mix the antiseptic dilution with defibrinated blood and allow this mixture to stand in the incubator at 37° C. At intervals samples are removed and the physiological state of the leucocytes is arrived at by comparing their capacity to ingest microbes with the phagocytic capacity of leucocytes from blood treated in exactly the same way with physiological salt solution 1. Lancet, 1915, i, 737, 787, 843, 897. G 2 100 substituted for antiseptic. Two volumes of the blood-antiseptic mixture is mixed with one volume of previously opsonized microbes and incubated for 10 or 15 minutes, after which the amount of phagocytosis which has taken place is estimated. The same is done with the control blood without antiseptic. By testing in this way at long and short intervals, a true picture of the action of any anti- septic on leucocytes is obtained. From Table XXXI it appears that when this method is employed to test the slow-acting antiseptics, such as fiavine and brilliant green, and 24 hours' contact is allowed, the latter dye is only one-tenth as toxic to leucocytes as flavine ; when, on the other hand, the same solutions are tested by 2 hours' contact in Wright's emigration tubes, ^ brilliant green is found to be 4 times as toxic as fiavine. It may be noted that Browning^ also, using the opsonic method and 20 minutes' contact of the antiseptic with the leucocytes, found brilliant green to be 4 times as toxic as fiavine. The table also shows that whereas carbolic acid and flavine manifested the same toxicity to leucocytes when acting for only 20 minutes, flavine was found to be 1,000 times as toxic as carbolic acid when the antiseptic was allowed to act on the cells for 24 hours. ■ Table XXXI. Showing the effect of Antiseptics on Leucocytes. Antiseptic. Method I.' Time of con- Method II. Time of con- Method III. tact oj anti- tact of anti- Concentration which reduced septic and septic and the phagocytic activity of the leucocytes leucocytes leucocytes by half. 20 minutes. 2 hours. Concentration Greatest Time of contact of antiseptics which rediKed concentration and leucocytes. the amount of which permitted phagocytosis by emigration of 2 hours. 5 hours. 24 hours. half. leucocytes. Carbolic acid 1 in 500 1 in 1,600 1 in 1,600 1 in 1,600 1 in 2,000 Mercuric chloride . . 1 in 7,000 1 in 16,000 1 in 16,000 1 in 16,000 1 in 20,000 Iodine 1 in 3,500 1 in 200 1 in 500 1 in 1,000 1 in 1,000 Eusol (available CI) 1 in 4,000 1 in 2,400 — — . Dakin's solution (available CI) . . 1 in 4,000 1 in 3,600 1 in 3,600 1 in 3,600 1 in 3,600 riavine . . . . 1 in 500 1 in 4,000 1 in 1,000 1 in 500,000 1 in 2,000,000 Brilliant green 1 in 2,000 1 in 16,000 1 in 5,000 1 in 20,000 1 in 160,000 Malachite green . . 1 in 7,000 1 in 16,000 1 in 6,000 1 in 12,000 1 in over 100,000 Crystal violet 1 in 7,000 1 in 16,000 — It is to be noted also that the migratory power of leucocytes provides a more delicate test than does their phagocytic activity. This is shown especially in connexion with the aniline dyes by a comparison of the second and third columns, where, in both cases, ■2 hours was the time of contact of the antiseptic and the leucocytes. With each of the dyes examined the greatest concentration in which ' Lancet, 1915, i. 737, 787, 843, 897. ' Browning, GuUbranseji, Kennaway, and Thornton, Brit. M. J., 1917 i 73 3 These figures are taken from an article by Browning, GuUbransen, Kennaway, and Thornton {Brit. Med. J., 1917, i. 73). • 101 emigration occurred was from 3 to 4 times weaker than the con- centration which reduced the amount of phagocytosis by half. This would seem to show that in i2 hours certain concentrations of these slow-acting substances damage the leucocytes to such an extent that they prevent active emigration, although they are still sufficiently alive to ingest microbes suitably offered to them. It must be remembered that phagocytosis is only a stage in the destruc- tion of the microbe, and it is probable that these damaged leucocytes would be incapable of completing the work of destruction which is only commenced by the ingestion of the bacteria. (10) The effect produced by certain antiseptics upon the exudation of fluid into the wound — and upon the properties o! that exudate. In spite of the failure to achieve the sterilization of any infected wound by a Single application of an antiseptic — or indeed to show that antiseptics exert any appreciable bactericidal effect when repeatedly applied — it has been widely maintained that the employ- ment of certain of them in the treatment of infected wounds has been beneficial. The question therefore arises — are these substances capable of exerting some beneficent effect distinct from a directly anti-bacterial action ? One of us (A. F.) ^ has suggested that, by virtue of a mild irritant action on the tissues, certain antiseptics provoke an increased flow of the normal exudate into the wound, and that to this exudate^- of fluid and leucocytes — should be ascribed any beneficial effect that is forthcoming. To such an indirect action of antiseptics Fleming has applied the term ' physiological '. With this conception in view the following experiments were carried out, in order to deter- mine : (a) The rate of exudation into the wound following the applica- tion of various antiseptics. (b) The albuminous content of such exudates. (c) The leucocyte content of such exudates. (d) The anti-tryptic value of such exudates. (a) The Eate of Exudation. Experiments with chloramine T solution. (1) The normal exuda- tion into the cup-shaped wound ' P ' having been first determined, as described on p. 74, a 4 per cent, solution of chloramine T was applied for varying periods, at the end of each of which the increase of fluid in the cavity was carefully estimated. The result is graphi- cally shown in Fig. XXXV. This chart shows that the rate of flow of the exudate for the first 5 and 10 minutes is practically the same as that into the empty cavity ; the amount exuded in 30 minutes shows a very marked increase. This is most probably due to the irritant action of the antiseptic solution, and such an assumption is borne out by the nature of the > Fleming, Brit. J,. Snrg., 1919, 7, 99. ■ 102 exudate. For instanee, the 5-minute specimen was slightly mucoid, this being due to the action of the antiseptic on the leucocytes. The 10-minute specimen was mucoid and slightly red in colour. The 20-minute specimen was thick, mucoid and red, while the 30- minute specimen was almost a jelly and stained deeply red. This red colour was due to red blood-corpuscles, the presence of which indicated a marked irritant action of chloramine T in 4 per cent, solution. (2) This experiment was carried out in the same manner on the wound ' P ', a 1 per cent, solution of the antiseptic being used. Minutes 10 20 30 Time fluid remained in wound. Kg. XXXV. Rate of exudation into a wound filled with chloramine T (4 per cent.). Contruuous line represents amount of exudate into cavity filled with chloramine T. Dotted line represents amount of exudate into empty cavity on the same day. Shaded area indicates the amount of exudate due to chloramine T. Amount of exudate C.C. 1 • 0-9 0-8 0-7 0-6 0-5 0-4 0-3 0-2 0- I MinuteslO 20 30 40 50 60 Time fluid remained in wound. Fia. XXXVI. Rate of exudation into a wound filled with chloramine T (1 per cent.). Continuous line = exudate into cavity filled with chloramine T . Dotted line = exudate into empty cavity on same day. — I 1 1 1 1 " / / L / . ' ' / I / / / y / / / / 1 — v^ 1/ // -y-^ // fj Jl // // — J(V A // u. _ Fig. XXXVI shows that the amount of exudate passing into the solution was practically identical with that into the empty cavity. The irritant action on the walls of the wound was, in this case, entirely absent. Experiments with Dakin's solution. (1) This Avas carried out as above but on the wound ' C '■ — and, in this case, the rate of exudation into the wound after withdrawal of the antiseptic was also estimated. The result is shown in Fig. XXXVII. This Fig. shows that during the application of Dakin's fluid for the first 5 or 10 minutes the rate of flow is identical with that into the empty cavity. When the Dakin's solution had been in the cavity 20 minutes the exudate was slightly increased ; this was also the case after 40 minutes' application. When the fluid has 103 been removed the flow in the next 40 minutes is again shghtly increased. (2) Dakin's solution was applied to the same wound for 75 minutes. Pig. XXXVIII shows the rate of exudation into the wound before, during, and after the application. It will be seen that not only was the exudate increased during the application, but continued in excess of the normal rate for at least 7 hours after the antiseptic had been withdrawn. After 24 hours it had, however, returned to the normal amount. (3) The wound was filled in with Dakin's fluid four times in 4| hours. The fluid was changed after 1, 2, 3, and 4|- hours. After the last sample had been withdrawn the flow of exudate into the empty cavity was estimated after 1 and 2 hours' interval. The results are shown in Fig. XXXIX. //icrease jn vo/ufne o/^ /Ju/'d. 0-25CC. - 0-2 cc ■ 0-/5'CC. O-/ CO. 0-OSCC. O abpJiira Pbn of/hk/ns so/ut/b/) . £xu o ^MMfe^D^wHM^JIHI^i^JHHLABtaJIH^^^J^^^^—^^^HHiH ili Fig. XLII. Rat ■p ( f flow of exudate per hour into the wound before, duimp and after the application of flavine solution 1 in l,(XiO Table XXXII. T mix, conditions, (t-c, under which the exudate Volmne of Bate of wn-s measured. exudate. fiowperhr. Flow into empty cavity 50 miu. before uitroductiun of tiavine 0-15 c.c. 0-lS c.c. Flow into flavine solution during first 45 min, . . 0-11 c.c. 0-15 c.c. ,, ,, ,, ,, following HO mill. 0-14 c.c. 0-14 c.c. „ " ihrs. . 0-38 c.c. 0-18 c.c. „ „ „ „ 4 hrs. 0-5 c.c. 0-125 c.c. ,, ,, ,, ,, ,, 15v iirs. . . '2-5 c.c. 0-16 c.c. Flow into empty cavity during first 21 hrs. after removal of flavine . . ... 0-14 c.c. 0-06 c.c. Flow into empty cavitj' during 4-J- lirs. on day following removal of flavine . . .... 0-37 c.c. 0-08 c.c. Flow into empty cavity during 4 hrs., two days after removal of flavine ... . 0-25 c.c. 0-06 c.c. From these results it Mill be seen that, after the application of flavine to the wound for 23|- hours, the amount of exudate into the empty cavity was reduced to less than half the amount, and this continued for at least two days after the removal of the solution. During the later period of the application of the flavine a thick 107 film of what may be described as sputum-like material was deposited on the walls of the wound cavity. This appeared to have but little inhibitory effect on the passage of fluid from the walls into the cavity as long as the flavine solution remained in the wound. When, however, the solution was removed, this membrane appeared to form a laarrier to the passage of fluids, so that less than half the amount of exudate passed into the empty cavity. The nature of the sputum-like material will be discussed more fully when dealing with the emigration of leucocytes. These results are shown in graphic form in Fig. XL II. (h) The Albumen Content of the Exudates Induced by THE Presence of Antiseptics. This was investigated only after the application of eusol and Dakin's solution respectively to the wounds ' P ' and ' C '. The amount of albumen in the several samples of fluid was estimated by pre- cipitation with salicylsulphonic or trichloracetic acids and subsequent ceutrifugalization in tubes of equal bore. The results were expressed as percentages of the precipitate obtained with a ten-fold dilution of serum. It was shown by each experiment — and is illustrated in Fig. XL — that increase of the amount of albumen in the fluid was roughly proportional to the increase of the exudate. This finding is in harmony with the conception that the increased exudation induced by the antiseptics is due to an irritation of the tissues — that it is, in fact, an inflammatory manifestation. (c) The Number of Leucocytes in the Exudates. This was determined by counting in an ordinary haemocytometer cell, the fluid being first diluted, in most cases, with a 1 per cent, solution of acetic acid tinted with neutral red. The control observa- tion in these experiments was obtained by determining the number of leucocytes emigrating into normal salt solution. Experiment ivith chloraniine T, 1 per cent, solution. The number of leucocytes passing into the fluid is shown in the adjoined table Table XXXIII. Time one. per cent, chloramine T Number of leucocytes per cubic milli- solution had been in wound. metre of antiseptic -\- exudate. 20 minutes 91,200 30 „ 336,000 50 „ 742,000 On comparing these figures with those in Table XXXV, which shows the result when normal saline solution was employed, it is seen that after 20 minutes this concentration of chloramine T does not hinder the emigration of leucocytes. Experiment with Dakin's solution. The results are given in the following table : 108 Table XXXIV. Length of time Dakin's solution had Number of leucoci/tes per cubic milli- remained in the wound. metre of antiseptic + exudate. 5 minutes 190 10 „ 480 20 „ 520 40 „ 4,770 These results appear to indicate that Dakin's solution inhibits the emigration of leucocytes as long as the concentration of hypo- chlorite remains above a certain level, and this supposition is con- firmed by a series of experiments in vitro which showed that the solution, in dilutions up to 1 in 32, completely inhibited the emigration of leucocytes into a fibrin network. These experiments would indicate that in 40 minutes the hypochlorite concentration of the fluid contained in the wound had fallen below even this figure. Exferiment ivith eusol. In samples of eusol withdrawn from the wound cavity after 5 and 10 minutes' application it was found to be quite impossible to make even an approximate count of the leucocytes which had been exuded from the walls of the 'wound, owing to the lytic action of the hypochlorite. Samples, however, removed after 20 minutes were found to contain, in the case of wound ' P ', 350,000 leucocytes per c.mm., and after 60 minutes, 480,000 per c.mm. These figures correspond closely to those obtained when normal salt solution was employed in place of eusol, and this later emigration of leucocytes appeared to take place when the hypochlorite, as is shown in section (4), p. 79, had been reduced to a negligible concentration through its interaction with the exudate and wound walls. Experiment ivith flavine, 1 per cent, solution. The number of leucocytes passing into the flavine solution gradually fell from 4,350 per c.mm. which were present after the solution had been in the wound for one hour to 850 per c.mm. in the specimen remaining in the wound for 4 hours. In the specimen which had remained in the wound for 15 J hours it was found impossible to make even an approxi- mate count, as after that period the wound had become lined with the sputum-like membrane which was found to consist of leucocytes in various stages of degeneration. After the solution had been removed very few leucocytes passed into the wound cavity for at least two days. (d) The Antiteyptic Value or the Exudates. Wright's work^ has emphasized the importance of the antitryptic power of the blood fluids and wound exudates in relation to microbic infection. The effect of ehminating this quality of the serum by additions of trypsin was well shown by experiments in which the amount of microbic growth was measured by gas formation as described in section (5). Fig. XLIII illustrates an experiment of this kind in which the two series of tubes contained infected serum whose antitryptic power was progressively diminished (from right to left) by increasing additions of trypsin. In the three or four tubes on the left of each series the serum thus treated is comparable to the ' corrupted lymph ' which is present in every infected wound in which 1 Wright, Lancet, 1913, i. 737, 787, 843, 897. 109 large uumbei'S of leucocytes are Jjeiiig disintegrated (witji liberation of trvptic ferment). The increased growth of the microlje in these tubes gives us warning as to the undesiraliihty of permitting any considerable reduction in the antitryptic value of the wound exudates. Fig. XLIV illustrates the same point in connexion witli the growth of Streptococcus in serum. In reality the importance of the antitryptic power of the exudates is greater than even these experiments indicate — for not only does the fluid in wdiich that (|u.ality is abolished become changed into a favouraljle culture medium, 1)ut its opsonic and other antiljacterial actions are also lost. It was with these considerations in view that the effect of anti- septics up(_in the antitiTptic \"alue of the exudat<-'S Avas investigated. («) B. spiirogrnes. [h) B. wMui. Vaseline Gas formed by growth of bacterium. Fio. XLIII. Showing the effect of a reduction in the antitryphic p^iwer of the serum on the growth of (a) B. sporoqenes, (b) B. welchii. The figure given below each tube indicates the anti-tryptic, or, as the case may be, the tryptic power of the serum employed in that tube. That labelled ' O ' has had its antitryptic power exactly neutralized by the addition of trypsin. The tubes on the left of this ime in each series contain free tryptic ferment in increasing Cfuantities, while those on the right become increasingly antitryptic, until the one on the extreme right represents unaltered serum with its normal antitrj'ptic power. (Equivalent tu a 1 : 30 dilution (jf the trypsin used.) Method of investigation. The fluid exuding into the wounds was collected before the application of the antiseptic, and at intervals after this had been withdrawn. It was first tested with a view to determining whether it contained free tryptic ferment. For this purpose a milk-clotting method, based on Mellanby's ^ work, was employed. One volume of the clear supernatant obtained by centrifuging the fluid was mixed with one volume of separated rnilk to which 0-5 to 1-0 per cent, of calcium chloride (cryst.) had been added. This mixture w-as drawn up into the stem of a capillary pipette and incubated at 50° C. for half an hour. A control iriixture, in wdiich the exudate Avas replaced l)y water, Avas also incul)ated, ' Mellanliy and Woollev, •/. Ph'i.^ioh, lOli?, xlv, 370. no in order to make sure that the milk had no tendency to clot spon- taneously. If, at the end of the incubation, the milk mixed with exudate had clotted, while that in the control tube remained unclotted, the exudate was judged to contain free tryptic ferment. Failing this result, it was necessary to proceed to a further test with a view to determining the antitryptic power of the exudate. For that purpose a series of tubules was filled in with (a) one volume of the exudate to be tested, (b) one volume of a series of progressive dilutions of a trypsin solution, (c) two volumes of milk to which 1 per cent, of calcium chloride had been added. The tubes were then placed in a water bath at .50° C. for half an hour and examined to ascertain in which tubes clotting of the milk had taken place. The antitryptic power was then expressed in terms of the strength of the trypsin solution which was just neutralized by the fluid. Fio. XLIV. Showing the effect of a reduction in the antitryptic power of the serum on the growth of tn^ptococcvs. For explanation of the figures below each column see description of Fig. XLIII. Experiment with Lakin's solution. The exudate was t'ested before and at intervals of 1 and 2 hours after withdrawal of the solution from the wound ' C ' — in which it had been allowed to act for 4^J- hours. The result is shown in Fig. XLY From this chart it will be seen that for at least 2 hours after the Dakin's fluid had been removed there was a very considerable drop in the antitryptic power of the serum. In connexion with this effect it may be recalled that the application of Dakin's solution has frequently been accredited with hastening the separation of sloughs in infected wounds. Experiment witJi eusol. The wound ' C ' was treated for 7.5 minutes with eusol, and the exudate collected at intervals of 40, 115, and 185 minutes after its removal, as well as before the application. The results are shown in Fig. XLVI. On examining this Fig. it ^vill be seen that during the first 40 minutes after the eusol had been removed the antitryptic power of the Ill exudate was veiTiiiucli diminished, aftia' 115 minutes it bad recovered to some extent, but even after 185 minutes it was still less tban before the application of the eusol. This diminished antitryptic power is most probably due to the breaking down of leucocytes and other cells in the walls of tlie wound, in consequence of whiclr the trypsin contained is set free and partially neutralizes the antitryptic power of the serum. Experiment uitli flnvine, 1 in 1,000 solution. Tliis antiseptic was y ///7-/C7 //>7 SO //o 6o //>? ro / m so / /n So / /r> /ao / /n /20 / /n /JO / W /40 ///7 /60 ///? /so ///7 J^OO 3 ■ //M 70 / /n -fO / in SO / /n 60 / m 70 / /n SO / /n S>0 //,7 /OO / m /20 ///7 /^O /,h /60 ///7 /SO y/n^OO Fig. XLV. Efjtct of DuknCi .iolutiou on the antitryptic poicti of the exudate. Dilution of trypsin neutralized by an equal volume of the exudate. The thick column indicates the antitryptic power of the exudate into the empty cavity before tlie application of the Daltin's solution. The thin columns indicate the antitrj'ptie power of tlie exudate into the empjty wound at intervals after the removal of the Dakin's solution. The times are given above the columns. Fig .XLVl. EfJ\ n^if eu .^ol o a tin: a nli- Iryptic poicer of the e.ntdate into the wound. Dilution of trypsin neutralized by an eijual volume of the exudate. The thick column indicates the antitr3'pjtic pjower of the exudate into the empty cavity before the application of the eusol. The thin column indicates the antitryptic power of the exudate into the empty cavity at intervals after the removal of tlie eusol. Tlie times the samples were taken are given above the corresponding columns. applied five times in the course of 24 hours to the wound ' C '. Tlie exudates collected before the apphcation and 2i hours after the solution had been removed were found to have the same antitry])tic power. The resiilt is somewhat surprising in view of the marked effect which a prolonged application of flavine has been shown to have on the leucocytes, massing them into a tenacious mucoid-like membrane adhering to the walls of the wound ; it is not improlialile, 112 however, that their contained trypsin remains bound to the sputum- like material as is the case in pneumonic sputum which similarly consists largely of leucocytes broken down into a mucoid-like material. (11) Experiments bearing upon the employment of hypertonic salt solutions in the treatment of infected wounds. As is now well known, Wright has recommended as a treatment for suppurating, slough-covered wounds, the application of a 5 per cent, solution of sodium chloride, with a view to (a) the rapid separation of sloughs by the agency of tryptic ferments, which he showed to be set free by the action of the solution on leucocytes, and (b) the pro- duction of an increased flow of lymph, wlrich, by virtue of its bacteriotropic properties, should kill off, or inhibit the growth of, many of the bacteria contaminating the wound and surrounding tissues. He further recommended that, when tire wound had become clean under this treatment, the hypertonic solution should give place to physiological salt solution (0-85 per cent.), the aim then being to obtain an abundant emigration of leucocytes into the wound and by means of these to achieve the destruction of such serophytic organisms as had not been eliminated from the wound by the increased lymph flow. To these recommendations certain objections had been raised ; it was put forward that the conditions of the experiments in vitro on which Wright based his teaching were not similar to those obtained in wounds — and, in particular, it was contended that the increased flow, caused by the drawing action of 5 per cent, salt solution, would consist, not of lymph, but of fluid containing little or no albumen. To see how far experiments in vivo would bear out Wright's contentions and in particular to obtain evidence as to the nature of the fluid drawn out by the salt solution, a series of observations similar in kind to those already described in connexion with the investigation of antiseptic solutions was made on the cup-shaped wounds 'P' and 'C. The data which we obtained may be set out under the following heads : (a) The effect of hypertonic salt solution on the rate of exudation into the wound. (h) The albumen content of the exudation induced by salt solution. (c) The number of leucocytes present in such an exudate, and also in that which takes place into physiological salt solution. (d) The effect of hypertonic salt solution upon the antitryptic value of the exudate. (a) The Eatb of Exudation. Experiment 1 . The normal rate of flow into the wound ' P ' having been determined, it was filled in with 5 per cent, salt solution, and measurements of the contents were made at frequent intervals. These were plotted out in Fig. XL VII. Experiment 2. The flow into wound ' C ' was estimated at intervals : (1) when the cavity was empty ; (2) when the cavity was filled with 113 physiological salt solution ; and (3) when it was fiUed with 5 per cent, salt solution. The results are shown in Fig. XL VIII. Minutes 10 20 30 40 Time fluid remained in wound. I'iG. XL\'II. Rate of exudation of fluid into a wound cavity filled witli .5 per cent, salt solution. The continuous line represents the amount of exudation. The dotted Ime represents the amount of exudation into the empty cavity of the wound on the same day. The shaded area indicates the amount of ex'iidation which is actually due to the salt solution. Fii;. XLVIII. Rate of exudation of fluid into a wound cavity [Expirlmiiit 2) : (a) when empty ; (6) when filled with normal salt solution ; (c) when filled with 5 per cent, salt solution. 1727 H 114 It will be seen from this Fig. that the flow into 0-85 per cent, salt is practically the same as that which takes place into the empty cavity, but that, as in Experiment 1, a much greater flow was induced by the 5 per cent, solution. Experiment 3. Five per cent, salt solution was left in the wound for 75 minutes. The amount of exudate before, during, and after this application is shown in Pig. XLIX. The new point brought out by this experiment is that the increased flow continued for at least 30 minutes after withdrawal of the salt solution ; and further, by estimating the salt content of the several exudates, it was ascertained that the fluid passing into the cavity alter removal of the salt solution was considerably richer in sodium OS 0-4- Oj oz o-/ c -5 S Fig. XLIX. Ratt of fiow of exudate into the wound hefore, during, and after the application of 5 per cent, sodium chloride solution. Increase in volume of fluid. The thick column represents the rate of flow of exudate per hour into the sodium chloride solution. The thin column to the left represents the rate of flow per hour into the empty cavity before the application of 5 per cent, sodium chloride solution. The thin columns to the right represent the rate of flow per hour into the empty cavity at different periods after the removal of the 5 per cent, sodium chloride. chloride than the normal exudate. The increased flow was most probably due to the passage outwards of the sodium chloride which had previously passed into the tissues from the 5 per cent, solution. In concluding this part of the investigation comparison may be made between the amount of exudation induced by salt solution and that which was obtained by the application of certain antiseptics to the same wound. For this purpose Figs. XXXVIII, XXXIX, XLI, XLII, and XLIX are here reproduced in combination, see Fig. L. It will be seen that the effect exerted by salt solution is by far the greatest. Reduction oj strength of the salt solution in the wounds. ^ Experiments 1 and 2 above showed that the increase of the exudate induced by the presence of hypertonic salt solution in the wound was most marked when this solution was renewed at frequent intervals. This result was, of course, to be expected, for it will be evident that the solution in the wound will progressively diminish in strength, both by dilution 115 with the incoming lynipli and by diffusion of the Hodiinn chloride into the tissues. Experiment 4 was carrifd out to measure the diminution in tlie strength of the solution. The wound ' P ' was filled in witli a 5 per cent, solution which remained for varying periods, e. g. 5, 10, 15 minutes, &c., as shown in Fig. LI. The concentration of salt in each sample was estimated as follows. A suspension of human red blood cor- puscles thoroughly washed free from serum was made in 0-85 per cent, sodium chloride solution. Twenty c.mm. of this suspension was then mixed with 20 c.mm. of the fluid which hatl been in the wound for a known length of time, rendered clear by centrifuging. Distilled water was now carefully added in sufficient quantity to cause com- plete haemolysis of the suspended corpuscles. The amount of distilled water necessary to produce haemolysis was carefully noted. A control series of experiments was then made in which, in place of solution. Dakin'w yohition. Flavine (1 in 1,000). .5 per cent, salt solution. Eusol. Fit;. L. Transudation of fluid per hour into a wound cavity before, during, and after the application of salt solution, eusol, Dakin's solution, and flavine. the fluid Avhich had been in contact A\-ith tlie wound, known strengths of salt solution were added to the suspension of corpuscles ; the amount of distilled water necessary to cause complete haemolysis in these mixtures was then estimated. By comparing the amount (if distihed water used in the two series the concentration of salt in the various samples was easily estimated. This method was suggested by Wright's technique ^ for estimating the amount of salt in the blood and urine. Using this technique, it was found that the rate at Avhich salt disappeai-ed from the solution in the wound was much more rapid in the early stages of the experiment than in the later ones ; for instance, in the first 10 minutes of the experiment the concentration feU from 5 per cent, to 3-01 per cent., a loss approximately of 2 per cent., while the difference between samples remaining in the wound 40 and 60 minutes respectively was only 0-36 per cent. (1-93 to 1-57 per cent.). After the solution had remaini.-d in the wound for 60 minutes the concentration had fallen to 1-57 per cent. The results are shown in Fig. LI. ^ \\Yi!iht,TcchHi(2i:cnf the Tt6h'/ndCripinarijGh:isiTnh,, London ((:on=it?Mlp),\d\2jn. Hi 116 (&) The Albumen Content of the Exudate Induced BY Salt Solution. This was estimated as described on p. 107. The wound ' C ' was filled in for varying periods with 0-85 per cent, salt solution, and later with a 6 per cent, solution. Fig. LII shows quite distinctly that the amount of albumen in solution which passed from the walls of the wound into the 5 per cent, solution was much in excess of that which passed into normal saline solution. S- /O /i- so J25 30 J5 -^ -45 so SS SO Time during which the salt solution had remained in the wound. Fig. LI. Rate of diminution of strength of 5 per cent, salt solution left in a wound. (c) The Number of Leucocytes Contained in the Exudate. _ This was investigated on several occasions when the fluid used in filling in the wounds was respectively normal saline solution and 5 per cent, sodium chloride solution. The results are given in the following table : Table XXXV. Time during which he fluid had remained in the wound- 5 minutes 10 20 30 40 60 WOTTND 'P'. Normal salt. 5 per cent. salt. 36,000 78,000 346,000 434,000 593,000 16,400 36,400 62,000 125,600 234,000 WOTIND ' C '. Normal salt. 5 per cent. salt. 5,160 16,600 20,000 28,000 1,400 1,440 1,660 2,000 117 This table shows conclusively that the leucocytes pass out much more readily from the granulation tissue into the cavity of the wound when this contains normal saline solution than when it contains 5 per cent, sodium chloride solution. It is also probably true that in the presence of normal saline solution the passage of the leucocytes is greater than when the cavity is left empty ; this, however, could not be estimated with sufficient accuracy, owing to technical diffi- culties, for definite figures to be given. {d) The Effect of Hypertonic Salt Solution upon the Antitryptic Value of the Exudate. No observations were made directly upon the hberation of tryptic ferments by the action of 5 per cent, salt solution on the /^at/a q/ amount O^Cllbu/nen tn f/ciid /o urnount m a. ten y^o/d dliu^/bn of serum . 0-7 0-6 OS 0-4 0-3 0-2 01 S% sj-Li: • Jolu C/on r fO /S JO SS JO ys -fO m/nu^-es Time during which the fluid had remained in the wound. Fig. LII. Showing the amount of albumen present in the exudate when the wound was filled with 5 per cent, and 0-85 per cent, salt solution respectively. leucocytes. The only experiment we carried out was for the purpose of testing whether the lymph passing into the wound after withdrawal of the salt solution had a diminished antitryptic power. Fig. LIU shows that for half an hour a slight diminution did occur, but that after 2 hours the antitryptic power was the same as before the application of the salt. (12) Summary and discussion of results. (1) Two simple cup-shaped wounds have served us as ' test tubes ' by means of which the action of certain antiseptics and of hypertonic salt solution — as well as the reactions of the tissues to these substances — could be studied in vivo. (2) The application of eusol, Dakin's solution, and solutions of chloramine T to these wounds, in such a manner as to give the 118 antiseptic tlie bent possible chance of exercising its bactericidal effect, caused no appreciable reduction in the bacterial flora of the wounds (vide Section 3). Owing to technical diificulties, the analogous experiment with flavine did not give a clear result, but it can be said that nothing like a complete bactericidal effect was obtained. (3) The effective strength of eusol and Dakin's solution is very rapidly dissipated by contact with the tissues when apphed even to I 5 '/50 '/40 '/so '/60 '/70 '/so '/90 '/ioo '//'fO //60 '//SO '/200 The times arc given above the columns. Flu. LIII. Effiirl of o ptr Ciiil. salt solution mi tin aiditryptk poiver of tin exudate. The thick column indicates the antitryptic power of the exudate into the empty cavity before the application of the 5 per cent, salt solution. The thiu columns indicate the antitryptic power of the exudate into the empty cavity at intervals after the removal of the 5 per cent, salt solution. a perfectly clean woiind. Within 1(1 minutes their hypochlorite concentration is reduced by at least 80 per cent, and their bactericidal vakie has become practically nil. Very much the same applies to chloramine T. (It is probable that a still greater loss of strength would have 1>een recorded if these antiseptics had been applied to wounds in which there was a large accumulation of leitcocytes, a condition which frequently obtains in surgical practice and, especially, wlieu Carrel's system of intermittent irrigation is em- ployed.) In the case of flavine a similar but slower reduction in strength 119 occurs as the dye becomes combined with the tissues of the wound ; and in this case — as with all other dyes tested — a further serious loss of effective strength occurs through fixation of the dye by the cotton and other fabrics which are habitually employed for dressing wounds. (4) In view of these serious drains upon the effective strength of antiseptics in a wound, the question naturally arose— Is there a sufficient amount of the antiseptic agent left in the wound to exercise a bactericidal effect ? This problem was investigated by determining the effect of various strengths of antiseptic solutions upon bacteria suspended in serum. In the case of eusol and Dakin's solution it. was found that a hypochlorite concentration, comparable to that which remains in a wound 5 minutes after its application, was not only incapable of restraining the growth of microbes in serum but actually stimulated the growth of certain types to a very marked degree. In the case of flavine, concentrations of 1 in 4,000 to 1 in 16,000 were found to be necessary to inhibit the growth of Staphylo- coccus, according to the number of cocci employed for the test — and it appears unlikely that such concentrations as this remain active in the wound for any length of time after the application of a 1 in 1,000 solution. The stimulation of microbic growth in serum was not confined to the hypochlorite solutions, being demonstrated also with carbolic acid, iodine, chloramine T, and malachite green solutions. It did not occur equally with all bacteria. (5) In Section 9 it is shown that all the antiseptics in use have a destructive action on the leucocytes, and this destructive action occurs in a lower concentration than is necessary for a lethal action on the bacteria. Wright, Fleming, and Colebrook ^ have demonstrated the very striking bactericidal efficiency of leucocytes when provided with the requisite conditions for their functioning. In the cavity of a wound to which an antiseptic has been applied such leucocytic destruction of microbes will be put out of action for a time varying with the rate of dissipation of the antiseptic, and in this way it may again happen that the balance of advantage, following the employ- ment of such an agent, will be with the bacteria rather than the patient. (6) It has been suggested that certain antiseptics, although incapable of exerting a directly bactericidal effect in the wound, may yet contribute something indirectly to the antibacterial processes by provoking a -physiological reaction on the part of the tissues. It has also been stated as a fact of clinical observation that the separation of sloughs in infected wounds is hastened by the use of Dakin's solution. In this connexion our experiments showed that the application of hypochlorite solutions resulted in an increased exudation of lymph, which sometimes lasted over a period of several hours — and also that this lymph differed from that normally passing into the wound in that its antitryptic power was much reduced. This latter result may help to explain how the antiseptic has pro- moted (sic) the separation of sloughs. In our experiments— which were done with clean wounds — its application led merely to a diminu- 1 Wright, Fleming, and Colebroolt, Lancet, 1918, i. 831. 120 tion of the antitryptic power of the exuded lymph (presumably by disintegration of leucocytes), but in a foul wound full of dead leucocytes, it is probable that the same process would make the discharges actively tryptic, or increase the amount of tryptic ferment already liberated in the wound, and thus lead to the digestion of sloughs. £i a wound already free from sloughs, on the other hand, it would seem that no useful purpose can be served by the increased flow of lymph of reduced antitryptic power obtained by this means. Wright has shown that what is required in such a wound is not so much a flow of lymph as an abundant emigration of leucocytes and the main- tenance of optimum conditions for their functioning. The use of antiseptic solutions is directly opposed to these aims. (7) The effects of introducing hypertonic salt solution into a wound were studied and found to be as follows : (a) An immediate and marked increase in the exudation occurring into the wound cavity, this increase being greater than that obtained by the irritant action of any antiseptic solution. (b) The exudate so obtained did not consist merely of watery fluid, as had been sometimes alleged, but was rich in albuminous substances. (c) The emigration of leucocytes into the wound was suspended for a time but gradually returned to normal as the sodium chloride concentration of the fluid fell away. An abundant emigration of leucocytes took place when the wound contained physiological salt solution. These findings agree in every particular with the results of experiments in vitro published by Wright in the early days of the war. He also demonstrated very clearly that the disintegrative action of 5 per cent, salt solution upon the leucocytes of pus led to a liberation of tryptic ferment, and upon this basis advocated the frequent application of the solution in the treatment of slough-covered wounds. Wright's work, together with the series of experiments here described, enable us to piece together the cycle of events occurring after the instillation of 5 per cent, salt solution into a wound, some- what as follows : First 20 minutes. Rapid exudation of lymph ; liberation of tryptic ferment by disintegration of leucocytes present on the surface of the wound, in sloughs, and free in the pus (this ferment, however, will not be able to exert its full digestive effect until the salt concentration has fallen considerably) ; emigration of fresh leucocytes almost entirely suppressed ; concentration of the salt solution falling roughly to one-half its original content. Second 20 minutes. Less rapid exudation of lymph, but the anti- tryptic power has been abolished owing to the disintegration of leucocytes by the salt solution, which has now diffused into the walls of the wound ; digestion of sloughs by the tryptic ferment in the cavity of the wound becoming very active ; emigration of leucocytes recommencing ; concentration of salt solution falling further — to 1-5 or 2 per cent. Third 20 minutes. Exudation again less abundant but still more 121 than normal owing to the high sodium chloride content of the tissues ; lymph distinctly tryptic ; digestion of sloughs proceeding at maximal rate ; emigration of leucocytes becoming more abundant; concentra- tion of salt solution nearing the isotonic level. Second hour. Little change in rate of exudation — the lymph less tryptic or becoming slightly antitryptic ; digestion of sloughs continu- ing but not quite so actively as before ; emigration of leucocytes at normal rate and some of them undergoing natural disintegration, so reuiforcing slightly the tryptic ferment. As the wound becomes progressively cleaner the serous exudate will tend to have a greater antitryptic value and will, therefore, be enabled to exert its anti- bacterial properties upon any sero-saprophytic bacteria that may be present. In addition to the investigations here recorded the Authors have reported elsewhere upon three subjects which arose out of the work, and particularly attracted their attention. As these papers are available for reference they will not — in view of the present heavy cost of printing — be here reproduced. They are as follows : 1. On the Question of Bacterial Symbiosis in Wound Infections. Lancet, 1917, i. 604. The findings of this paper indicate that symbiosis may play an important part in regard to the prohferation of bacteria in wounds. The growth in vitro of pure cultures of the anaerobic bacteria commonly found in wounds is much less rapid and less abundant than is the case when either streptococci, staphylococci, diphtheroid bacilli, B. proteus, B. pijocyaneus or coliform bacilli are grown in association with them. Similarly the growth of streptococci is greatly facilitated by association with diphtheroid bacilli. Diphtheroid bacilli and many other bacteria are shown to absorb oxygen during their growth, and it is suggested that the symbiotic effects obtained may be ascribed in part to that process. 2. On tile Orowth of Anaerobic Bacilli in Fluid Media iinder apparently Aerobic Condi- tions. Lancet, 1917, ii. 530. The presence of some porous substance is shown to favour the growth of anaerobic bacteria in a fluid medium. Growth is shown to occur first in the interstices of such a substance where, presumably, the bacteria are able to create partially anaerobic conditions by absorption of the oxygen. 3. On Skin Grafting. Lancet, 1917, ii. 5. Some 33 transplantation operations are reported — a large number of them being successful. Small grafts were employed and the raw surfaces from which they were taken were afterwards closed with sutures. Local anaesthesia (by nerve blocking) was usually employed instead of general anaesthesia. The importance of dressing the grafted wound daily is emphasized, and certain recommendations made with regard to the technique of dressings. In the general treatment of the cases the authors had the advantage of being able to consult the Surgical and Medical Staff of St. Mary's Hospital, and they take this opportunity of recording their thanks to Mr. V. Warren Low, Mr. Clayton Greene, and Mr. Ktzwilliams for their advice and for the performance of some operations, and to Dr. Wilfred Harris for examining several cases of nerve injury. Their thanks are also due to Dr. M. M. Bird, the Resident Medical Superintendent, St. Mary's Hospital, who was kind enough to undertake the temporary charge of three patients whom it was thought desirable to isolate. They would thank Mr. G. V. Davis and Mr. B. Thomas who, during the period of the research, were house-surgeons to the wards of the Inoculation Department, and to Mr. T. M. Davis who, for some weeks, undertook these by no means light duties in addition to his other hospital work. They would also record with thanks the help received from Dr. D. G. Sutherland, in respect to carrying out some routine bacteriological investigations, and to the late Dr. J. B. Xias for his help in the wards, at a time when the work was more than usually arduous. Lastly, they wish to place on record their appreciation of the way in which the Sister-in-charge of the Inoculation Department Wards, Miss Winson, carried out her duties. Her devotion to the patients, quickness to grasp new methods, and the carrying out of many instructions foreign to her usual duties, greatly assisted the work. APPENDIX CLINICAL ACCOUNT OP THE 61 CASES, WITH BACTEEIOLOGICAL NOTES Case No. 1. Wounded (bullet) 1.7.16. Admitted 16.7.16. Dwcharged 26.9.16. History. The bullet had entered the front of the right thigh about 4 inches above the patella, causing a fairly clean fracture of the bone, and making a large ragged exit wound. On July 4th, on his arrival at the Base hospital, the leg was found very septic, and incisions were made evacuating large quantities of pus ; a quantity of sloughs and gangrenous tissue was also removed. Condition on admission (16th day). The patient's general condition was fair. On examination, a very extensive wound was found on the posterior surface of the right leg, the skin and intermuscular tissue of the whole of the posterior aspect of the thigh being destroyed, except for a narrow bridge of skin from the gluteal fold to the middle of the calf. The muscles lay practically separated from one another. The femur was fractured between 5 and 6 inches above the knee and large quantities of pus were being discharged, especially from the seat of the fracture. The wound of entry had healed. Treatment. The limb was splinted and intermittent irrigations of 5 per cent, salt solution were given. The local condition improved considerably, and granulations were formed, rapidly binding the muscles together and filling up the deeper parts of the wound, while a very rapid growth of epithelium took place, especially at the upper part of the wound. The average evening temperature was, at first, about 102-5° F., falling later under treatment with small doses of vaccine made from M. tetragenus and Streptococcus isolated from the wound to 101° F. In spite of all care a bed sore formed and the patient's general condition became gradually worse, so the leg was amputated through the middle of the thigh on August 11th by a^ racquet-shaped incision, no attempt being made to close the wound. Recovery was uninterrupted and very rapid, the stump being at first dressed with wet dressings of 5 per cent, saline, later with normal saline, the dressings being separated from the raw surface by a sheet of perforated oiled silk to prevent them sticking. Early extension was applied to the skin, and the whole wound was healed with a firm scar and well covered bone by September 25th (45 days after amputation). Bacteriological Notes. On admission, July 16th. B. sporogenes, B. tertius, B. hutyricus. An anaerobic tetrad. Streptococci (liquefying faecalis type). Coliform and diphtheroid bacilli. Staphylococci (a few). July 24th. Film only. Tetrads very numerous. Streptococcus, Gram-positive bacilli. Diphtheroids. August 2nd. B. sporogenes. A coliform bacillus and Streptococcus equinus. August 16th. B. sporogenes. B. proteus and principally Streptococcus faecalis. August 18th. No anaerobes. September 18th. Streptococcus faecalis and B. proteus. Note. The anaerobes did not disappear from the wound as quickly as usual, a very few being found {B. sporogenes) as late as August 2nd. After the ampu- tation they reappeared, and it may here be noted that a few days after any opera- tion, unless the wound is practically sterile, the opening up of areas of umnfected tissues always leads to an immense increase of the bacterial flora. As regards the streptococci, it is noteworthy that this is one of the very few cases which remained throughout the whole of the course of treatment without Streptococcus pyogenes ever being found, and it is considered that the absence of this organism accounts for the extremely favourable way in which the amputation wound healed up. Case No. 2. Wounded (shell) 11.7.16. Admitted 19.7.16. Discharged 27 1 17 History. At the Casualty Clearing Station the wounds were opened up and much muscle was excised for gas gangrene. Condition on admission (8th day). General condition good. There were two 123 extensive wounds on the calf of the right leg, one on the inner side, the other on the outer, with much loss of skin and muscle. The outer wound was roughly oval in shape, about 5 inches long by 4 inches broad, and included an extensive comminuted fracture of the fibula, of which bone a considerable portion was missing. The anterior tibial and peroneal nerves were completely divided. Much muscle tissue had been destroyed. There was a deep pocket passing from the lower portion of the wound downwards between the tibia and fibula. The wound on the inner aspect, which was separated from the outer one by a bridge of healthy tissue and skin, was roughly oval, being about 6 inches long and 4 inches broad, and extended into the calf muscles. Treatment. With intermittent application of hypertonic salt solution the wound surfaces rapidly became clean and healthy looking but the pocketing was very troublesome, and the temperature remained high until small doses of streptococcus vaccine were given, when it rapidly became normal. In connexion with this case observations were made as to the stimulant action of malachite green on the growth of epithelium. A 1 per cent, solution of malachite green in spirit was used as a dressing to the upper half of the inner wound, the lower half being left as a control. Measurements made by tracing the area of the wound through a sheet of clear oellulloid showed that the portion of the wound spra^-ed with the malachite green developed epithelium much more rapidly than the control portion ; the average increase of the epithelial edge in one week being 0-59 centimetres for the malachite green treated portion and 0-38 centimetres for the control portion. He was seen some months later walking almost perfectly in a special boot to prevent foot-drop. Bacteriological Notes. On admission. B. sporogenes, B. tertius, Streptococcus pyogenes, B. proteus, Staphylococci (a few). Diphtheroids. In films : Tetrads also seen. Jidy 25th. B. sporogenes, B. tertius. Streptococcus pyogenes. Staphylococcus (a few). Diphtheroids. July 31st. No anaerobes. Streptococcus pyogenes (only). August 21st. Streptococcus pyogenes (only). September 1st. Streptococcus pyogenes, most abundant. A few B. pyocyaneus and B. proteus. Case No. 3. Wounded (bullet) 7.7.16. Admitted 21.7.16. Discharged 26.10.16. History. At the Base a considerable portion of the right quadriceps was removed for local gas gangrene. Condition on admission (14th day). His injuries were : (1) an almost healed bullet wound of left forearm, with a fracture of the ulna li inches above the wrist. This wound showed no evidence of sepsis and was almost healed. (2) On the anterior aspect of the right thigh there was a large wound extending over its whole surface deeply into the muscles. Much muscle had been removed for gas gangrene. (3) On the posterior surface of the left thigh was a more super- ficial wound, the skin and deep fascia only being involved. These two latter wounds had fairly clean granulating surfaces. General condition good ; no fever. Treatment. The left forearm was put up in plaster and gave no further trouble, the bone uniting in good position and no limitation of movement resulting. On August 6th the wound of the right thigh was sutured, but the tension on the stitches was very great owing to the amount of soar tissue that had been formed in the depths of the wound, and, in consequence, the stitches had to be removed , early; limiting the success of the operation. Healing progressed slowly but without any set-backs. Bacteriological Notes. On admission. Ko anaerobes were found. Staphylococcus, diphtheroids, B. proteus, Streptococci (a few), M. tetragenus, and a ooliform bacillus. July 28th. B. proteus and Staphylococcus. August I9th. Staphylococcus and a coliform fermenting glucose with produc- tion of acid but having no effect on lactose, dulcite, and mannite. As regards cane-sugar a slight amount of acid was produced in the first 24 hours, but the broth shortly after again became alkaline. August 21st. Very few microbes seen in films. Staphylococcus and diphthe- roids isolated. 124 September 18th. Right thigh : Staphylococcus and diphtheroids. Left thigh : Staphylococcus, diphtheroids, and a few Streptococcus equinus. Note. This case is again remarkable for the absence of Streptococcus pyogenes throughout the whole period of observation ; the progress of the case was quite uneventful. Case No. 4. Wounded (shell) 1.7.16. Admitted 25.7.16. Discharged 18.12.16. Condition on admission (24th day). The patient was very pale and debilitated, but without fever. Wounds. There was a large, unhealthy looking, granulating surface extending from the point of the shoulder 4 or 5 inches down the upper arm. This was continuous with a granulating surface involving the axilla and anterior axillary fold. There was a separate wound on the outer border of the scapula which coEtimunicated with the axillary wound. X-rays showed a large number of minute pieces of metal in axillary region, arm, and side of chest. The humerus was fissured, but there was no displacement. There was extensive bruising of the nerves of the brachial plexus, giving rise to severe neuralgic pain and partial paralysis of the arm, forearm, and hand. Treatment. An attempt was made to reduce the size of the wound on August 3rd by suturing, without much success. Treatment largely consisted of moist saline dressings. The progress was slow but satisfactory. He was dis- charged, almost completely healed, on December 18th, the nerve lesions being still troublesome but improving. Bacteriological Notes. On admission. B. sporogenes, a tetanus-like organism, B. sphenoides, Strepto- cocci, Staphylococci, B. proteus, diphtheroids, and M. tetragenus. August 19th. B. proteus and B. pyocyanev^, very numerous. August 24th. B. proteus, numerous, and others. September 18th. B. proteus, B. pyocyaneus, and a ooliform bacillus, which produced acid in glucose broth but had no action on lactose, saccharose, dulcit«, or mannite. Remarks. The outstanding feature of this case was the unhealthy aspect of the granulations ; they were pale, soft, and bled easily. The most constant organism present. was B. proteus. Case No. 5. Wounded (shrapnel) 7.7.16. Admitted 26.7.16. Discharged 17.12.16. History. At the Base he was operated on for secondary haemori'hage, the left common interosseous artery being tied. No history of gas gangrene. Condition on admission (19th day). On the right upper arm there was a clean granulating surface about 3 inches long on the inner side, not extending below the deep fascia. On the right side of the chest, a little below the clavicle, was another clean granulating surface, no injury of the deeper tissues being found. There was a through and through wound of the left forearm with a compound fracture of the ulna. Both entry and exit wounds were discharging freely. The right median nerve had been bruised, and there was severe neuralgic pain in the palm of the hand and much wasting of the hand muscles, especially the interossei. Treatment. The fracture was put up in plaster with windows for dressings as soon as the discharge lessened under wet saline dressings. Progress was uneventful, there being no fever throughout the progress of the case ; the right median nerve was cut down on by Mr. Low,- after the wound over it had healed soundly. It was found involved in scar tissue, from this it was freed and a piece of fascia stitched over it. This only relieved the pain for a time, and the patient was still suffering considerably when he left the hospital. The fractured ulna united soundly in good position. Bacteriological Notes. On admission. Pus from the .left forearm showed many microbes,- Gram- negative bacilli. Streptococci, diphtheroids, and Gram-positive bacilli. Cultures from forearm gave a few B. sporogenes and B. tertius, many Streptococcus pyogenes, and a coliform bacillus. Some of the superficial wounds gave cultures of Streptococcus pyogenes, B. proteus, and a diphtheroid. August 4th. Chest wound gave Staphylococciis only. August 13th. Chest wound gave Staphylococcus only. 125 August 21st. The wounds connected with the fracture of the left ulna showed large numbers of Streptococcus pyogenes only. Case No. 6. Wounded (bullet and shrapnel) about 18.7.16. Admitted 29.7.16. Discharged 17.11.16. History. The patient lay out for 3 days after being wounded. Amputation of the left leg in the middle of the thigh was performed at the casualty clearing station. Condition on admission. 1. Small scalp wound : suppuratuig but healing. 2. Left hand, wrist, &c. : many small wounds cleaning and healing. 3. Right forearm : large flesh wound, exposing the tendons ; this wound was cleaning satisfactorily. 4. Right upper arm : large flesh wound exposing the muscles ; this wound was graniilatuig satisfactorily. 5. Right foot : a perforating bullet wound which had caused a comminuted fracture of the third meta-tarsal, rather septic. 6. Amputation stump : the bone was much exposed, the marrow fungating, and the muscles and skin tending to fall away ; there was much suppuration but no extensive sloughs. General condition was very fair, not much fever (maximum 100° F.). He complained greatly of indigestion. Treatment. Wounds dressed with 5 per cent, saline until clean, afterwards with normal saline. Extension applied to the soft parts of the stump. Progress was very rapid and without any set-backs. When the stump was nearly healed a secondary amputation was performed, the wound being loosely closed and dressed from the beginning with wet dressings of 5 per cent, saline. There was very little constitutional disturbance and healing was very rapid, a good stump remaining. Some sequestra were also removed from the right foot. Bacteriological Notes. On admission. Films from stump showed diplococci and a long Streptococcus, coliform bacilli, and a Gram-positive bacillus. No anaerobes could be isolated from this situation. Streptococcus, mostly of the faecalis type, but pyogenes also present. Films from the foot showed Streptococcus (long chained) and B. sporo- genes. August 25th. Cultures from the foot showed B. proteus, diphtheroid bacilli. Streptococcus pyogenes, and B. sporogenes. October 19th. Three days after secondary amputation practically pure culture of Streptococcus pyogenes with a few diphtheroids. Case No. 7. Wounded (bullet) 9.7.16. Admitted 29.7.16. Discharged 28.12.16. History. Patient was operated on at the Casualty Clearing Station for sepsis. Condition on adynission (20th day). General condition good. Woimds. The entry wound was near the acromion on the right side, which communicated by a sinus about 4 inches long with a wound near the inferior angle of the scapula ; there were two other wounds lower down on the back, in the right side of the upper lumbar region, which were evidently the result of incisions made for drainage. The scapula was fractured. X-rays showing commi- nution of the axillary border. A fair amount of rather thin pus. Treatment. Wet saline dressing. Progress was rather slow but uneventful. On October 18th the remaining wound at the inferior angle of the scapula was explored, some detached pieces of bone removed and drainage improved. Bacteriological Notes. On admission. Films showed nothing but Streptococcus. Cultures showed large numbers of Streptococcus pyogenes and an occasional Staphylococcus. No anaerobes. August 4th. Pure Streptococcus pyogenes. August 22nd. Streptococcus pyogenes and Staphylococcus. October 2nd. Streptococcus pyogenes and Staphylococcus in about equal numbers. Case No. 8. Wounded (shrapnel) 19.7.16. Admitted 30.7.16. Discharged 12.12.16. History. At the Base extensive incisions were made for gas gangrene. Condition on admission (11th day). General condition was fairly good, and there was no great degree of fever. Wounds. Over the right parietal bone there was a scalp wound about li inches in length. This extended down to the bone, which was bare of periosteum, and the outer table of the skull was fractured. No cerebral symptoms were present. 126 There was a compound fracture of the proximal phalanx of the little finger of the left hand. The wound was granulating but some bare bone could be felt. The prmcipal wound was situated in the left thigh ; originally the projectile entered somewhere in the region of the anterior superior iliac spine and passed out a few inches lower down the thigh, and the wounds about to be described were almost entirely the result of incisions made to combat the gas gangrene. The largest wound extended from the anterior superior iliac spine down the front of the thigh to just above the knee, being about 5 inches broad in the upper part of the thigh. In the upper and outer region of the thigh woimd there was a considerable loss of tissue. There were several smaller wounds, linear incisions situated over the calf and the posterior aspect of thfe thigh, and, lastly, there was a linear wound about 4 to 5 inches long in the abdominal wall over the outer part of the left iliac fossa. Most of these wounds were very clean and there was no pocketing or sloughing ; and the smaller ones were healing rapidly. Treatment. Wet saline dressings. It was in this case that perforated celluloid was first used to prevent the dressings from sticking, a device introduced on account of the excessive pain caused by the removal of gauze which stuck to a raw surface. The result was moat satisfactory. Progress. On the whole, it was very rapid and satisfactory ; the only event worth recording in the earlier stages of the case was an attack of erysipelas, which commenced at the upper part of the wound, which had been dressed with ointment, and slowly spread down the leg. It was of a very mild type. The wounds were later successfully skin-grafted, and when the j^atient was discharged on December 12th there remained only a sinus' in the scalp leading down to necrosed bone. Bacteriological Notes. On admission. Films showed enormous numbers of bacteria, ooliform bacilli, Streptococcus, tetrads, and sporing bacilli. Cultures : B. sporogenes, B. tertius, B. proteus, and Streptococcus pyogenes, and an unnamed anaerobic bacillus. August 21st. Culture made from various situations showed Streptococcus pyogenes in the upper ]3art of large wound. Diphtheroids only in the lower unhealthy portion. August 22nd. Culture shows pure Streptococcus pyogenes. August 24th. Streptococcus pyogenes and B. pyocyaneus. August 30th. Streptococcus pyogenes and Staphylococciis. September 5th. Chiefly Streptococcus pyogenes and a few Staphylococcus. .November 1st. Streptococcus pyogenes and a few Staphylococcus. Case No. 9. Wounded (bullet) 19.7.16. Admitted 30.7.16. Discharged 6.1.17. History. Gas gangrene developed in right thigh, which was treated by large incisions. Condition on admission (llth day). General condition was not very good, the patient's colour being earthy and his pulse rather feeble. Wounds. There was a bullet wound of the right elbow region, the entry wound being over the head of the radius and the exit one over the internal condyle with much comminution of the bones arid' suppuration of joint and both wounds, but no surrounding inflammation. X-rays showed fracture of the humerus, ulna, and (most probably) the radius, just below the head. The right thigh had a single very extensive wound on the outer and posterior aspect, extending from the great trochanter to the lower border of the popliteal space. This wound extended deep into the intermuscular planes. It was about 5 inches broad at its widest part, and showed a great tendency to sag open. The surface was clean and the suppuration moderate. Treatment. Elbow splinted on an angular splint and saline dressings applied both to thigh and elbow region. Four days after admission the thigh wound was brought together by deep and superficial stitches, large drainage tubes being inserted into the deep muscular plains. This considerably lessened the area of the wound in the central portion of the thigh and, although all the stitches did not hold, the general result of the operation was very satisfactory. Healing was complete by the middle of October. At first the wounded elbow-joint remained very quiescent, but later drainage became unsatisfactory and the inflammatory condition became more acute. Incisions were made into the joint, but drainage remained unsatisfactory and the 127 patient's general condition became daily worse. On September 3rd the arm was amputated above the elbow. After this the patient's general condition rapidly improved and the stump healed satisfactorily, except that a small ring of necrosis of the distal end of the humerus caused a sinus to persist until December. On the removal of this necrosed bone the sinus rapidly closed. The patient was discharged on January 6, lOlV, with perfect movements of the leg, and the right arm had a sound stump. Bacteriological Notes. On admission. B. sphenoides. Streptococcus pyogenes, diphtheroids from the thigh, B. proteus, and Streptococcus pyogenes from the elbow. August 3rd. B. proteus and a coliform bacillus giving fermentation reaction of B. coli communis and Streptococcus pyogenes from the elbow. Thigh : B. sphenoides, B. proteus, Staphylococcus, and Streptococcus pyogenes. August 11th. Streptococcus pyogenes and a few Staphylococcus from thigh. August 21st. From elbow many Streptococcus pyogenes, a, few Staphylococcus and B. pyocyaneus. September 4th. Only Staphylococcus and B. pyocyaneus. October 1st. Thigh : diphtheroids and B. pyocyaneus. Arm : Staphylococcus and Streptococcus. October 16th. Wound of thigh almost healed, anaerobic cultures. Strepto- coccus pyogenes and a few tetrads. Case No. 10. Wounded (bullet) 23.7.16. Admitted 31.7.16. Discharged 25.9.16. History. Five days after the injury he was operated on for cellulitis, an incision being made in the front of the thigh, from which the bullet was extracted and pus evacuated. Condition on admission. Patient had a superficial wound about 3 inches long situated on the right buttock close to the anus. This wound was cleaning well. Opposite this was a penetrating wound of the left gluteal region, the entry wound being quite close to the anus and admitting a probe for about 4 inches deep to the gluteal muscles ; there was a scanty discharge of blood-stained pus. In the front of the thigh situated near the anterior border of the tensor vaginae femoris was an incised wound ; from this wound a probe could be passed for 2 or 3 inches deep to the muscles in the direction of the wound of entry. The l&ft buttock was swollen, but there was no extensive inflammatory induration. A considerable amount of deep haemorrhage had apparently taken place, the skin over the front and outer aspect of the thigh being much discoloured by effused blood. X-rays showed no injury to the bone, and no foreign body could be detected. Progress. After the first ten days the progress of the wound was quite unevent- ful. The patient was discharged on September 25th, 1916, with all the wounds healed and but little temporary limitation of the movements of the hip-joint. Bacteriological Notes. On admission. Films showed many streptococcus, some Gram-negative bacilli, and a very few bacilli resembling B. welchii. Cultures gave a few colonies of B. welchii, many of Streptococcus and B. proteus. . August 8th. Streptococcus pyogenes and B. proteus. August 24th. Streptococcus pyogenes, diphtheroid and a few B. pyocyaneus. August 29th. A few colonies of Staphylococcus and Streptococcus Case No. 11. Wounded (bullet) 24.7.16. Admitted 31.7.16. Discharged 5.10.16. Condition on admission. General condition good. The patient had a through and through wound in the right upper arm, midway between the axilla and the elbow ; and a drainage tube had been passed right through beneath the biceps. The wound situated on the inner aspect of the arm was considerably the larger and more lacerated. Both wounds were fairly clean, but a quantity of thin blood-stained discharge was escaping. X-rays showed the humerus to be uninjured and that no foreign bodies were present. Treatment. At first 5 per cent, saline packs, later normal salt was used. Healing was uneventful. Bacteriological Notes. On admission. Films only showed streptococci. Cultures in addition to Streptococcus pyogenes proved the presence of B. welchii. 128 August 1st. Streptococcus 'pyogenes together with a few B. pyocyaneus and- a coliform organism. August 28th-29th. Streptococcus pyoge7ies. September 18th. Streptococcus pyogenes and also a few colonies of B. welchii. Case No. 12. Wounded (shell) 29.7.16. Admitted 3.8.16. Discharged 27.9.16. History. Operated on for gas gangrene at Boulogne two days after the injury. Condition on admission. The wound was situated on the right deltoid region, being roughly oblong in shape, about 7 inches long and 5 inches broad. There was much loss of tissue, the humerus being exposed but quite uninjured in the centre of the wound. The base of the wound was very dirty, covered in many places with sloughs, and the skin edges were very irregular, a large flap hanging down posteriorly. The dressings were excessively painful. Treatment. Five per cent, saline packs changed 2-hourly, the wound surface being protected by a sheet of perforated celluloid which entirely eliminated the pain of the dressings. In two days under this treatment practically all the sloughs had disappeared and the surface looked like clean raw meat. Then the posterior flap was brought over the wound and fixed in position with strapping. With simple normal saline dressings the granulations became oedematous, but when pressure was applied with a firm bandage they took on a healthy appearance. The subse- quent progress was uneventful, and perfect healing resulted. There was no limitation of the movements of the arm. Bacteriological Notes. On admission. Films showed enormous numbers of bacteria, principally diplococci, large Gram-positive and Gram-negative bacilli, and coliform bacilli. Cultures, B. sporogenes, B. tertius. Streptococcus pyogenes. Streptococcus salivarius, coliform bacilli, Staphylococcus. A bacillus exactly like B. tetani was also seen in a trypsin-blood-broth culture, but was not isolated. August 17th. Films showed only cocci and diphtheroids. Cultures, Staphylococcus albus and aureus and Streptococci. August 19th. Streptococcus pyogenes and B. pyocyaneus. August 29th. Streptococcus pyogenes. September 1st. Streptococcus pyogenes and a few Staphylococcus. Case No. 13. Wounded (shell) 30.7.16. Admitted 3.8.16. Discharged 28.12.16. Condition on admission (4th day). There was a large sloughing wound with irregular ragged edges, corresponding roughly in size to the area of the right scapula, over which it was situated. The supra and infra spinatus muscles were in large part destroyed. The scapula was extensively fractured, most of the spine being broken away, and there was a considerable number of loose pieces only partially covered with periosteum. No fracture of the underlying ribs could be detected, and the right humerus and clavicle were both sound. General condition was by no means good, the patient looked very thin and hectic, there was a considerable degree of continued fever, the evening tempera- ture usually being 102-5° F. with a daily morning remission of about 2 degrees. There was bronchitis with constant coughing and a good deal of muco-purulent sputum. Examination of the chest, however, did not suggest any intra-thoracic injury. Treatment. The wound was dressed with 5 per cent, saline packs, the wound surface being protected by a sheet of perforated celMoid. The bronchitis was treated with a vaccine made from B. influenzae and Pneumococcus, both of which micro-organisms were present in the sputum in enormous numbers. A blood culture having given a growth of an unclassified Streptococcus, in one tube out of ten, a vaccine was prepared and administered, seemingly -without the slightest effect. Small doses of a mixed Streptococcus vaccine were also given. Progress. In spite of the continued fever the wound cleaned rapidly, but healing was slow, neither contraction of the surface nor epithelial growth at the edges being at all marked. Several pieces of scapula were removed as sequestra. The surface of the wound was not very good, the granulations being very smooth 129 and shiny and the discharge scanty and thin, a dusky red blush extending from the edges. Although the bronchitic condition improved, the fever became rather higher and more intermittent at the end of the first fortnight. August 29th. B. pyocyaneus having been found abundantly in the wound and in almost pure culture, a small dose (2 million) of a B. 'pyocyaneus vaccine was given. The temperature at once dropped and in a few days became normal, remaining about the normal line for the remainder of the patient's stay in hospital. The condition of the wound rapidly improved. October 18th. For some time past the healing process had been very slow, portions of the wound often being covered by a membrane and depressions with smooth shiny indolent-looking bases appearing on the granulating surface. Cultures showed the presence of an unclassified Streptococcus (see bacteriological notes) ; a vaccine having been made from this coccus small doses (3 to 5 million) were administered, a very marked improvement occurring as a result of this treatment, November 25th. The wound had a very healthy bacterial free surface, but contracture and formation of epithelium were both very slow. The unhealed portion of the wound was, therefore, skin-grafted by Steele's method (see Douglas, Fleming, Colebrook, Lancet, 1917, ii. 5). The result was most satisfactory, the whole surface being rapidly covered with epithelium, but the scar is very adherent to the underlying bone. December 28th. The patient was discharged. In April 1917 the patient was again seen — the ultimate result was very satisfactory, movements of the arm and shoulder being almost perfect. The scar was quite sound, except at one small point where several small pieces of dead bone had worked out. The skin grafts were still quite visible, appearing as white islands in the surrounding reddish brown scar, which was remarkably soft. Bacteriological Notes. On admission. Films showed enormous numbers of bacteria, chiefly diplo- cocci, clumps of Gram-positive cocci, tetrads, and large Gram-positive bacilli resembling B. ivelchii, also some free spores. Cultures gave two varieties of Streptococcus, one the mannite fermenting type of pyogenes, the other an unclassified type resembling salivarius but fermenting salioin ; Staphylococci, Gram-negative streiitobacilli, and coliform bacilli. Anaerobic cultures showed B. welchii and B. sporogenes to be present. A blood culture was positive — a streptococcus with the following characteristics being found in one tube out of the ten into which the blood was distributed. Individual cocci usually round, but some irregular in shape ; chains, brevis type ; broth culture showed even turbidity ; saccharose broth made acid, lactose, rafiuiose, mannite, salioin, and milk unaffected. Neutral red broth, anaerobic culture gave fluorescence. Sputum : large numbers of Pneumococci and B. influenzae both isolated. August 21st. Film showed very few organisms, chiefly Streptococcus and a few diphtheroids. Cultures gave B. sporogenes, Streptococcus pyogenes (mannite fermenting type), Staphylococcus, and a Gram-negative bacillus. August 25th. Blood culture negative. August 26th. From a deep pocket near the axilla a nearly pure and abundant culture of B. pyocyaneus was obtained ; the more superficial portion of the wound giving only a few Staphylococci and diphtheroids. August 30th. Deep pocket. B. pyocyaneus and Staphylococcus, no Strepto- coccus isolated ; the cleaner portion showing only three colonies of Staphylo- coccus. September 13th. Deep pocket. B. pyocyaneus and a few coliform organisms. September 18th. From the deep pocket anaerobic cultures gave no strict anaerobes, but only 5. pyocyaneus. Streptococcus, diphtheroids, and a few coliform bacilli which produced acid from glucose and slightly from saccharose but had no effect on lactose, dulcite, or mannite. The streptococci isolated on this date were an unclassified type having the following characteristics : individual cocci, usually round but oval forms not uncommon ; chains, brevis type ; broth culture, a granular deposit was formed leaving the broth clear. Fermentation reactions. Acid produced in lactose and salicin broths ; saccharose, raffinose, and mannite unaffected ; acid and clot was produced in milk cultures ; neutral red broth culture incubated anaerobically gave no fluorescence. October 4th and 5th. Films made from membranous portion showed only 1727 I 130 Streptococcus ; anaerobic cultures gave streptococci of the same type as were isolated on September 18th, and one or two colonies of Staphylococcus. Culture (aerobic) from the pocket gave pure Sta-phylococcus aureus. From the membrane anaerobic cultures gave Streptococcus of the same type isolated on September 18th, a few tetrads, diphtheroids, and Staphylococcus. November 9th, before grafting, only three or four colonies of diphtheroid bacilli. Case No. 14. Wounded 19.7.16. Admitted 3.8.16. Discharged 27.9.16. Condition on admission (14th day). General condition good ; temperature practically normal. Two wounds were present : ( 1 ) the smaller consisted of a superficial wound about 4 inches long by 2 inches broad, situated over the right costal margin ; (2) the larger and more severe was situated over the left scapula. In shape and size it was roughly oblong, being about 10 inches in length and the maximum width being about 6 inches. The scapula was exten- sively fractured but was covered by a clean granulating surface. The lower edge of the wound tended to sag badly. Treatment. At first 5 per cent, saline packs were applied ; later normal salt was used. The edges of the wound were brought together with strapping. Progress was rapid and practically uneventful. The wound over the scapula healed rapidly, and the bone united perfectly. Bacteriological Notes. On admission. Films from the wound over right costal margin showed only Strejitococci and diphtheroids. Cultures gave Streptococcus pyogenes and a few Staphylococcus. Films from the wound over the left scapula showed only Streptococci and diphtheroids. Cultures gave a few colonies of B. sporogenes and many of Streptococcus pyogenes and Staphylococcus. August 12th. Film from the larger wound, only a few Streptococci seen. August 17th. Cultures from the larger wound. Streptococcus pyogenes and Staphylococcus. From the front wound — Streptococcus pyogenes only. August 19th. Cultures from wound of back. Streptococcus pyogenes and a few B. pyocyaneus. August 28th. Cultures from wound of back. Streptococcus pyogenes. August 30th. Cultures from wound of back : (i) from an unhealthy looking portion of surface, many Streptococcus pyogenes, (ii) From a healthy looking portion of the surface, a few colonies of Streptococcus pyogenes. September 18th. Wound of back. Plate culture gave only one colony of Streptococcus pyogenes. Trypsin-blood-broth, Staphylococcus, Streptococcus, and diphtheroids. Heated pus, sterile. Case No. 15. Wounded (hand grenade) 2.7.19. Admitted 3.8.16. Dis- charged 26.9.16. History. The right leg was amputated below the knee at the Casualty Clearmg Station, the wound being sewn up. The left leg was amputated about 4 inches above the knee for sepsis at the Base, the guillotine method being employed. Condition on admission (32nd day). The right stump 4 inches, below the knee joint had healed. The left stump showed a circular wound about 4 inches in diameter which was granulating well, the bone being just covered. Just above the right elbow there was a marked deformity which X-rays showed to be due to a fracture of the external condyle of the humerus ; there was no note as to how this had been treated, but union was firm. Progress was quite uneventful, and when the patient was discharged the left stump was quite healed except for a minute spot. Bacteriological Notes. On admission. All the cultures taken were overgrown with B. proteus. August 17th. CSiltures gave pure Streptococcus pyogenes. August 31st. Streptococcus pyogenes and Staphylococcus. September 13th. Staphylococcus and diphtheroids in about equal numbers. Case No. 16. Wounded (shell) 19.7.16. Admitted 3.8.16. Discharged History. Patient arrived at the Base on July 21st, where he remained until August 3rd, suffering from a left haemothorax in addition to the wounds. Condition on admission (15th day). The patient's general condition was 131 good ; shortness of breath was the only symptom of the haemothorax. The chest signs were very slight, some impairment of resonance and of the breath sounds on the left side being all that could be made out. His wounds were both situated high up on the shoulders, on the right side there was a deepish track leading towards the clavicle from the supra-spinous fossa. On the left side the wound, which was in a similar situation, was apparently more superficial. X-rays showed the shadow to be increased over the whole of the left side, but the dia- phragm moved normally and the dome was not depressed. The heart was somewhat pushed over to the right side. There were two or three small pieces of metal embedded in the right shoulder. Progress was rather slow, owing to the presence of the foreign bodies, three of which were removed and drainage improved by counter openings. The haemothorax gave no further trouble and was seemingly completely absorbed. Bacteriological Notes. On admission. Culture gave Streptococcus pyogenes and Staphylococcus, also some very minute colonies of a thin curved bacillus, which failed to grow in subculture. Films also showed diphtheroids. August 22nd. Cultures gave a few colonies of Staphylococcus and one colony of Streptococcus. September 5th. Left shoulder almost healed — Staphylococcus only. PJght shoulder — Streptococcus pyogenes in pure culture. October 16th. Eight shoulder — many Streptococcus pyogenes and a few Staphylococcus. ' Case No. 17. Wounded 12.7.16. Admitted 9.8.16. Discharged 4.9.16. History. At the Base two or three days after being wounded, he had secon- dary haemorrhage on two occasions and a severe attack of erysipelas which was treated with streptococcus vaccine. Condition on admission (28th day). The wound of entry, in front of the lobe of the right ear, quite small and healed; the exit wound behind the sterno- mastoid was larger and more ragged but quite clean. All signs of erysipelas had disappeared. Progress. Rapid and very satisfactory, the wound healing rapidly from the bottom. Bacteriological Notes. On admission. Streptococcus pyogenes, diphtheroids and B. proteus. August 18th. Staphylococcus only. August 19th. Staphylococcus only. August 22nd. Staphylococcus only. Case No. 18. Wounded (bullet) 19.7.16. Admitted 21.8.16. Discharged 14.11.16. History. At the Base on July 22nd, amputation was performed through middle of right thigh (flapless method). Condition on admissimi (32nd day). The general condition was good. Wounds. Amputation stump of the right thigh was clean, but the bone was projecting considerably and the tissues had lost much of the elasticity owing to the formation of scar tissue. There was a small sinus on the posterior internal aspect of the stump about 2 inches long. Left leg. There was a perforating wound about the middle of the calf, both apertures being small. No injury of the bones or nerves could be found. A moderate discharge of pus was escaping from the wounds. Progress was almost uneventful, occasionally there were slightly inflamed spots about the stump, but on the whole healing was rapid. A ring of bone separated from the end of the femur about the middle of Sejjtember but in spite of this the bone still protruded beyond the soft tissues. By this date the left leg had healed. In the middle of October a secondary amputation was performed, about 3 inches of the shaft of the femur being removed through an irregular incision which was almost completely closed by sutures. The stump was dressed from the first with very wet hot 5 per cent, saline packs and under this treatment healing took place rapidly with but little suppuration. The patient was discharged on November 14th, 1916, just four weeks after the secondary amputation, with a soundly healed stump. Bacteriological Notes. On admission. Cultures from the left calf gave Streptococcus pyogenes and 12 132 a heat-resisting Streptococciis, while the stump gave B. pyocyanens, diphtheroids, and a Gram-negative bacillus. September 5tli. Staphylococcus, Streptococcus pyogenes, diphtheroids, and B. proteus were grown from the stump. (Ttus was one of the wounds from which a bacillus apparently identical with B. diphtheriae was isolated.) September 18th. Gave a number of colonies similar to the above. October 5th. The cultures were overgrown with B. proteus. October 20th. After the secondary amputation Streptococcus pyogenes was found in considerable numbers. Case No. 19. Wounded (shell) 29.7.16. Admitted 21.8.16. Discharged 14.11.16. History. Gas gangrene developed at the Base and was treated with multiple incisions. Condition on admission (23rd day). General condition was good. The wounds were situated on the posterior and outer aspects of the thigh, the principal one being on the posterior aspect and extending ihto the intermuscular planes. In addition to this there were some twenty-five other wounds, evidently incisions for the treatment of gas gangrene, most of them extended through the deep fascia. Progress was at first rapid and by September 15th all the smaller incisions had healed, but the principal wound showed some tendency to digestion of the epithelial edge and the granulating surface. Local applications of nitrate of silver and brilliant green, however, improved the condition, and the patient was discharged on November 14th, completely healed. Bacteriological Notes. On admission. Films from the large wound showed Streptococci and Staphylo- cocci. Cultures gave colonies of Streptococcus pyogenes and B. pyocyaneus. October 5th. Plates overgrown with B. proteus. October 9tli. Plates overgrown with B. proteus. October 10th. Diphtheroids only. When the large wound showed areas of digestion of the surface and epithelial edge, films were made from these situations and compared with films made from portions of the surface where the granulations had a more healthy look. On staining and examining these a very marked difference was found in respect to the number of organisms seen, the films made from the unhealthy looking portion were crowded with streptococci and coliform bacilli (most probably B. proteus) while those made from the healthy surface contained practically no micro-organisms. Case No. 20. Wounded 12.6.16. Admitted 22.8.16. Transferred 23.6.17. History. Tetanus having developed, the left leg was amputated at the Base through the middle of the thigh by a guillotine operation. The stump remained in a very septic condition until continuous irrigation with hypertonic saline was employed, when considerable improvement was noticed. Condition on admission (61st day). The left leg stump had almost healed over, but the bone was protruding about 2 inches beyond the soft parts. Right leg. There was a compound comminuted fracture of the tibia and fibula just below the knee. Over the fracture of the tibia was a deep hole, opening on the inner side of the leg ; stretching from this towards the outer side was a granulating surface surrounded by a considerable area of scar which was hard, inelastic, and very adherent to the deeper tissues. This scar extended over the region of the neck of the fibula to almost behind the knee. The fracture showed very little evidence of union. The external popliteal nerve had been divided just above the head of the fibula. The patient's general condition was poor. There was a large splint sore on the heel. X-ray showed that the whole of the tibia had been rarefied by osteomyelitis. Progress. At first it was very slow and unsatisfactory, as after healing considerably the soar would break down ; the hole over the head of the tibia showed no signs of healmg. If the patient had not lost the other leg amputation undoubtedly would have been the proper treatment. In the beginning of October the wounds were explored and some counter openings made to improve drainage. After this the patient's general condition began to improve. In November a more extensive operation was performed, several pieces of dead bone being 133 removed and drainage made as free as possible. Until February 1917 very- little of importance took place, the patient's general condition steadily improved, and a radiogram after bismuth injection showed that a considerable amount of new bone had been formed and that sequestra were separating inside the shaft of the tibia. Towards the end of February a mild attack of erysipelas intervened, and shortly afterwards a new sinus formed about 3 inches above the internal .malleolus. Further X-ray photographs pointed to the whole of the original shaft of the tibia having necrosed and formed a sequestrum lying inside the new bone. About the middle of April the tibia was, therefore, opened up, and the sequestrum was removed from the whole length of the shaft through two incisions, one being an enlargement of the original hole, on the inner side of the head and shaft of the tibia, the lower one being above the internal malleolus. After this operation healing was rapid, the lower wound healing very quickly. Improvement continued until the patient was transferred on the closing of the Inoculation Department wards. The condition at that time was as follows : The upper woiind over the head of the tibia had almost healed, the epithelium spreading downwards into the large cavity in the head of the bone, but there was a sinus over the head of the fibula leading to a small sequestrum. The patient's general condition was excellent. Bacteriological Notes. On admission. Films showed very numerous bacteria amongst which were large capsulated Gram-negative bacilli, Streptococci (two varieties), diphtheroids tetrads. Cultures gave two varieties of coliforms, Streptococcus faecalis and Streptococcus pyogenes, B. pyocyaneus. Staphylococci, M. tetragenus, diphtheroids, B. tertiiis, and B. sporogenes. August 26th. Films showed Streptococci, diphtheroids, a coliform bacillus, tetrads, and a long slender Gram-positive bacillus. August 31st. B. sporogenes and B. tertius. Streptococcus pyogenes, B. proteus. September 1 8th. Streptococcus pyogenes, Staphylococcus, and diphtheroids. November 8th. From sequestra removed at operation, B. sporogenes, B. sphenoides, and B. tertius. Streptococcus pyogenes and B. proteus were all obtained by culture and in addition to these some tetrads. Staphylococcus, and Gram-positive bacteria were seen in films. November 13th. Streptococcus, diphtheroids, Staphylococcus coliform bacilli, and B. proteus, also B. sporogenes and B. tertius from the heated pus. April 13th, 1917. From a sequestrum removed at the second sequestrotomy, B. sporogenes, B. tertius. Staphylococcus, Streptococcus, and B. proteus. April 18th. From the pus, Streptococcus, Staphylococcus and B. proteus were isolated. May 5th. Streptococcus and Staphyloccus, diphtheroids, and B. proteus. Case No. 21. Wounded (bullet) 12.8.16. Admitted 27.8.16. Discharged 14.11.16. History. Operated on at Casualty Clearing Station for gas gangrene. Condition on admission ( 15th day). General condition good, except for marked nervousness and dislike of noise. The patient had a large ragged wound about 9 by 6 inches in the front of the left thigh, extending deeply into the muscles. The femur was uninjured. This wound had a fairly clean surface, but one or two sloughs were still attached. In addition there were two linear wounds about 5 inches long, the result of incisions on the outer side of the same thigh. On the right thigh there were two small wounds, one about | inch in diameter in front of the great trochanter, apparently almost healed, the other about 1 inch long, situated in the right groin. This wound had a track leading from it directly backwards and from it a little thick pus was oozing, but there was no surrounding inflammation. Progress was rapid, especially at first, the large wound filling up well, and the smaller ones healing very rapidly. Later the healing became much slower, but by November 14th all the wounds were healed. The movements of the leg were seemingly in no way impaired, the quadriceps muscles acting well and strongly. Bacteriological Notes. Films from large wound showed enormous numbers of bacteria, Gram- positive cocci, single cocci, diplooocci, and tetrads, diphtheroids, Gram-negative bacilli, and Gram-negative diplococci. 134 Cultures gave B. sporogenes, B. sphenoides, Streptococcus 'pyogenes. Strepto- coccus equimis, and an' unclassified Streptococcus, whicli produced acid in saccha- rose and raffinose broths but had no action on lactose, mannite, or salicin. Milk was neither acidified nor clotted and neutral red broth (anaerobic) was unaffected. The individual cocci were round and regular and the chain short. B. proteus, a coliform bacillus, and diphtheroids were also found. September 4th. Streptococcus pyogenes. September 6th. Streptococcus pyogenes. October 5th. Cultures showed chiefly Staphylococcus and a few B. pyocyaneus. Case No. 22. Wounded (bullet) 13.8.16. Admitted 11.9.16. Discharged 24.6.17. Ow admission (29th day). General condition was good, temperature only slightly and intermittently raised. There was a wound about 7 inches long and 4 inches broad over the outer side of the middle of the thigh leading down to the bone, which was fractured in two places. Treatment. Extension was continued with a modified Thomas splint, the wound being intermittently irrigated with 5 per cent, saline and drainage secured by means of a water suction pump. Progress. Under this treatment the condition steadily improved, the bone uniting in very fair position, and the wound healing slowly but with the scar somewhat adherent to the bone, and a persistent discharge of pus from a cavity containing sequestra. In February 1917, and again in June, some of these sequestra were removed by open operation, but probably not with complete success, since after the patient had left the hospital with the wound healed it again broke down. In view of the fact that such operations are almost invariably followed by increased suppuration associated with an abundant proliferation of all the bac- teria in the wound, it was decided to follow up the second operation immediately with applications of flavine on gauze. The course of events was, however, in no way changed for the better, for the bacteria proliferated exactly as they had done before and a slight attack of erysipelas develojied in the affected limb a day or two after the operation. Bacteriological Notes. On admission. Films showed only a moderate number of bacteria. Streptococci and a coliform bacilli being noted. Cultures gave Streptococciis salivarius, a Gram-negative coccus, and a coliform bacillus. September 25th. Streptococcus salivarius and Streptococcus equinu-s, also B. proteus. November 8th. Streptococcus pyogenes and Staphylococcus. November 23rd. B. proteus. Streptococcus pyogenes, and diphtheroids. February 9th. B. proteus and Staphylococcus only. Februarj' loth. B. proteus and Staphylococcus only. February 28th. (Two days after operation.) Films showed very large numbers of Streptococci and diphtheroids. March 1st. (After operation.) Films again show many Streptococci, diph- theroids, and a Gram-negative bacillus. Cultures gave Streptococcus pyogenes, Staphylococcus, and B. proteus ; a few B. sporogenes were also isolated by anaerobic cultures. March 9th. Pure cultures of Streptococcus pyogenes. March 27th. Film showed only Streptococci and diphtheroids. Cultures gave B. proteus. Staphylococcus, and Streptococcus. April 27th. Streptococcus pyogenes. May 3rd. Film showed only a few Streptococci. Case No. 23. Wounded (shell) 3.9.16. Admitted 12.9.16. Discharged 28.12.16. ® History. On arrival at the Base patient had developed gas gangrene, and this was treated with multiple incisions. Condition on admission (9th day). There was a wound over the right buttock, midway between the great trochanter and the natal cleft ; this had evidently been enlarged by a deep cross-shaped incision and was kept open by a large drainage tube which passed about 3i inches into the buttock. The wound surface was fairly clean, but there was a considerable amount of blood-stained stinking pus from the depths. In the left buttock, over the gluteus maximus 135 was a wound from which a sinus extended about 4 inches in the direction of the anus. Over the upper part of the left thigh and buttock there were nine other wounds the result of incisions, each about 3 inches long and, for the moat part, only extending through the skin and subcutaneous tissues. One, however, in the neighbourhood of the wound caused by the projectile was deeper, extending well into the muscles. There was also a wound in the natal cleft close to the anus. These wounds were all fairly clean. The patient's general condition was good but there was some intermittent fever. Progress. For the first three or four weeks the patient had irregular attacks of fever, due to temporary failures of drainage, but after that period there were no constitutional symptoms. Various treatments were employed to encourage the growth of epithelium, such as a 1 per cent, solution of brilliant green, red lotion, &o., and in the end the wound healed very satisfactorily. The patient was discharged on December 28th. Bacteriological Notes. On admission. Kims showed Streptococci, diphtheroids, ooliform bacilli, and spore-bearing bacilli. Cultures gave B. sporogenes. Streptococcus pyogenes, B. proteus, a coliform bacillus, and diphtheroids. October dth. Films showed many Streptococci. Cultures, practically pure Streptococcus pyogenes. October 31st. Cultures gave Streptococcus pyogenes, diphtheroids, and Staphylococcus. Case No. 24. Wounded (shell) 27.8.16. Admitted 15.9.16. Discharged 24.1.17. On admission (18th day). General condition was not very good, the patient being rather worn out with pain, sepsis, and haemorrhage. There was a large, deep, ragged wound, midway between the point of the right shoulder and the root of the neck, roughly measuring 5 by 4 inches. The clavicle and scapula were both extensively fractured and several pieces of bone were present in the wound, the surrounding muscles being much lacerated. The wound was granulating and showed little suppuration. Progress. During the first fortnight there was a considerable amount of fever and a, dusky red blush spread from the wound down the back to as low as the iliac crests. 'This blush had not the appearance of erysipelas and soon subsided, after which the wound rapidly filled up and healed. Several pieces of bone were removed from time to time, the clavicle united in fairly good position. Bacteriological Notes. On admission. Films showed many Streptococci, tetrads or Staphylococci, a coliform bacillus, and a few Gram-positive bacilli. Cultures gave Streptococcus pyogenes, Staphylococcus, a coliform bacillus, and an anaerobic bacillus (? Vibrion septique). September 20th. Film showed Streptococci only. September 21st. Film showed diplococci and some diphtheroids. Cultures gave colonies of Staphylococcus, Streptococcus pyogenes, diphtheroids, and a coliform bacillus which produced acid and gas in mannite broth but had no effect on glucose, lactose, saccharose, or dulcite. November 8th. Film showed coliform bacilli. Streptococci, and diphtheroids. Cultures gave a coliform bacillus. Streptococcus pyogenes, and diphtheroids. Cultures from heated pus were sterile. Case No. 25. Wounded (shell) 9.9.16. Admitted 16.9.16. Discharged 28 12 16. On admission (7th day). The patient had a flesh wound on the outer side of the lower half of the left thigh, in the upper part of which the muscle had herniated through the fascia lata. The surface of the wound was fairly clean, but there were a few sloughs still adhering. -,• • • u j Progress Under treatment the wound rapidly cleaned and was diminished in size considerably by strapping. Later as healing became very slow the w-ound was skin-grafted by Steele's method. This at first was very successful, all the crafts adhering, but later the epithelium failed to spread as well as was expected. The patient was discharged on December 28th, the wound then being soundly healed. 136 Bacteriological Notes. On admission. Films showed only diphtheroids and cocci. Cultures gave two varieties of diphtheroids. Staphylococcus, a few Streptococci, and a few Gram- negative bacilli. Anaerobic cultures from heated pus gave B. sporogenes. October 18th. Staphylococci and diphtheroids were isolated from the patient's urine. October 31st. Some days after skin grafting only a few Staphylococci and diphtheroids were found in cultures taken from the wound. Case No. 26. Wounded (bullet) 1.9.16. Admitted 17.9.16. Discharged 28.1.17. History. Gas gangrene developed at Casualty Clearing Station, which was treated by excision of the gangrenous muscle. Condition on admission (16th day). There was a large wound over the front and inner side of the right thigh commencing about the level of the apex of Scarpa's triangle and measuring 7 by 4 inches. Just to the outer side of this wound was an operation wound. Over its whole area the large wound extended deep into muscles which had been freely excised, the wound surface was covered with fine clean granulations. The patient's general condition was very good. Progress. The wound healed steadily and satisfactorily, the edges being approximated by strapping. There was, however, some fever for the first fort- night or three weeks. He was much troubled by attacks of severe pain, com- mencing about the level of the knee and passing down the inner side of the leg ; these became more and more frequent and were found to follow the distribution of the internal saphenous nerve. Eucaine solution injected over the course of this nerve in the wound temporarily stopped the pain. On November 27th the internal saphenous nerve was cut and the attacks of pain ceased and did not recur. On December 20th, healing having become rather slow, the remaining area was covered in with Steele's grafts, the pieces of skin being taken from the anaesthetic area which resulted from the division of the internal saphenous nerve. These grafts took very satisfactorily. Later the thigh was massaged daily as a very extensive and hard mass of scar tissue had formed which apparently so diminished the blood supply as to bring the healing process almost to a standstill. This daily massage was quite painless and appeared to have a very satisfactory result, the remaining small areas being rapidly covered with epithelium. The patient was discharged quite healed and with full movements of the leg on January 28th, 1917. Bacteriological Notes. On admission. Films showed many Streptococci, Staphylococci, and a Gram- positive bacillus. Cultures gave Streptococcus pyogenes. Staphylococcus, and a few coliform bacilli. Cultures made from heated pus were sterile. October 3rd. Films showed very few bacteria, Streptococci, and diphtheroids. Cultures gave Streptococcals pyogenes, diphtheroids, B. pyocyaneus and a few Staphylococci. October 26th. B. pyocyanexis was isolated and was by far the commonest organism present. December 16th. Cultures taken before skin grafting gave a few colonies of Streptococcus pyogenes. Case No. 27. Wounded (shell) 6.8.16. Admitted 18.9.16. Discharged 29.1.17. Condition on admission (43rd day). (1) There was a long narrow wound, almost healed, about 10 inches in length, running obliquely across the back. (2) In the lumbar region was a small depressed wound also almost healed from which a piece of shell weighing 2^ ounces had been removed. (3) Just above the right external malleolus was a small round hole leading down to the tibia the diameter of the orifice being about | inch. (4) On the dorsum of the right foot was a small wound, situated between the metatarsal bones of the great and second toes. (5) Lastly there was a wound over the radial side of the left forearm also nearly healed. X-rays demonstrated a fracture of the left radius, grooving of both the tibia and fibula, and foreign bodies in the neighbourhood of the wounds in the right leg and right foot. Progress was rather slow, some of the scars tending to break do-ivn ; all the fractures united soundly Patient was discharged on January 28th, 1917, 137- with all the wounds healed except that over the lower part of the right tibia where there was still a small sinus. Bacteriological Notes. On admission. Pilma showed Streptococcus only. Cultures gave B. proteusj B. pyocyaneiLs, Streptococcus pyogenes, and a heat-resisting Streptococcus. September 26th. B. proteus. Streptococcus pyogenes, and diiDhtlieroids by culture. October 5th. Cultures were overgrown with B. proteus. Case No. 28. Wounded (shell) 20.8.16. Admitted 18.9.16. Discharged 27.12.16. Condition on admission (28th day). Left stump was healing satisfactorily. A wound of the right thigh 3 inches above the knee measured about 5 by 4 inches and involved considerably more than half the depth of the muscles. The surface was clean and the epithelium was beginning to spread from the edges. Progress. On November.25th the wound of the right thigh was skin-grafted with Steele's grafts and ' section grafts ' ^ with a most satisfactory result, the whole surface being almost healed in three weeks and quite sound in four weeks. Patient was discharged on 27.12.16 all the wounds soundly healed, the stump being fairly good, but the bone was adherent to the soar. The wound of the right thigh was quite soundly healed. Bacteriological Notes. On admission. Kims showed very few bacteria, Streptococci, and Staphylococci. Cultures gave B., proteus and Streptococci. October 5th. Culture made but they were all overgrown with B. proteus. October 6th. A few colonies only of diphtheroids and cocci. Case No. 29. Wounded (shell) 16.9.16. Admitted 29.9.16. Discharged 23.4.17. History. Large masses of the gluteal muscles had been excised at a Base Hospital in France for localized gas infection and later practically the whole of the right sacro-iliac joint was excised. Condition on admission (13th da^jr). The patient's general condition was good considering the size and situation of the wound. This was situated in the right buttock and was roughly oval in shape, the long axis measuring about 15 inches, the short axis about 8 inches. Practically the whole of the gluteal muscles had been removed and at its centre the wound was very deep, extending down between the sacrum and ilium so that the abdominal contents bulged into the wound when the patient coughed. The great trochanter lay towards the outer and lower part of the wound covered only by granulations. There was also in the centre of the wound a large mass of necrosed ilium, with tracks going deep to it from the wound surface. The hip-joint was unaffected. The jiatient was much troubled with cough, large quantities of sputum being brought up, which on examination was found to be crowded with Pneumococci and B. influenzae. There was a bed sore over the left great trochanter. Progress. For the first six weeks there was a considerable amount of fever, probably due in part to the bronchial condition. On October 7th a large piece of necrosed bone was removed from the neighbour- hood of the sacro-iliac joint, which greatly improved the drainage of the deeper portion of the wound. After this the wound progressed wonderfully quickly, granulations springing up from the bottom and filling up the cavity, the epithelium also spreading in from the edges. When, later, the contraction of the wound and the spread of epithelium became slow, some forty-five Steele's grafts taken from the patient's thigh were applied to the granulating surface, no special arrange- ment being adopted. Although several useful centres of epithelial growth were established by this operation, the result was not so good as it would probably have been had the grafts been arranged peripherally and near each other. The advantage of such an arrangement had not, however, been learned at that time. A second skin-grafting operation two months later, when the formation of scar tissue had become very pronounced, and the granulations in consequence very bloodless, was an almost complete failure. However, under massage to loosen the scar tissue and to promote circulation, healing progressed slowly but surely, and the patientwas discharged with the wound healed on April 23rd, 1917. ' Douglas, Fleming, and Colebrook, Lancet, 1917, ii. 5. 138 Seen some months later the patient appeared in perfect health, the wound was perfectly sound, the sacro-iliao joint being quite firmly united. Movements of the hip were quite perfect and the patient could walk, run, or jump without any difficulty. The only point noted was that the leg on the injured side was slightly longer than that on the uninjured side. This was corrected by a thick boot sole on the sound side. Bacteriological Notes. On admission. Films showed long and short chained Streptococci, long and short Gram-negative bacilli, long and short Gram-positive bacilli, tetrads, 8taphi/lococci, and diphtheroids. Cultures gave B. sporogenes and B. tertius, Streptococciis salivarius and Streptococcus equinus, M. tetragentis, diphtheroids, and a coliform bacillus. October 6th. Kims showed Streptococci and diphtheroids, there were but few bacteria. Cultures gave Streptococcus pyogenes. Streptococcus salivarius, and B. proteus. October 7th. Films from the necrosed bone 'showed diplococci, tetrads, diphtheroids, coliform bacilli, and a large Gram-positive bacillus and a long thin bacillus with an end spore, also many free spores. Cultures gave B. welchii, B. sporogenes and B. tertius, Streptococcus pyogenes, and some coliform bacilli. Sputum gave an abundant culture of Pneumococci and B. influenzae. November 7th. Film showed only a few Streptococci and tetrads. November 30th. Film showed very few bacteria, one or two cocci and diph- theroids being seen. Cultures gave a few colonies of Streptococcus pyogenes, diphtheroids, and B. proteus. After this, although many observations were made, the wound was always practically sterile. It may here be mentioned that cultures taken from the bone that had been removed some months after it had been kept in a dry state gave cultures of Streptococci, B. welchii, and B. sporogenes.^ Case No. 30. Wounded (shell) 14.9.16. Admitted 30.9.16. Discharged 27.2.17. History. After being wounded patient lay out for twenty-four hours. He arrived at the Base on September 16th where Ee was operated on for gas gangrene. Condition on admission {16th day). The patient had an extensive wound in the region of the left calf. The left gastrocnemius and soleus had both been almost entirely dissected away, except for the upper one-fourth, together with the skiQ covering them, on account of extensive gas gangrene ; the long bones were quite uninjured and the wound surface was clean. The foot was in excellent condition. Progress. At first healing took place rapidly but later it became very slow, the soar becoming adherent to the bones. Later, the treatment adopted was that which has been used for very callous varicose ulcers, layers of gauze being bandaged on from the toes to the knee, each layer having a coat of Unna's paste painted over it. About four layers of gauze and paste were put on and this dressing left for about one week. Under this treatment the epithelium steadily extended, and when the wound had become almost linear a row of Steele's grafts was applied with good results. The ultimate result was that when the patient was discharged on February 27th the wound was scarred over except for a very minute spot. The foot was in excellent position. Bacteriological Notes. On admission. Films showed many bacteria. Streptococci, Gram-negative bacilli, and diphtheroids. Cultures gave Streptococcus pyogenes and B. pyocyaneus, and B. sporogenes. October ith. Streptococcus, B. pyocyaneus. Staphylococcus, diphtheroids, and a coliform bacillus which produced acid in glucose broth but had no effect on lactose, dulcite, or mannite. Saccharose broth was rendered slightly acid after some days. October 25th. A blood culture, made in consequence of a sudden rise of temperature, was negative. October 31st. Streptococci, diphtheroids, and a coliform bacillus by culture. December 7th. A few B. pyocyaneus and diphtheroids. ' Cultures were again made from the same piece of bone three years after Its removal, with the result that the B. welchii and Streptococcus faec'alis were both isolated- 139 Decembsr 27th. Practically no bacteria seen in films ; the cultures, however, gave B. proteiw. Case No. 31. Wounded 26.9.16. Admitted 5.10.16. Discharged 6.1.17. Condition on admission (9th day). The patient had through and through wounds of both thighs. The entry wound of the right thigh had been enlarged by operation, and measured about 4 by 2J inches. It was situated about the middle of the thigh and had a drainage tube of large size passing horizontally backwards to the exit wound. The left thigh had also two wounds situated about the middle, the entry wound being to the front and inner aspects, the exit wound at the back and outer side ; a small drainage tube was inserted into the wound of entry. The patient was discharged on January 6, 1917, healed and with practically no limitation of movement. Bacteriological Notes. On admission. Right thigh. Films showed a considerable number of bacteria, diphtheroids, and Streptococci. Cultures gave Streptococcus pyogenes, Staphylo- coccus, diphtheroids, and coliform bacillus, which produced acid in glucose broth but did not affect lactose, saccharose, dulcite, or mannite. Left thigh. Films showed Streptococci and diphtheroids. Cultures gave Streptococcus pyogenes, Staphylococcus, and diphtheroids. October 18th. Right thigh. Cultures gave pure Streptococcus pyogenes. December 5th. Left thigh. A small abscess having formed, pure Streptococcus pyogenes was isolated from the pus. Case No. 32. Wounded (shell) 7.8.16. Admitted 7.10.16. Discharged 31.3.17. History. The left foot was amputated at the CCS. a little above the malleoli, antero-iDosterior flaps being used. He arrived at the Base on August 10th with severe sepsis of the right foot and ankle-joint ; the ankle-joint had to be exten- sively opened up and much bone removed. While at the Base there was exten- sive septic thrombosis of the external saphenous vein requiring incisions on the outer side of leg. Condition on admission (61st day). The stump of the left leg was almost healed. The right ankle-joint was extensively damaged, the os calcis and astra- galus being much comminuted ; there was a large wound on the inner side of the joint from which a tube was passed to the outer side emerging below the external malleolus and rather posterior to the joint. On probing much dead bone could be detected. There was also an incised wound about 2|- inches long on the outer and posterior aspect of the riiiddle of the leg. Progress. At first there was very gradual improvement in the patient's general condition. On November 23rd the wounds were opened up and much necrosed bone, chiefly pieces of the astragalus and os calcis, were removed, together with much unhealthy granulation tissue ; the wound was then packed with gauze to stop the haemorrhage which was free. This packing was removed the next day under gas and the wounds were dressed with 5 per cent, saline packs and 3 per cent, saline baths were employed daily. Under this treatment im- provement was very rapid, the suppuration becoming less and less. Unfortunately a troublesome splint sore developed over the heel. The ultimate result was, however, excellent, the bones uniting solidly with the foot in good position and all the external wounds healing. The patient was discharged on March 31st, 1917. Bacteriological Notes. On admission. Cultures from right ankle gave Streptococcus pyogenes, Strepto- coccus anginosus. Staphylococci, and B. proteus. B. sporogenes and B. cochlearis were isolated by anaerobic methods. November 7th. Films from right foot showed Gram-negative bacteria. Gram- positive bacteria, and a few Streptococci. Cultures gave B. proteus, Streptococci, and a coliform bacillus which fermented glucose with the production of acid only, saccharose was also slightly acted on, a trace of acid being produced after several days ; dulcite, lactose, and mannite were all unaffected. B. sporoge7ies was also found in a culture made from heated pus. November 8th. B. proteus, Streptococcus faecalis, and Streptococcus equinus. December 3rd. Cultures overgrown with B. proteus. December 23rd. Almost pure Streptococcus anginosus. 140 January 10th. B. proteus, Streptococciis pyogenes, and diphtheroids. February 11th. Streptococcus pyogenes. Case No. 33. Wounded 28.9.16. Admitted 9.10.16. Discharged 22.1.17. History. Localized gas infection which developed in the region of the shoulder necessitated excision of much of the left deltoid. Coiidition on admission (11th day). The right elbow had a wound over the external condyle of the humerus about the size of a 5-shilling piece; at the bottom of this the head of the radius could be seen, the joint surface being quite exposed. There was a large wound of the right shoulder and upper part of the arm, with much loss of the deltoid muscle, the humerus could be seen at the deepest part of the wound. Most of the sldn, &c., had been destroyed, but there was a small flap left in the upper part. The wounds were all very clean. Progress. Healing took place rapidly, the right elbow joint closing up with little suppuration and no pain.' The extensive wound of the right shoulder was slower in healing, several pieces of bone having to be removed from the humerus. When the wound had partially filled in and the granulations were in a satis- factory condition Steele's skin grafts were applied, but considerable difficulty was encountered in satisfactorily dressing the grafted surface and many of the grafts were destroyed in consequence. The patient was discharged on January 22nd, 1917, all the wounds being soundly healed. The right elbow jouat was ankylosed. Bacteriological Notes. From the shoulder wound films showed Streptococci of two types, oval and round in shape, a coliform bacillus, and large and small Gram-positive bacilli, also diphtheroids and Staphylococci. Cultures gave B. proteus, Streptococcus pyogenes, and a Streptococcus which fermented lactose, saccharose, mannite, and salicin, but did not affect raflSnose ; milk was rendered acid and clotted ; neutral red broth cultures incubated anaerobically were rendered florescent, gelatine and coagulated egg were liquefied. Diphtheroids and B. sporogenes. From the elbow films showed only very few bacteria. Streptococci, coliform bacilli, and diphtheroids being seen. Cultures gave B. proteus. Streptococcus pyogenes, and diphtheroids. The heated pus from the elbow gave no growth. October 11th. Pus from the elbow gave a few colonies of B. proteus and Streptococcus. October 13th. A similar result was obtained. October 17th. Pus from the shoulder. Films showed very few microbes, Staphylococci, diphtheroids, and Streptococci. October 18th. Pus from the elbow gave cultures of Streptococcus pyogenes, Staphylococcus, and B. proteus. October 27th. Cultures from the shoulder wound gave B. pyocyaneus and an unknown anaerobe. November 8th. Cultures from the elbow gave Streptococcus and a few coliform bacilli (no B. proteus). From the shoulder wound B. proteus only. Case No. 34. Wounded (shell) 4.10.16. Admitted 12.10.16. Discharged 27.2.17. History. Muscle excised at Casualty Clearing Station and again at the Base in France for gas gangrene. Condition on admission (8th day). There was a large gaping wound on the outer side of the right leg which extended from the knee to the ankle. There was a very extensive loss of skin, fascia, and the muscles of this region, the fibula was fractured, and the outer part of the tibia was exposed in the lower part of the wound for about two inches. In the upper part of the wound was a tube which passed through the wound between the bones, emerging at the back of the calf. The wound had still many sloughs adherent to the surface. Progress. Under treatment the wound rapidly cleaned and its area was considerably diminished by drawing the edges together with strapping. Later the healing process became very slow, the soar being firmly adherent to the bone in places. At the end of November the wound, then reduced to a long narrow strip, was skin-grafted with but very little success, healing, however, continued to progress and the patient was discharged on February 27th, 1917^ with the wound healed and the fibula firmly united. 141 Bacteriological Notes. On admission. Films showed Streptococci, tetrads. Staphylococci, and a coli- form bacilli. Cultures gave a typical form of B. irelchii and a bacillus resembling B. cochlearis, also Staphylococcus and a coliform bacillus which produced acid and gas in broths containing glucose, lactose, dulcite, saccharose, and mannite. October 19th. Films showed many bacteria, diphtheroids, and Staphylococci. October 26th. A pyo-sero culture gave pure Streptococcus pyogenes. November 8th. Film showed many organisms, Streptococci, Staphylococci, coliform bacilli, and diphtheroids. Cultures gave pure Streptococcus pyogenes. Cultures made from heated pus remained sterile. Case No. 35. Woimded (grenade) 13.10.16. Admitted 20.10.16. Dis- charged 7.3.17. History. Operated on at a Casualty Clearing Station and again at the Base for gas gangrene. Condition on admission (7th day). There was a very extensive wound of the left thigh about 12 inches in length commencing from the fold of the buttock down the thigh ; it extended deeply into the intermuscular planes, the muscles being widely separated ; there was, however, no great loss of skin. There were two other wounds on the left buttock, one of them having an area of about 5 square inches. The femur was uninjured. These wounds were cleaning, but some sloughs still remained attached. There was also a large sloughy wound on the left forearm roughly oval in shape and about six inches long extending deeply into the intermuscular tissues ; the bones were not fractured. The fingers of the left hand were much contracted and flexed, being in the position of main en griffe ; the interossei and other small muscles were much wasted ; pain was complained of in the palm of the hand, thumb, index, middle, and radial side of the ring fingers. This condition was evidently due to bruising of the median nerve. In addition, there were signs of low grade arthritis in the joints of the fingers, the wrist, elbow, and shoulder joint of the right arm. The patient stated that before he had been wounded these joints were quite normal. Progress was uneventful, the arm wound healed without any troubles develop- ing, the healing being very much hastened by a number of Steele's grafts, all of which took and rapidly formed epithelium round them. The thigh wounds pro- gressed very rapidly in the early stages and everything went well until the begin- ning of January 1917, when the surface became unhealthy and the newly-formed scar tended to break down. This condition rapidly improved with the use of wet 5 per cent, saline packs, and the patient was discharged on March 7th with all his wounds soundly healed. There was, however, still some pain in the palm of the hand and the fingers supplied by the median nerve, and in spite of careful splinting the fingers were still somewhat contracted ; the chronic arthritic condition of the left shoulder, elbow and wrist, and smaller joints of the fingers still persisted, although the lesions were no longer progressive. Bacteriological Notes. On admission. Cultures from the thigh wound gave B. proteus, Staphylo- coccus aureus, coliform bacilli. Streptococcus faecalis, and diphtheroids. Cultures from the heated pus were sterile. Films made from pus from the wound of the forearm showed many bacteria. Staphylococci, Streptococci, and diphtheroids. Cultures from arm gave Staphylococcus, diphtheroids, B. proteus, but no colonies of Streptococci could be found. Anaerobic cultures gave B. sporogenes. October 25th. Cultures from the thigh gave Staphylococcus and Streptococcus. November 8th. Films from the thigh showed a very few Streptococci. Cul- tures were overgrown with B. proteus. November 20th. The wound of the forearm before being grafted gave cultures of Staphylococcus and a diphtheroid. December 5th. Wound of forearm — Culture pure Staphylococcus. ■ January 10th. Wounds of buttock and thigh, which were at that time not doing at all well, gave abundant cultures of B. pyocyaneus and Streptococcus. Case No. 36. Wounded (shell) 8.8.16. Admitted 15.11.16. Transferred on closing of wards, 23.6.17. Condition on admission (100th day). Several large healed scars were present, on the outer side of the thigh below the trochanter and over Scarpa's triangle. There was a sinus leading down to the neighbourhood of the fracture from about 142 the middle of Scarpa's triangle and another one also leading to the same part and opening behind the great trochanter. X-rays showed the fracture with the lower fragment drawn up behind the upper one, which was considerably flexed. The shortening of the limb amounted to 3J inches. There were also very many small pieces of metal scattered in the tissues. The fracture was firmly united. Progress. The condition remained practically stationary for some weeks, a good deal of difficulty being encountered in maintaining efficient drainage. Two operations were performed to promote drainage and for the removal of sequestra, with the result that the wounds were almost healed when he was discharged from hospital. Bacteriological Notes. On admission. Kims showed Streptococci only. Cultures gave pure Strepto coccus pyogenes. December 20th. Streptococctis pyogenes and a few B. protens. Films and cultures were made from time to time ; the cultures always gave Streptococcus pyogenes and on one occasion Streptococcus anginosus in addition. B. protev^ and diphtheroids were also occasionally present. Case No. 37. Wounded (shrapnel) 18.10.16. Admitted 9.11.16. Discharged 1.1.17. History. Pieces of shrapnel were removed from left elbow and left knee at the Base, where he had a slight secondary haemorrhage from a small vessel in the right axillary fold. Condition on admission {21st day). The patient had a clean wound about 4 inches long extending from the right anterior axillary fold down the arm. There were two small granulating flesh wounds on the front of the left arm just above the elbow joint. There was also a clean wound on the front of the right side of the chest a little below the clavicle, and two wounds on the inner side of the left knee which did not open into the joint. Progress. The wounds on the chest, left arm, and left leg healed rapidly; the large wound on the right arm was skin grafted, which considerably shortened the patient's convalescence. The patient was discharged on January 1st, 1917, with all the wounds soundly healed. Bacteriological Notes. On admission. Pus from the large wound of the right shoulder and arm ; films showed Streptococci, Staphylococci, coliform bacilli, and diphtheroids. Cultures gave growths of B. proteus. Streptococcus pyogenes, Staphylococcus, and diphtheroids. From the small wound on the knee only Staphylococcus. November 11th. Cultures from the wound on chest gave Streptococcus and B. pyocyaneus ; from the wounds near left elbow and left knee Streptococcus pyogenes. Case No. 38. Wounded (bomb) 13.11.16. Admitted 21.11.16. Discharged 3.2.17. History. He was kept at a Casualty Clearing Hospital for four days and was there operated on for commencing gas gangrene. Condition on admission (8th day). General condition was good. There was a large, deep, irregular wound in the region of Scarpa's triangle which roughly measured 8 by 6 inches; much skin, fascia, and muscle had been lost. The surface of the wound was very dirty and many sloughs were still adhering. Progress. This was at first extremely rapid, the wound cleanmg in a very few days and at first contracting very rapidly. Later the healing process became very much slower, the scar tissue forming a hard dense mass. In the early part of January the surface was skin grafted with but little success owing probably to the dense scar tissue cutting off practically all the lymph supply. Healmg, however, continued and was seemingly very distmctly assisted by vigorous massage of the mass of scar tissue, a procedure which was quite painless. The patient was discharged on February 3rd with good and powerful movements of his leg and no lameness. Bacteriological Notes. On admission. Films showed very many bacteria. Streptococci and diplococci, tetrads, small Gram-positive bacteria, some very large Gram-positive diplococci^ large Gram-positive bacteria and large Gram-negative bacteria, coliform bacilli' diphtheroids, and Staphylococci. Cultures, anaerobes, B. welchii, B. tertius, and B. sporogenes. Streptococcus pyogenes and diphtheroids were also grown. 143 December dth. Film only showed many diphtheroids and a few Streptococci. Cultures gave diphtheroids. December 27th. Very few bacteria seen in films, and all were diphtheroids. Films made from the wound on various occasions after this date never showed many bacteria, usually only one or two diphtheroids could be seen lying inside phagocytjs. Case No. 39. Wounded (shell) 13.11.16. Admitted 21.11.10. Died 26.11.16. History. The patient was operated on at a Casualty Clearing Hospital for gas gangrene. Condition on admission (8th day). The general condition was not very bad. He had a single large wound on the posterior aspect of the thigh roughly measuring 8 by 6 inches ; this was in a very foul condition, being covered with sloughs ; the skin edges were thickened and everted, the muscles were much torn and digested away, there was a considerable loss of skin and subcutaneous tissue, but parts of the wound did not involve the muscles. Progress. Under treatment the wound began to clean rapidly and the sloughs to separate. His general condition gave no anxiety until November 25th, when the pulse rate began to rise. The wound showed no signs of developing gas gangrene, erysipelas, or any cellulitis. The patient died quite suddenly and unexpectedly on Xovember 26th ; there were no preceding signs of embolus or haemorrhage to account for the sudden death. Post-mortem gave no further indications as to the cause of death except that the heart showed slight fatty degeneration of the muscle, which w-as very flabby. Bacteriological Notes. On admission. Films showed very many bacteria. Streptococci and diplococci, diphtheroids, coliform bacilli, tetrads. Gram-negative diplococci, long Gram- negative bacilli, some of which had oval spores, large and small Gram-positive bacilli. Cultures, anaerobic, gave B. welchii, B. sporogene-s, B. tertius, B. sphenoides, and a bacillus resembling B. tetani. Streptococci and coliform bacilli were also isolated. Post-mortem. The heart's blood was found to be sterile. Case No. 40. Wounded (shell) 12.11.16. Admitted 4.12.16. Discharged 11.3.17. History. The patient remained in a Casualty Clearing Station for seventeen days and during that time was operated on six times for gas gangrene. Corulition on admission (22nd day). The patient's general condition was good, but any noise or sudden start always brought on pain in his injured leg. There was a superficial partially healed wound of the back just below the left scapula. The principal wound was situated at the back of the right thigh and extended from the back of the knee upwards for about 10 inches. There was a very extensive loss of skin, fascia, and muscles over the whole area. There was marked foot-drop and frequent attacks of pain in the outer side of the leg and dor- sum of the foot evidently due to the injury to the outer part of the sciatic nerve Progress. Under treatment the thigh wound very rapidly commenced to heal, and it was then seen that so much skin, &c., had been lost that unless some plastic operation was performed a very large area of scar tissue would have to form and healing would take a very long time. On December 20th, two flajjs were dissected up, one on each side of the wound, and brought over to meet each other across the wound. This operation was successful, the flaps remaining in position and no sloughing taking place, the wounds being dressed from the earliest stages with hypertonic salt solution. On January 1st the raw areas from which the flaps had been removed were skin-grafted by Steele's method with a very successful result, a large portion of the wounds being quickly covered by epithelium. Another grafting was performed on February 17th, with equally good results on one side of the thigh, but with less success on the other side, owing to movement of the grafts. However, when the patient was discharged on March 11th, all his wounds were healed, and there was no contraction of the knee, which had full and strong movements. The periodic attacks of pain and the foot-drop were both very much improved. Bacteriological Notes. On admission. Films showed diphtheroids. Staphylococci, some Gram- negative cocci and Gram-negative bacilli, but no Streptococci. Cultures gave 144 B. pyocyaneus, diphtheroids, and Gram-negative bacilli ; Streptococci were also grown. From the wound of the back cultures gave diphtheroids and Staphylococci. December 12th. Pyo-sero-cultures gave Streptococcus pyogenes. December 21st. The day after the operation, cultures gave numerous colonies of Streptococcus and some diphtheroids. December 22nd. Cultures gave many colonies of Streptococcus pyogenes and diphtheroids. December 27th. Films from an unhealthy-looking spot showed many bacteria, Streptococci, diphtheroids, and Staphylococci. Cultures gave Streptococci, diphtheroids, and Staphylococci. December 28th. Heated pus cultures were sterile. December 29th. Film showed practically no bacteria. February 15th. Culture from the portion of the wound of thigh dressed with brilliant green was sterile, while one made from the part dressed with ' Comfrey ' gave six colonies of Staphylococcus. Case No. 41. Wounded (shell) 13.11.16. Admitted 15.12.16. Discharged 31.3.17. History. At the Base, twelve days after the injury, the right leg was ampu- tated for sepsis through the knee joint. Condition on admission (32nd day). The patient's general condition was very fair. The skin and soft parts had contracted away from the condylar end of the femur, leaving a large granulating surface and a sinus opening on the posterior edge. Progress. Under treatment with wet 5 per cent, saline packs and later normal saline the surface cleaned rapidly. Extension of the soft parts was applied and the granulating surface successfully skin-grafted on three occasions. The stump was soundly healed when discharged, but reamputation may have been necessary later. Bacteriological Notes. On admission. Films from the granulating surface of the stump showed Gram-positive cocci and diphtheroids. Cultures gave growths of Streptococci, Staphylococci, and a ooliform bacillus which fermented with the production of acid and gas glucose, lactose, and mannite, but had no action on dulcite or saccharose. Films from the sinus showed many bacteria, large Gram-positive cocci, diplococci, and diphtheroids. Cultures gave colonies of Streptococcv.s, Staphylococcus, and diphtheroids. January 10th. Cultures showed an almost pure growth of Streptococcus. Case No. 42. Wounded (shell) 16.12.16. Admitted 29.12.16. Discharged 31.3.17. History. Patient had flesh wounds of the left thigh, thoracic wall, and left shoulder. Condition on admission (13th day). The patient's general condition was not satisfactory. There were at times slight gastro-intestinal disturbances with abdominal pain, also pain in the feet, and for several weeks there was some irregular fever. Four small granulating wounds were present in the left upper arm and thorax and a large lacerated wound in the lower tliird and outer aspect of the left thigh. This wound was also beginning to granulate, but showed a considerable amount of discharge and its surface did not look quite healthy. Progress. The healing of the wounds was apparently much delayed by the persistent fever for which no adequate cause could be found. A blood culture was made about January 22nd with a negative result. The stools were examined bacteriologically on January 25th, and B. dysenteriae, Flexner, was isolated. There were no symptoms or history of true dysentery, but the patient complained of abdominal pain. Three later bacteriological examinations of the stools were all negative. The patient began to improve rather suddenly as regards general symptoms about February 9th, and after that continued to convalesce steadily. He was discharged on March 31,1917, with all his wounds healed and his general condition quite satisfactory. Bacteriological Notes. On admission. (Notes lost.) January 10th. Cultures from the wound of the thigh gave B. proteus, Strepto- cocci, and B. pyocyaneus. 145 January 22nd. Blood culture negative. January 25th. B. dysenteriae (Flexner) isolated from faeces. This bacillus was identified by (1) its fermentation tests — ^it produced acid in glucose and mannite broths, but had no action on lactose, dulcite, saccharose, or maltose^ and (2) by it being agglutinated to full titre by a standard serum. On February 8th, 9th, and 12th, the stools were again examined, but no B. dysenteriae could be found. February 17th. A film made from the wound only showed a very few cocci. Case No. 43. Wounded (shell) 1.11.16. Admitted 4.1.17. Discharged 23.6.17. ■ ® History. The patient's injuries were: (1) Compound fracture of the skull in the right frontal region. (2) Compound fracture of the right tibia with division of the anterior tibial nerve. (3) Flesh wounds of both legs, the chest wall, and right hand. At the CCS. portions of the fractured skull bones were removed and pieces of shrapnel from his other wounds. At the Base pyaemic foci developed in the left elbow and wrist joints as well as several pressure and bed sores which also suppurated. On admission (65th day). His general condition was bad, with great emacia- tion and anaemia. The temperature was constantly raised. All the wounds were suppurating freely, including the pyaemic foci which had been incised, and cerebral pulsations were very marked in the frontal wound. The left elbow especially gave occasion for anxiety. Treatment. His general condition was treated by small doses of Streptococcus pyogenes vaccine which appeared to quickly control his temperature and no further pyaemic abscesses developed. Later small doses of staphylococcic vaccine were given in addition. The left elbow was slung in a counterpoised splint made of perforated celluloid which enabled intermittent irrigation to be easily carried out. Progress. Under this treatment the patient quickly improved ; it was, however, necessary on January 19th again to incise freely the left elbow joint to establish efficient drainage, but the inflammation of the left and right wrist joint subsided without further surgical interference. After this date improve- ment was continuous both as regards the general condition and the healing of the various wounds. Several pieces of bone separated from the skull and were removed from time to time. On March 28th, a skiagram having indicated the presence of a sequestrum in the right tibia, the wound was opened up and the dead bone was removed. This wound then slowly healed, a considerable cavity in the bone having been made. The patient was discharged on June 23rd, 1917,, his condition then being that the head wound had soundly healed but required a protecting plate as the cerebral pulsations were still very distinct. The wound of the right leg had almost completely healed and there was no indication of any necrosed bone. The left elbow had healed completely and soundly, but the joint had very little movement ;' the left hand had healed, but the movements of the left wrist were greatly impaired. The wounds about the right wrist had also healed, but again the movements of this joint were limited ; both wrist joints were improving, however, under massage. Bacteriological Notes. On admission. Pus from the wound of the head. Films and cultures showed Staphylococcias and Streptococcus pyogenes. Pus from the left hand gave a pure culture of Streptococcus. January 16th. Films from a membrane which had formed over the left elbow showed practically nothing but diphtheroids of the short type. In culture two types of diphtheroids grew, some of the colonies showing a short bacillus, others a longer bacillus. Neisser's stain was practically negative. Five colonies were subcultured and their fermentation reactions tested, all gave acid in glucose and saccharose broths, none of the other sugars, &c., which were tested showed any change. Cultures from the pus of the elbow joint taken on the same day gave a pure culture of Streptococciw pyogenes, and films made from_it shov/ed nothing but Streptococcus. January 19th. Pus from the right leg. Films showed a few cocci only, Cultures gave Streptococcus pyogenes and Staphylococcus. Heated pus was sterile, January 21st. Pus from elbow. Films showed but few bacteria, chiefly diphtheroids, but also a few Streptococcus. 1727 K 146 January 31st. Cultures from pus from the head. Aerobic plate gave Staphj/lococciis only. Anaerobic broth cultures gave Streptococcus pyogenes and Staphylococcus. February 15th. Film made from pus from the elbow showed no bacteria. March 29th. Cultures made from the sequestrum removed by operation from the right tibia on March 28th, when grown in broth anaerobically, gave Gram- positive bacilli resembling morphologically B. welchii. Gram-negative bacilli, and a Streptococcus. Pus taken the day after the operation showed in films Streptococci and a bacillus resemblmg B. welchii. Cultures, aerobic and anaerobic, gave Staphylococcus, Streptococcus pyogenes, and a coliform bacillus. March 30th. Cultures made from the pus (second day after the operation) gave only Staphylococci and Streptococcus pyogenes. April 2nd. Film made from the pus (fourth day after the operation) showed Staphylococci and Streptococci. Cultures gave Streptococcus pyogenes and Staphylococcxis. April 3rd. Film from the pus (fifth day after operation) again showed Streptococci and Staphylococci. Cultures gave Streptococcus pyogenes and Staphylococcus. April 12th. Pus from the same wound "(fifteen days after operation). Films showed diphtheroids, Staphylococci, and Streptococci. Cultures gave Staphylo- coccus and Streptococcus pyogenes. Case No. 44. Wounded (shell) 10.1.17. Admitted 22.1.17. Discharged 24.3.17. History. Gas gangrene had developed round a wound in the thigh and had been treated by extensive excision of the infected tissues. Condition on admission (12th day). There was a large clean wound on the inner side of the right thigh. There was also a counter opening for drainage on the posterior aspect. The surface was quite clean. Two days after his admission an attempt was made to close the wound by secondary suture ; so much soar tissue had, however, formed in the depth of the wound that great tension on the sutures was necessary to bring the skin edges together ; the result therefore was not very successful. Progress. In spite of the failure of the secondary suture to close the wound, healing at first was very rapid ; later it became very much slower and skin- grafting was employed, again without much success, owing most probably to the presence of a dense mass of scar tissue beneath the granulating surface. The final result was, however, quite satisfactory, and the patient was discharged on March 24, 1917, the wound being quite healed and the movements of the thigh excellent. Bacteriological 'Notes. On admission. Films showed very few bacteria, a few Streptococci and coliform bacilli being seen. Cultures gave a few colonies of Streptococcus and a coliform bacillus. January 29th. Cultures made after the operation were overgrown with B. protects. February 9th. Culture made from a boil which developed near the edge of the wound grew Staphylococcus only. March 8th. Culture made 10 days after the grafts were applied gave B. proteus. Films showed no bacteria. Case No. 45. Wounded (bullet) 10.1.17. Admitted 23.1.17. Discharged 17.3.17. History. At a Casualty Clearing Station the wound had been thoroughly opened up. Condition on admission (13th day). The patient's general condition was good ; the wound was situated rather to the inner side of the right thigh and penetrated through to the posterior surface, neither the bone nor any important vessel or nerve had been injured ; the anterior wound was the largest, being roughly 6 inches long and 3 inches broad ; from it two tracks passed between the muscles towards the bone. On January 29, six days after his admission the anterior wound was brought together by secondary suture, the wound having been previously treated with 5 per cent, saline packs to complete the cleaning of the surface. This was very successful, as but little tension was necessary to bring the edges together, and by treatmg the sutured wound from the beginning 147 • with 5 per cent, saline packs which were kept constantly wet by means of tubes arranged in the dressing the resulting sepsis was very slight and healing was very rapid. Later progress was quite uneventful, and the patient was dischai-ged on March 17 completely healed and with full movements of his legs. Bacteriological Notes. On admission. Films showed very few bacteria which consisted chiefly of Gram-negative bacilli. Cultures gave B. welchii, Staphylococci, B. pyocyaneus, and diphtheroids. February 2nd. Four days after the secondary suture of the anterior wound pus taken from the sutured portion showed in films chiefly diplococci (most probably Streptococci) ; the cultures were overgrown with B. proteus. Case No. 46. Wounded (bomb) 12.11.16. Admitted 14.1.17. Died 14.2.17. History. The left foot was amputated for gas gangrene, and as the gangrene recurred in the stump, a further amputation was performed below the knee. Pyaemia then set in, the right knee filling .with pus and several other abscesses forming, one over the right tibia being subperiosteal and another inter-muscular in the right thigh. Periostitis of the left great trochanter also developed and an incision down to the bone was made in this region. A large bed-sore also formed over the sacrum. Staphylococcus and B. pyocyaneiis were found in all the wounds. , On admission (62nd day). The patient's condition appeared quite hopeless. The temperature was high, the pulse rapid and irregular, and there was very marked wasting and anaemia. He was very drowsy. The wounds of the right foot had healed, and the amputation stump was healing slowly, but both leg and thigh on the right side and the trochanteric region on the left were very widely excavated by the pus tracking from the original pyaemie abscesses, drainage was also very imperfect. An almost solid oedema was present in both legs up to the groin. Over the sacrum was a large deep bed sore. Treatment. Treatment consisted of the removal of sloughs and the main- tenance of efficient drainage from the various pyaemie abscesses. Progress was steadily down hill ; further pyaemie abssesses developed in the right thigh and leg, and an extensive septic thrombosis either of the vena cava inferior or the common iliac vein of the right side occurred. Pleurisy with efiusion also developed on the left side shortly before the patient's death on February 14th, 1917. There was no post-mortem examin3,tion. Bacteriological Notes. Staphylococci and B. pyocyanevs had been isolated from the wounds at Boulogne. On admission. Films from the wound below the right knee showed many Staphylococci and a few smaller diplococci which might have been Streptococci. Films from a slough taken from the wound over the left great trochanter showed many bacteria, principally Staphylococcus, Streptococcus, diphtheroids, and a Gram- negative bacillus. Cultures of the pus from the right knee grown aerobically and anaerobically gave colonies of Staphylococcus only. Cultures from the left trochanter region gave B. proteus and B. pyocaneus in addition to Staphylococcus and Streptococcus. Heated pus from the right calf was sterile. January 1 7th. A blood culture remained sterile. February 8th. Pus from a freshly opened pocket in the region of left great trochanter gave Staphylococcus, Streptococcus, and diphtheroids. February 10th. A pyo-sero-oulture from the left great trochanter gave Staphylococcus and diphtheroids only. Remarks. The patient appears to have been practically the only case in which Staphylococcus played the most important role in the bacterial infection. In the secondary abscesses, which were embolic in nature. Staphylococcus was always present, and although Streptococci could usually be demonstrated they were very much fewer in number and their presence was not constant. Case No. 47. Wounded 13.1.17. Admitted 28.1.17. Discharged 26.3.17. History. At the Base on the 3rd day he was found to be suffering from local gas infection, which was treated by incision and 5 per cent, salt packs. Condition on admission (15th day). The patient's general condition appeared quite satisfactory, although he had a considerable amount of fever. His wound was situated on the front and inner aspect of the right upper arm, the skin being K 2 » 148 destroyed over a very considerable area together with extensive laceration of the muscles beneath. The main vessels and nerves were not involved, and the bone was uninjured. The wound surface was cleaning, some sloughs still adhering to the surface. Progress. On the fourth day after the patient's admission he had a rigor and the temijerature rose to over 104° P. ; at the same time a rather dusky red blush was noticed round the wound, which rapidly spread from the arm to the body and then downwards over the back and side until a very considerable area of skin was involved. There were no bullae. This was at first thought to be an attack of erysipelas and small doses of vaccine made from Streptococcus pyogenes were administered, but the temperature remained high, 104° P. to 105° F. every evening, dropping to almost normal in the morning. The general condition remained very good, the pulse was never rapid, 96 per minute being the highest recorded and more often it was from 80 to 88. Respiration varied from 18 to 2-t per minute. The rash soon began to gradually fade, the tempera- ture becoming more intermittent from 104° F. in the evening to 96° F. in the morning. With the fading of the rash desquamation commenced from the whole area of the trunk occurring far beyond the limits of the rash. The temperature fell to normal on the eleventh day after the rigor which had ushered in this condition. During the attack there was a marked ' loss of voice ', the patient only being able to speak in whispers. A blood culture taken the day after the rigor remained sterile. In many ways this attack was clinically unlike erysipelas and far more resembled the disease which has been described as dermatitis exfoliativa. The subsequent history was quite uneventful ; the granulating wound was skin-grafted on two occasions with most perfect results, a large surface of about nine square inches being completely healed in just over three weeks. The patient was discharged on March 26th, 1917. Bacteriological Notes. On admission. Cultures of the pus on glucose agar plates gave no growth ; these plates were incubated aerobically. The appearance of the pus on the dressing indicated the presence of B. pyocyaneus. January 30th. Glucose agar plates incubated first anaerobioally then aerobically gave only one or two colonies of Staphylococci. A glucose broth tube in which was a small piece of potato after 3 days' incubation gave a culture of Staphylococctis. January 31st. (The day the rash first appeared.) Culture on glucose agar plate first incubated anaerobically then aerobically gave no growth whatever. February 1st. Film showed a considerable number of Gram-positive diplo- cocci. Culture on glucose agar plate gave two colonies of Staphylococcus only. Blood culture negative. February 2nd. Agar cultures aerobic and anaerobic gave numerous colonies apparently all Staphylococcus. February 4th. A film of the pus shows only a few Gram-positive cocci, mostly diplococci. Culture on agar incubated aerobically gave a fair number of colonies of Streptococcus pyogenes and a few Staphylococcus. February 23rd. Two films taken before skin grafting, one showed no bacteria, the other a very occasional Staphylococcus. March 7th. Film taken before second skin-grafting operation showed no bacteria. Culture on agar gave a single colony of B. pyocyaneus. The repeated failure to obtain cultures caim.ot be explained. The media cannot have been at fault, as numerous other cultures were being made daily on samples from the same stock. As the wounds were being dressed with saline solutions it cannot have been due to antiseptics. Case No. 48. Wounded (bullet) 5.1.17. Admitted 28.1.17. Died 9.2.17. History. The wound was ' excised ' at the Casualty Clearing Station. At the Base the wounds progressed slowly, but the patient suffered from bronchitis and his general condition was stated not to be good. Condition on admission (23rd day). The general condition was fair, but the patient seemed rather exhausted by the journey, and the temperature was considerably raised. His wounds were (1) a granulatmg area over the right calf about 4 by 4 inches extending into the muscles, parts of which were lost ; (2) two dirty wounds one on each side of the lower part of the thigh just above the knee 149 , These wounds were about 3 inches long and opened into the popliteal space (a former note stated that the popliteal vessels were exposed in the wound). There was a considerable amount of unhealthy pus coming from both wounds. Progress. The day after the patient's admission, severe secondary haemor- rhage occurred, the inner wound was, therefore, opened up and the bleeding point sought for, but the tissues were so infiltrated with blood and inflammatory exudation that it could not be found, the leg was therefore amputated by an oblique incision, no attempts being made to close the wound. The patient rallied well after this, the stump was cleaning well and the temperature was daily taking a lower level, when on the eleventh day after the amputation a very severe secondary haemorrhage took place in the middle of the night, and the patient died within half an hour of its discovery. Bacteriological Notes. February 1st. Films from the stump showed enormous numbers of diph- theroids, a few chains of Streptococci, and some diploeocci. February 2nd. Film from the stump : Gram-negative bacilli the most numerous, but large numbers of diphtheroids, chains of Streptococci, and diplo- eocci also present as well as Gram-positive bacilli. Cultures gave numerous very small colonies of a strepto bacillus, also two varieties of Streptococci, one of which was faecalis, the other pyogenes, and a large bacillus. Anaerobic cultures gave B. welcTiii and Streptococcus faecalis. February 3rd. Film from stump shows fewer bacteria; diphtheroids and Gram-negative bacilli, are still the most numerous, the anaerobic types are much rarer, the cocci, both Streptococci and Staphylococci, are relatively increased. February 4th. Film from stump which is cleaning, but had still a good deal of pus lying in the intermuscular planes, shows fewer bacteria, chiefly diphtheroids, Gram-negative bacilli and Streptococci. A film taken from near the skin edge shows comparatively few bacteria, diphtheroids and cocci only being seen. February 5th. Films from stump. Bacteria still numerous, diphtheroids, Streptococci, Gram-negative bacilli, a few anaerobe-like t^'pes. February 6th. Film from stump still shows numerous diphtheroids, Strepto- cocci, and Gram-negative bacilli. February 7th. Film shows far fewer bacteria, diphtheroids, and Streptococci February 8th. Film shows still fewer bacteria, diphtheroids. Streptococci, Staphylococci, and Gram-negative bacilli were still noted. Cultuses were over- grown with B. proteus. February 9th. Film shows much fewer bacteria, chiefly Streptococci. Case No. 49. Wounded (shrapnel) 22.12.16. Admitted 28.1.17. Transferred 31.3.17. History. On December 24 some foreign bodies were removed ; on the 26th multiple incisions were made in the lower part of the upper arm and the upper part of the right forearm. On December 31 the elbow joint (right) was opened up and a resection was performed. Condition on admission. On admission (38th day) the patient's general condition was good ; the main wound was on the outer side of the elbow joint with which it communicated by several tracks lined with soft granulations. There were several almost healed incisions about the upper arm and forearm. No swelling or signs of cellulitis. Progress. Quite uneventful, but rather slow. The patient was discharged on March 31st, 1917. Bacteriological Notes. On admission. Films from the larger wound showed a few bacteria only. Streptococci and a Gram-negative bacilli. Cultures gave colonies of a C4ram- negative coliform bacillus, a diphtheroid, Staphylococcus, and B. pyocyaneus. "January 31st. Culture gave Staphylococcus, diphtheroids, B. pyocyaneus, and Streptococcus of the faecalis type. February 8th. Cultures gave colonies of B. proteus, Staphylocmxus, B. pyocyaneus, diphtheroids, and a coliform which fermented glucose, saccharose, mannite and maltose producing acid but no gas and had no effect on lactose, and duloite. It -was non-motile. In broth it produced a scum on the surface and the colonies on agar were quite unlike B. dysenteriae. February 15th. Cultures gave Streptococci and Staphylococci only. ; 150 Case No. 50. Wounded (shrapnel) 10.1.17. Admitted 29.1.17. Discharged 7.5.17. History. The patient remained in a Casualty Clearing Station for 8 days and was then transferred to the Base, where he stayed for 12 days. His con- dition when in the Casualty Clearing Station was described as follows : He had a severe comminuted fracture of the left ulna 2 inches below its upper end. From a wound 7 inches long which commenced near the external condyle of the humerus and passed down the ulnar line of the forearm the head of the radius protruded ; the bone and cartilage was quite uninjured, but the ligaments of the elbow joint were extensively torn and there was severe laceration of the extensor muscles. The posterior interosseus nerve was damaged. The wound was cleaned, the radial head was returned and sutures put in. The wounds of the left leg were excised and drained. Five days later the elbow wound had to be opened up and part of the joint capsule was cut away, the head of the radius was then returned. B.I.P.P. applied to the wound, which was then sutured. Condition on admission (19th day). The general condition was good, the wound of the elbow had completely opened up, a few remaining sutures coming away at the first dressing ; the head of the radius was lying quite on the surface of the wound, which was suppurating freely. It was decided not to perform any operation but to allow the wound to heal and, after an interval of some months, reset the joint. A wound of the left thigh was quite superficial and granulating satisfactorily. Progress was quite uneventful and rapid ; the wound was skin-grafted on two occasions with striking success. The sinus leading to the site of the fractured ulna healed rather more slowly as some small bone fragments came away. The patient was discharged on May 7th with the elbow quite healed, but the head of the radius projected markedly, the scar being adherent to it. The joint was fixed at a right angle. He was recommended to have a resection of the joint performed some months later. Bacteriological Notes. On admission. A film from a pustule on the thigh showed Staphylococcus. Cultures from the same source gave Staphylococcus only. Film from the elbow showed Staphylococcus, diphtheroids, and Streptococcus. Cultures gave Staphylo- coccus and diphtheroids. Cultures from heated pus gave a Gram-positive anaerobic bacillus with a subterminal spore, ? B. sporogenes. No subcultures were made. February 9th. Pus from the thigh wound. Films showed diphtheroids and a few Staphylococcus. Cultures gave Staphylococcus, diphtheroids, and B. pi-oteus. February 12th. A pyo-sero culture from the elbow gave only Staphylococcus. Case No. 51. Wounded (shrapnel) 11.1.17. Admitted 4.2.17. Transferred 23.6.17. History. Multiple shrapnel wounds of the right leg and foot, the largest one involving the popliteal space and upper calf region. Here the muscles were much lacerated, the external popliteal nerve divided, the internal popliteal nerve exposed. After cleaning up the wounds at the CCS. the divided nerve was sutured, but, owing to sepsis, the sutures cut out after a few days. Condition on admission (24th day). The patient's general condition was good. There were several very small wounds of the right foot which were almost healed and a small superficial wound on the outer side of the calf. The large wound was cleaning, but there was much loss of skin, so that the whole popliteal space was a granulating surface. There was no power to flex the ankle or extend the toes ; periodical attacks of pain down the leg and in the foot were frequent and very troublesome. Progress was very slow, the wound surface was constantly becoming unhealthy in patches and after scar tissue had formed it would here and there break dowii. On May 2oth skin-grafting was attempted, about 50 per cent, of the Steele's grafts being successful and forming a very good scar. There was some little trouble to keep the knee extended. When transferred on June 23rd, the wound had very nearly healed, but there was marked foot-drop and loss of sensation corresponding to the distribution of the external popliteal nerve. The paroxj'smal pains in the leg were much better but still occurred at times. Bacteriological Notes. On admission. Films from the large wound showed Streptococci and diplo- 151 cooci. Cultures gave Streptococcus pyogenes (mannite fermenting type), Staphylo- coccus, and a few B. proteiis. No anaerobes could be grown. February 7th. Film from large wound showed many cooci. February 8th and 9th. Films from the same wound showed a few cocci only. February 10th. Two films taken from different parts of the wound, one showed very many cocci, the other very few. February 15th. Films from different parts of the wound all showed cocci and diphtheroids. Culture gave mostly colonies of Streptococcus and a few Staphylococcus. February 22nd. Films showed only a few cocci. February 27th. Surface of wound much healthier looking. Films showed very few cocci. March 19th. Film from a wound just above the ankle showed chiefly Strepto- coccus. Case No. 52. Wounded (shrapnel) 25.1.17. Admitted 10.2.17. Transferred 23.6.17. History. Multiple shrapnel wounds of the left arm, leg, and foot, one also of the abdominal wall, and one of the right leg. Several pieces of shrapnel were removed from the left leg at the CCS. , At the Base the left great toe was amputated and numerous incisions made to combat cellulitis of the leg, forearm, and upper arm. The left ankle was opened up and pieces of shattered bone removed. Condition on admission (15th day). The patient's general condition was fair, but his temperature was raised, and he showed a great deal of nervous apprehension and restlessness. There were some twenty-five wounds altogether and practically all of them suppurating freely. Of these one, about the size of a shilling, was on the abdomen, five or six were on the left arm, and the remainder on the left lower limb. The foot was much swollen, with pus tracking downwards ; between the metatarsal bones from the site of the toe amputation the ankle joint was severely infected, and the leg muscles much dissected up. There was no infection of the knee or the elbow joints, nor were there any complete fractures of the long bones. Skiagram showed very numerous foreign bodies in the left thigh, leg, and foot. Treatment. The leg was by far the most troublesome to treat, as it was extremely difficult to maintain free drainage of the many suppurating pockets which had resulted from former incisions. A special trough splint was made from a stout sheet of perforated celluloid which, after having been softened in 5 per cent, carbolic acid, was moulded to the requisite shape on a Thomas frame, the foot being kept at a right angle. By this arrangement it was possible to carry out intermittent irrigation of the many wounds through Carrel tubes, 3 per cent, salt solution being employed at first and, later, normal saline or dry dressings. There was for many weeks a most obstinate pustular condition of the skin of the leg and thigh, due apparently to Staphylococcus. Inoculation of staphy- lococcus vaccine failed to control the condition, but applications of a solution of flavine appeared to have a very beneficial action. Two operations were performed on the leg and one on the arm to promote better drainage, and ultimately, after somewhat slow progress, the patient was transferred with most of the wounds soundly healed, his temperature normal, and general condition enormously improved. Bacteriological Notes. On admission. Pus from the forearm. Films showed many Streptococci. Cultures gave »■ pure culture of Streptococcus pyogenes. Heated pus was sterile. Pus from the abdominal wound. Films showed no bacteria. Pus from the wounds round the ankle. Films showed very large numbers of bacteria, diph- theroids, large Gram-positive diplococci, and Streptococci. Cultures gave Streptococcus pyogenes and Staphylococcus. Heated pus gave a long Gram- positive bacillus with a terminal oval spore which grew aerobically and anaerobi- cally, and a heat resisting Streptococcus. February 1 1th -12th. Culture from the wound of the knee gave a pure growth of Streptococcus pyogenes. Pus from the stump of the great toe. Film showed many Streptococci and some Staphylococci. Cultures gave colonies of Staphylococcus and Streptococcus. Pus from the knee wound. Cultures from heated pus were sterile. 152 February 15th. [ The large Gram-positive spore-bearing aerobe was again isolated from pvis from the ankle joint. Films showed fewer bacteria than on the nth, only diphtheroids and Streptococci being seen. February 18th. Pus from a small sinus which opened about this date near the right ankle joint showed in iilms large numbers of Streptococci. Cultures gave Streptococci and Staphylococci. March 9th. Pus from a fresh opening about the middle of the leg. Films showed only Streptococci. Cultures gave a practically pure growth of Streptococcus pyogenes. March 10th. A small opening on the back of the thigh discharged pus in films which showed Staphylococcus and Streptococcus. March 29th. Pus from the ankle. Films showed only Streptococcus. Cultures gave colonies of Streptococcus and a coliform bacillus. April 10th. Pus from the right leg. Plates were overgrown with B. proteus. April 17th. Film from a sinus about the middle of the right leg showed diphtheroids and Streptococcus. Cultures from the acne-like pustules of the skin of the leg gave pure Staphylococcus. April 24th. A small collection of pus which formed in the left upjier arm. Film showed only a few Streptococci. Case No. 53. Wounded (shell) 9.2.17. Admitted 22.2.17. Transferred 23. 6. 17. History. At the CCS. the adductor longus and semi-membranosus muscles were excised and several large incisions made for the treatment of gas gangrene. Condition on admission (13th day). The patient's general condition was fair. There was a large irregular wound of the left thigh which commenced just below the gluteal fold and passed down the whole of the posterior aspect of the thigh as far as the bend of the knee. There was also a smaller incision wound about 8 inches long lying to the inner side of the larger wound, and a shallow oval wound about 2 inches in its long diameter just below the great trochanter. The largest wound had several tracks passing under the skin edge and often penetrating deeply into the tissues ; these were not easily seen and it was only by searching carefully with a probe that they could be discovered. The general surface of the wound was clean and granulating satisfactorily. Progress. Progress was slow owing to the size of" the wound and to the presence of the irregular tracks mentioned above. On several occasions the newly developed epithelium and scar tended to break down in patches. The wound was skin-grafted with partial success on two occasions. Finally the result was most satisfactory, all the wounds being healed when he left the hospital. Move- ments of the leg were perfect. Bacteriological Notes. On admission. Films made from pus from the finger showed numerous bacteria, Streptococci, Staphylococci, diphtheroids, and coliform bacilli. Cultures from the finger were overgrown with B. proteus, but Staphylococci and Streptococci were also present. Films made from the surface of the thigh wound showed many Streptococci and also many large bacilli resembling the anaerobes. Cultures gave Streptococcus pyogenes and a bacillus resembling B. welchii. February 24th. Films from pus obtained from a sinus leading from the large wound showed nothing but Streptococci. Cultures from pus on the inner side of the thigh gave colonies of B. pyocyaneus, diphtheroids, and Staphylococcus. The B. pyocyaneus was atypical, giving a brownish opaque colony on agar, the culture medium becoming green. THs bacillus fermented glucose producing acid, but had no action on lactose, dulcite, cane sugar, or mannite. Case No. 54. Wounded (bullet) 29.1.17. Admitted 24.2.17. Transferred on closing of wards 23.6.17. History. On arrival at the Base on the third day, gas-gangrene developed which was treated by very extensive incisions and excision of some of the a,dductors and the vastus extemus. Owing to the extent of the gas infection it was not found practicable to apply extension to the leg. A bed sore developed over the sacrum. Condition on admission (26th day). The patient's general condition was not very good ; pain, constantly raised temperature and loss of sleep were beginning to show their effects. There was considerable wasting. The femur was fractured at the junction of the upper with the middle third, the upper fragment being tilted forward and adducted so that it could be felt under the skin at the apex 153 of Scarpa's triangle. There were six large deep wounds round the left hip joint and over the left buttock, also some incisions down the front and inner side of the thigh. Posteriorily was a large deep wound leading forwards to the fracture ; this was occupied by a large size drainage tube. There was also a deep ulcer over the left buttock near the middle line which had the appearance of a deep sloughing bed sore. The patient suffered much from paroxysmal attacks of pain due most probably to some injury to the sciatic nerve. Treatment. A special splint was made by which the leg was slung with the hip joint and knee joints bent so as to bring the ends of the bone as much as possible into line, the limb being slightly abducted. Extension was applied to the lower fragment both from above the knee and from the leg. Progress. For the first seven weeks the temperature remained high, efficient drainage being very difficult as pus was frequently being pent up. This condition was becomiug serious, the patient beginning to show signs of exhaustion, when after three small doses of pyocyaneus vaccine had been administered in addition to a vaccine of Streptococcus pyogenes, the temperature fell rapidly to normal, where it remained for the rest of his stay in hospital. With the fall in temperature the general condition improved very rapidly, but the anterior wounds, after having almost completely healed, again broke down superficially. Healing was, however, again rapid, and when the patient was transferred on the closing of the wards the bone had united soundly in a very fair position, but with about 2J inches of shortening, and all the wounds were healed except two sinuses, one leading to the fracture from the anterior surface, the other leading to the same site from the posterior surface. The attacks of pain over the area supplied by the sciatic nerve, which were so troublesome on his arrival, gradually became fewer and less severe. The patient has since been seen (December 1917), when he was walking without any apparent lameness and appeared to have completely recovered. Bacteriological Notes. On admission. Cultures from the large wound in the back of the thigh gave colonies of Staphylococcus, Streptococcus faecalis, B. pyocyane us, and B. proteus. Anaerobic cultures gave B. welchii. March 14th. Film of the pus from the large posterior wound showed many Streptococci and diphtheroids. j&arch 27th. Film of the pus from the posterior wound showed many bacteria, coliform bacilli. Streptococci, and other cocci. Cultures gave colonies of Staphylo- cocci, Streptococci, diphtheroids, and B. proteus. April 3rd. Films from the same wound showed very many bacteria, Staphylo- cocci, Streptococci, diphtheroids, and a coliform bacilli. Cultures gave colonies of Staphylococci, Streptococci, B. pyocyaneus, and a coliform bacillus. April 17th. Kims from the same wound still showed many bacteria ; diph- theroids. Staphylococci, Streptococci, and coliform bacilli. May 1st. Films from the same wounds showed a moderate number of bacteria, chiefly diphtheroids, but Streptococci and a coliform bacillus were also present. Case No. 55. Wounded (shell) 23.1.17. Admitted 27.2.17. Transferred 23.6.17. ^ , History. Patient had a very severe compound comminuted fracture of the right tibia and fibula, great laceration of the muscles, and complete division of the anterior tibial nerve. On arrival at the Base on the eighth day the wound was described as ' very dirty and pouring pus ' and amputation was very seriously considered. Two large counter-openings for drainage were made on the outer and posterior aspects of the leg, and in the following three weeks there was some improvement ; but the condition became complicated by a very large pressure sore on the dorsum of the foot, which quite prohibited the use of an efficient extension. Condition on admission (35th day). Patient s general condition was good. The large anterior wound was discharging very freely and the tibial fragments lying exposed and overriding badly. There were several very large infected bullae on the foot and ankle ; the whole leg was oedematous. Treatment. Treatment was at first directed towards the improvement of drainage and the lessening of the suppuration ; the limb was supported m a splmt lined with perforated celluloid moulded to the shape of the limb, and the wounds were dressed with 5 per cent, saline packs. Only slight extension could be 154 applied from the sole of the foot owing to the presence of the pressure sore on the dorsum. Progress. Under this treatment the patient improved both in general condition and also as regards the amount of suppuration, but there was no sign of any return of function of the nerves, and it was found quite impossible to bring the ends of the bone into good position without extensive operative interference. Amputation was, therefore, performed on April 13th, the bones being divided at the level of the tuberosity of the tibia and two short lateral flaps being made ; the stump was left almost open, only two or three sutures being put in to close the vertical incisions. The wound was then dressed with 5 per cent, saline which was frequently renewed through tubes buried in the dressings. Healing was rapid and the constitutional disturbance very slight, the temperature becom- ing normal on the sixth day after the operation. Later a few skin-grafts were applied. The patient was transferred on June 23rd, the stump being completely healed and well covered. Bacteriological Notes. On admission. A film of the pus of the anterior wound showed chiefly Streptococci. Cultures were overgrown with B. proteus. March 1st. Pilm of the pus collected in the anterior wound showed enormous numbers of Streptococci ; some Staphylococci and diphtheroids were also present. Cultures were overgrown with B. proteus. No anaerobes were found. March 3rd. Cultures of the pus from the site of the fracture gave Streptococcus, Staphylococcus, and a Gram-negative bacOlus, most probably B. proteus. March 8th. A film of the pus still showed large numbers of bacteria ; diph- theroids most numerous but Streptococcus and Staphylococcus also present. Cultures gave B. proteus. Staphylococcus, and Streptococcus. Kims were made frequently after the amputation, the discharge being taken from the surface of the stump. April 16th. Films showed very large numbers of bacteria, the most numerous being diphtheroids ; but Streptococci were also abundant. April 18th. A very large number of bacteria present in the films. Gram- negative bacilli, diphtheroids, and cocci. April 19th. There was a great reduction in the number of bacteria seen in the films ; Gram-negative bacilli were the most numerous, but there were also many diphtheroids and a few Streptococci. Culture of the pus diluted 1 in 1,000 gave numerous colonies of B. proteus and B. pyocyaneus and an occasional colony of a diphtheroid bacillus. The temperature fell markedlj' on April 20th, "and became normal on the 21st. April 21st. Not so many bacteria were present in the films ; diphtheroids the most numerous. Streptococci also present. Cultures from pus diluted 1 in 1,000 gave colonies of B. pyocyaneus and Staphylococcus. April 23rd. Culture from undiluted pus gave colonies of Streptococci, B. proteus, and Staphylococcus. Culture made from pus diluted 1 in 500 gave no growth. April 24th. Bacteria were present in films only in small numbers, a few Streptococci and long diphtheroids being seen. May 8th. Film from a membrane that had formed on the end of the stump showed a moderate number of cocci. May 24th. Film from the stump showed a few diphtheroids and cocci. Case No. 56. Wounded (bullet) 25.1.17. Admitted 25.2.17. Transferred 23.6.17. History. Bullet wound of left leg carrying a very severe compomid com- minuted fracture of tibia and fibula but without injury to large vessels or nerves. The leg was freely incised for gas gangrene both at the CCS. and also on the 6th day at the Base. Condition on admission (31st day). The patient's general condition was only fair. There were several large suppurating wounds on the leg. The fragments of both bones were overriding but union had already begun, so that the position could not then be corrected. Treatment. Drainage was carefully maintained ; the wounds were at first dressed with 5 per cent, saline packs, later with normal saline. The limb was slung in a perforated oellulloid trough on a Thomas frame. Progress. Under this treatment the patient progressed slowly but at first 155- steadily the discharge becoming less, the swelling being much reduced, and the wound healing steadily. Firm union also occurred. At the end of March the patient had a mild and very local attack of erysipelas, after which improvement was very rapid, and this continued until May 11th, when he developed the following symptoms : May 11th. On this day he noticed commencing stiffness of the jaws : he had had a molar extracted about one month previously ; this was followed by a certain amount of inflammatory reaction which had completely subsided. May 12th. His jaws became stiffer so that swallowing was difficult, for instance fluids could be swallowed but not solids. He complained of sore throat, but on examination nothing could be found. Seen at 10 p.m. he could open his mouth about one inch. Abdominal muscles were definitely rigid, but no weakness or rigidity of the limbs could be discovered. He complained of a feeling of stiffness at the back of the neck and in the back, but examination did not support these sensations. Twice during the evening the injured limb became drawn up. Sweating was considerable and he had an anxious expression, but his mind was quite clear and there was no mental irritability. His temperature was abnormal. Thinkmg the condition might have been due to commencing tetanus,_at 10.30 p.m. 15 c.c. (3,000 units) of antitetanic serum (Lister Institute) were injected intrathecally after 15 c.c. of the cerebro-spinal fluid had been drawn off. The cerebro-spinal fluid was sterile and showed no cells. May 13th. Two hours after the injection of serum the patient complained of severe headache and vomited two or three times. At 6 a.m. he became unooflscious and remained in this condition until the next day. His temperature was 102° F. At 9 a.m. violent movements commenced and continued at inter- vals ; when quiet he appeared to be sleeping. When seen at 9 a.m. he was lying on his left side with his head retracted and definitely rigid, making rotary move- ments with his right arm. Later the convulsions became general so that by midday he had to be forcibly restrained. Risus sardonicus was definitely present in the morning. He also suffered from retention of urine. May 14th. He continued extremely violent during the night of the 13tli- 14th, but was calmer on the morning of the 14th. His neck muscles were more rigid. Towards the evening consciousness began to return. May 15th. He was much better and slept for longer periods ; his temperature was normal. Later he quickly recovered, the attack leaving no signs either mental or physical. He had had two former doses of antitetanic serum, one on January 26th or 27th and the other on February 21st. During his attack a plate of false teeth in a very foul condition was found in his mouth and removed ; his teeth were very decayed. Sir Frederick Andrewes, who saw the case during the attack, wrote concerning it : ' I regard the condition as one of hysteria, verging on hystero-epilepsy, but what relation it had to the intrathecal dose of serum I do not know — the diagnosis of tetanus becomes more shaky.' After this attack the patient progressed favourably, the amount of discharge from his leg becoming very small ; skiagrams, however, showed several pieces of necrosed bone which would require removal. The bones had united firmly and the external deformity was not at all marked. Bacteriological Notes. On admission. Films of the pus showed chiefly Streptococci, but Staphylo- coccus was also present. Cultures gave colonies of Streptococcus pyogenes. Staphylococcus, and B. welchii. March 7th. Cultures gave colonies of Streptococcus in large numbers and a few of Staphylococcus. Case No. 57. Wounded (shell) 4.1.17. Admitted 27.2.17. Transferred 23.6.17. History. The patient was wounded in the head, left shoulder, and both feet on January 4th ; he remained at a Casualty Clearing Station for 4 days where some of the wounds were excised and some pieces of shrapnel were removed. He developed gas gangrene at the Base 4 days after injury pn January 8th ; here he was found to have gas gangrene in the neighbourhood of the right ankle and a slight infection of the left ankle. X-rays showed the bones of both feet to be uninjured ; the wounds were freely opened up. On February 8th a large piece of shell was removed from the left shoulder and this wound was drained 156 by a counter-opening. On February 12tli he was trephined and a foreign body was removed from the temporal region. The dura mater was not opened. At the same time the right ankle joint was again widely opened up. Condition on admission (54th day). The patient's general condition was fairly good, there being but very little fever. On the left side of the scalp in the temporal region were 3 sinuses leading down to the fracture in the skull ; sup- puration was abundant and the scalp was very boggy and the drainage not very efficient. No dead bone could be felt. There were no cerebral symptoms. Over the left shoulder there were several small wounds which had almost healed. There was also a small suppurating wound over the point of the left elbow. The right ankle joint had been freely opened up by lateral incisions which were discharging freely, and the drainage appeared to be fairly satisfactory ; but the foot and the lower part of the leg were rather swollen. Movements of the joint were very painful. Round the left ankle incisions had also been made, but these did not appear to have opened the joint. Progress. Progress was very satisfactory although not very rapid ; the swelling over the area of the injury to the skull required incising, and improved rapidly after the removal of a buried drainage tube. When transferred on June 23rd all the wounds were practically healed, one small sinus still persisting m the scalp ; the wounds round the ankle joint had healed but the movements of the right jolut were considerably impaired. Bacteriological Notes. On admission. Films of the pus from the head showed Streptococci and Staphylococci. Cultures gave Streptococcus and Staphylococcus. Film of the pus from the ankle showed Staphylococcus and Streptococcus. No anaerobes could be found. March 16th. Film of the pus from the head showed only Staphylococcus. Culture gave Staphylococcus only. Film of the pus from the right ankle showed practically no bacteria. Cultures gave colonies of a large coliform bacillus. Streptococcus, and Staphylococcus. Case No. 58. Wounded (mine explosion) 3.2.17. Admitted 4.3.17. Trans- ferred 23.6.17. History. At the Base the right leg was amputated in the lower third as dry gangrene of the foot had supervened on a Potts fracture. This was followed by some gas gangrene of the stump, which had to be incised freely. The condition of the left leg was as follows : A simple impacted fracture of the tibia just below the knee joint ; the fibula was also fractured high up. The great toe and the two toes next it were gangrenous. At one time there was marked tense oedema of the left leg, but this subsided without treatment. Condition on admission (29th day). The general condition of the patient was not at all good, there being marked disturbance of the nervous system. During the day he was morose and as a rule quiet, but in the night he was sleep- less, uneasy, the condition resembling a commencing attack of delirium tremens. • This condition became rather more marked, so that it was found necessary to remove him to an isolation ward for about 10 days ; after this period his general condition steadily improved and his nervous system became quite normal. The stump of the right leg, which had been amputated about 3 inches above the ankle joint, was still very septic and had some sloughs adhering to the surface ; the tibia was projecting from the graniilating surface ; the end of the fibula was, however, covered. There were several linear incisions, partially healed, leading upwards from the granulating surface. The surface was intensely tender. The left leg was much shortened and the upper portion below the knee a good deal deformed. X-rays showed a fracture of the tibia, the lower fragment being firmly impacted into the cancellous tissue of the head of the bone. No fracture of the fibula could be made out. The patient complained of great pain which came on at frequent intervals and passed down the leg from the seat of the fracture. The great toe and the two toe.s next to it were the seat of dry gangrene, and a line of demarcation was forming. Progress. On March 30th, a distinct line having formed below the gangrenous tissues, the three toes were amputated, the head of the metatarsal bone of the great toe being saved, although the amount of skin available for a flap was very limited. These wounds were dressed with 5 per cent, saline packs immediately 157 after the operation. The stump of the right leg healed very slowly, but the process was very considerably hastened by skin-grafting on three occasions. ultimate result was very fairly satisfactory ; the wounds remaining after the toes had been amputated healed soundly, leaving a very good foot. The stump was almost completely healed and the bones were covered over, a ring of bone having separated from the tibia. The patient's nervous system had become quite normal. He was transferred on June 23rd, 1917. Bacteriological Notes. On admission. Films of the pus from the toes showed a few Staphylococci and Streptococci and many diphtheroids. Cultures were overgrown with B. protects and B. pyocyaneiis. Films of the pus from the stump showed enormous numbers of bacteria, mostly Staphylococci and Streptococci. Diphtheroids were also very numerous. A few Gram-negative bacilli were also seen. Cultures were overgrown with B. proteus and B. pyocyaneii^, so that the other varieties could not be ioslated. March 7th. Films of the pus from the stump, bacteria not nearly so numerous ; Staphylococci, Streptococci, Gram-negative bacilli, and diphtheroids. Cultures gave colonies of B. proteus. Streptococcus, Staphylococcus, and diphtheroids. March 27th. Films of the pus from the stump showed Staphylococci, Strepto- cocci, diphtheroids, and Gram-negative bacilli. Cultures gave colonies of Staphylo- coccus, Streptococcus, and diphtheroids. April 5th. Films of the pus from the stump showed only a few bacteria, which consisted chiefly of Gram-negative bacilli. Cultures gave only B. proteus. April 20th. Cultures gave colonies of a few B. proteus and diphtheroids. Case No. 59. Wounded (bullet) 15.2.17. Admitted 11.3.17. Discharged 5.5.17. History. At the Casualty Clearing Station he was operated on for gas gangrene, the wound being widely excised, clothing and other foreign bodies removed. Condition on admission {24th day). The patient's general condition was good. He had three wounds on the left thigh, two of which consisted of linear incisions which were practically healed, and the other of a large wound situated on the anterior and inner surface. This wound was about 15 inches long and 5 broad ; there was considerable loss of the skin, fascia, and much muscular tissue ; there were a few sloughs of fascial tissue still adhering to the surface. There was a good deal of pus confined under the skin edges on the outer side of this wound. Progress. At first the wound cleaned rapidly, so that after the patient had been treated for about 10 days the upper part of the wound was closed by secondary sutures. Tliis was accomplished without much tension on the sutures, and free drainage was maintained by. opening up a short section of one of the linear incisions. The wounds were dressed with 5 per cent, saline packs and the result was excellent, the sutures holding ; and the upper portion of the wound healed completely in a very short time. Three weeks later the remaining granulating surface was skin-grafted with striking success, the whole wound being soundly healed three weeks later, and the patient was discharged on May 5th with perfect movements of the leg. Bacteriological Notes. On admission. Films of the pus taken from the surface of the exposed muscle showed no bacteria. Those taken from the pus collected under the skin edge showed a moderate number of Streptococci and diphtheroids. Cultures gave colonies of B. proteus and Staphylococci. March 18th. Films from the upper part of the wound showed only a few diplococci. March 19th. Cultures gave Streptococci and B. proteus. April 10th. No bacteria could be seen in films. Cultures gave Staphylo- coccus only. Case No. 60. Wounded (shell) 43.17. Admitted 11.3.17. Discharged 23.6.17. History. No information could be obtained of Ms earlier treatment. Condition on admission (7th day). The patient's general condition was fairly good. His wounds consisted of (1) a ragged tear of the left great toe, the nail and terminal phalanx being partially severed. This wound was rather unhealthy. (2) A large wound on the back of the right thigh, just above the 158 knee, measuring roughly 8 by 6 inches. The skin, fascia, and muscles in this region had been extensively destroyed. The sciatic nerve had also been slightly injured, paroxysmal attacks of pain being frequent and very troublesome. The surface was mostly clean but no granulations had formed, and in places sloughs were still adherent. Progress. At first the wound cleaned rapidly and granulations formed, but the growth of epithelium and contraction of the surface were very slow. He was skin-grafted on five occasions with somewhat disappointing results ; but the ultimate result was satisfactory, as when he was transferred only an area about the size of a postage stamp remained uncovered with epithelium. There was some little trouble in keeping the knee from becoming flexed, but when dis- charged he could fully extend the joint. Bacteriological Notes. |0n admission. Films made from the discharge taken from the clean surface of the wound showed only a few bacteria. Staphylococci and Streptococci. Films made from the pus that had collected showed large numbers of bacteria, Strepto- ■cocci. Staphylococci, Gram-negative bacilli, tetrads (Gram-positive), and a few diphtheroids. Cultures gave colonies of Streptococcus, a coliform bacillus having the fermentation reactions of B. coli communis and Staphylococcus. Anaerobic ■cultures gave in addition a Gram-positive bacillus resembling B. welchii. The Streptococcus which was isolated did not belong to any classified group, but had the following characteristics : Individual cocci were round and regular, the chains were long, in broth cultures a granular deposit was formed, the super- natant broth being clear. Lactose and saccharose were fermented, acid being formed, but raffinose, mannite, and salicin were unaffected. March 12th. Films of the pus from the left great toe showed very numerous bacteria. March 1.3th. Films from the surface of the large wound showed very few bacteria, only an occasional coccus being seen. March 17th. Film from the surface of the large wound showed only a few Streptococci. March 18th. Cultures gave colonies of Staphylococcus. Films showed a few •cocci only. March 19th. Anaerobic cultures gave Streptococcus and Staphylococcus. April 13th. Films show no bacteria. Cultures gave a few colonies of B. pyocyaneus and diphtheroids. April 30th. Surface rather unhealthy looking. Culture gave many colonies ■of a coliform bacillus having the following characteristics : Motility marked. It fermented with the production of acid only glucose, and to a very slight degree .saccharose. Lactose, dulcite, and mannite were unaffected. May 3rd. After two days' treatment with flavine 1 in 2,000, during which period much of the new epithelium had digested away, films showed a fair number of Streptococci. Cultures gave an almost pure growth of a Streptococcus, having the characters of that isolated on admission. May 8th. A film showed a moderate number of diplococci. May 11th. After the wound had been dressed with brilliant green for 4 days, film shows practically no bacteria. Cultures gave a few colonies of Staphylo'- coccus and a Gram-negative bacillus. May 24th. Films showed a few Streptococci. Cultures gave colonies of B. pyocyaneus and a coliform bacillus with similar characters to that isolated ■on April 30th. May 26th. Films showed very few bacteria. Culture gave a few colonies of B. pyocyaneus. Case No. 61. Wounded (shell) 20.2.17. Admitted 9.4.17. Transferred History. At the Base the patient developed a psoas abscess, which pointed below Poupart's ligament and which was opened, a pint of pus containing Streptococci bemg evacuated. He had a previous history of having had his left testicle removed 6 years previously. Condition on admission (47th day). The patient was very wasted and anaemic There was a severely comminuted fracture of the humerus in the upper third of the shaft, with a wound roughly measuring 3 by 3 inches at the bottom of which lay the exposed bone. This wound was fairly clean but was discharging freely 159 On taking the arm off the Thomas splint in which he had travelled, it was found that there were very extensive pressure sloughs about the left hand ; both the thenar and hypothenar eminences were deeply destroyed ; there was also an abscess pointing in front of the wrist. Just below Poupart's ligament on the left thigh was a sinus which passed up towards the psoas region. X-rays showed the sinus, when injected with bismuth, to be somewhat tortuous and branched ; but the abscess appeared to be draining well as the patient had no temperature. Progress was fairly rapid, the hand cleaned and healed very quickly, the fractured humerus united in good position after several pieces of bone had been removed, and the wound healed. The psoas abscess was still discharging when the patient was transferred on June 2.3rd. Bacteriological Notes. April 10th. Pus from the psoas abscess showed in films a few cocci. Cultures gave a few colonies of Streptococcus. Pus from the shoulder. Films showed Streptococci and Staphylococci. Cultures gave colonies of Streptococcus pyogenes. Staphylococcus, and B. proteus. April 11th. Pus from the hand. Films showed Staphylococci and Streptococci. Cultures gave colonies of Staphylococcus and Streptococcus. April 17th. Pus from the hand. Films showed only a few diphtheroids. May 10th. 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