A BACTERIOLOGICAL INVESTIGATION OF EPIDEMIC CEREBRO-SPINAL MENINGITIS.’

Ry THEODORE SHENNAN, M.D., F.R.C.S.E., and W. T. RITCHIE, M.D., F.R.C.P.E.

(PLATE XLIX.)

DURING the past year epidemic cerebro-spinal meningitis has been prevalent in Scotland, and in the cases which came under our observation we made a close study of the disease in its pathological aspects. In the present communication we shall confine ourselves, in the main, to a consideration of some of the biological and biochemical characters of the causal micro - organisni Micrococcus meninyitidis cerebrospinalis, otherwise known as B@lococczu intracellularis menin- giticlis, iMicrococcus intracell alaris, or, briefly, the meningococcus. The etiological relation of this micro-organism to epidemic cerebro-spinal meningitis being now conclusively established and universally recognised, it might a t first sight appear that a record of our observa- tions upon the characters of the meningococci which we isolated would necessarily be a simple restatement of facts already known. A glance at the literature of the subject, however, reveals quite a number of vexed questions concerning some of the cardinal biological and biochemical features of the meningococcus, upon which the opinions expressed by different writers are still a t variance.

Although cocci had been detected in meningeal exudates by Klebs (1875), Eberth (1881), von Leyden (1883), Marchiafava and Celli (January 27, 1884), Leichtenstern (1885), Senger (1886)) and other investigators, no one had described the cocci in sufficient detail to permit of their exact identification, until in 1886 Fraenkel and then Weichselbaum brought forward conclusive evidence that acute leptonieningitis might be caused by Diplococcus pneunzonicr. These observations were extended by the researches of Fo& and Bordoni- Uffreduzzi (1886, lS88), and of Netter (1887), and it was soon fully recognised that pneumococci might be the exciting cause of acute cerebro-spinal menin- gitis either in its primary or its secondary forms.

I n 1887 Weichselbaum recorded his discovery of Micrococcus meningitidis eerebrospimlis in six cases of acute cerebro-spinal meningitis. The description which he then gave of the coccus in its morphological, tinctorial, cultural, and pathogenetic aspects proved that it was a micro-organism fundamentally

[Received February 19, 1908.1

From the Pathological Department of t h e Edinburgh Royal Infirmary.

EPIDlZMIC CEREBR 0-SPINAL MENINGITIS. 457

different from Diplococcus pneunaonice, and the essential accuracy of Weichselbaum’s observations and conclusions have been repeatedly verified and corroborated during the twenty years which have elapsed since he first described the meningococcus. Weichselbaum described the cocci as being usually in pairs, or tetrads, with their apposed surfaces flattened so that each coccus appeared to be of semicircular form. As the cocci were Gram-negative and usually lay within the cytoplasm of pus cells, they resembled gonococci. Their cultural characters upon agar, upon solidified serum, and in bouillon, a t 37” C., were described. It was emphatically affirmed that no growth occurred at 20” C. I n cultures the individual cocci presented striking differences in size and in the intensity with which they took the stain, small feebly stained and devitalised involution forms being very numerous. The adjacent sides of diplococci from cultures were flattened, whereas isolated cocci were round. As the cultures never retained their vitality for more than six days, and frequently lost it sooner, Weichselbauni advised that, in order to preserve any given strain, subcultivation should be carried out every second day. The intrapleural or intraperitoneal injection of the coccus into white mice, which were found to be the most susceptible animals, caused their death in thirty-six to forty-eight hours ; whereas the effect of inoculating mice subcutaneously was always negative. The diplococcus was also patho- genetic, although somewhat feebly so, t o guinea-pigs, rabbits, and dogs. These observations were amplified by Goldschrnidt (1887), and by Weichselbaum himself (1888), who found Micrococcus meninyitidis cerebrospinalis in three other cases.

The years from 1890 until 1893 witnessed a brisk controversy upon the identity of Eononie’s ‘‘ Streptococcus ineningitidis ” (1890), the “ Menin- gococcus ” of Fo& (1891, 1893), and DipZococcus pneumonia. Bordoni- Uffreduzzi (1890), who took an active part in the controversy, was correct in regarding Bonome’s ‘ I Streptococcus meningitidis” as a variety of pneumococcus, but he fell into the error of regarding Weichselbaum’s Diplococcus intra- cellularzs as another variety of the same micro-organism. This egregious fallacy survived for a number of years. For example, in Baumgarten’s Jahresbericht it was not until 1902 that the literature upon Diplococcus intracellularis was reviewed in a separate section from that upon Frankel’s pneumococcus ; and as recently as 1907 the Bordoni-Uffreduzzi fallacy is re-echoed in a communication by Elder and Ievers.

I n 1895 Jaeger published his first observations based upon a study of cultures of cocci isolated from genuine cases of cerebro-spinal meningitis, and his investigations led to Micrococcus meningitidis cerebrospinalis being almost universally recognised as the causal virus of epidemic cerebro-spinal meningitis, but the micro-organisms which he then cultivated and described as Diplococcus intracellularis differed in certain important features from Weichselbaum’s diplococcus. Jaeger found that the cocci in films from the exudate had a fairly well defined capsule; while in films from the cultures they formed chains which, although usually short, in two instances contained from twenty to thirty members. I n films from exudates and from cultures Ihe cocci were Gram-positive, but they were Gram-negative in sections of the tissues. Further, Jaeger’s cultures had much greater viability than those of Weichselbaum. I n subsequent communications Jaeger (1899, 1901) amplified, and in certaiii respects modified, his original views on the menin- gococcus. Thus he discarded his belief in the coccus being capsulated; he asserted that it was at times Gram-positive, at others Gram-negative, and that Gram-positive and Gram-negative examples might be seen in the same film. He also stated that growth might occur on gelatin at the room temperature.

Although the observations of Jaeger were based upon undoubtedly faulty technique, his work was soon widely accepted, and the coccus which he described came to be regarded as the causal virus of the disease. But the

458 THEODORE SHXNNAN AND W. T. RITCNIE.

work of all those who adhered to Jaeger’s views and supported his conten- tions was likewise faulty. The cocci described by Heubner (1896, 1902), Urban (1897), Kamen (1898), Vanzetti (1901), Lazarus-Barlow (1901), and Weyl (1905) differed not only from Weichselbaum’s diplococcus, but also from one another. Some of the cocci described as ‘ I Meningococci ” grew at 15-20” C., others liquefied gelatine; and the general opinions as to the essential and characteristic features of the meningococcus became involved in the utmost chaos. Pfaundler (1899) recognised two types of meningococci -(1) the ‘‘ Weichselbaum type,” having the classical characteristics as described by Weichselbaum, and (2) the “ Heubner type,” which forms chains and clusters, stains by Gram’s method, grows freely on agar, and also grows on gelatine. Pfaundler concluded that epidemic cerebro - spinal meningitis might be due to cocci of either “type,” or to some intermediate form, such as Jaeger’s coccus.

Until 1901, as is exemplified in the writings of Hunter and Nuthall (1901) and of Lazarus-Barlow (1901), the general conception of the menin; gococcus coincided in the main with the view expressed by Pfaundler, and although the observations of Kiefer (1896), E s t e r (1 896), Kischensky jl896), Still (1898), Councilman, Mallory and Wright (1898), and many other writers t o whom Weichselbaum (1903) refers, accorded with the views he himself had expressed, it was really the exhaustive and convincing research of Albrecht and Ghon (1901) which led to a general recognition of Weichselbaum’s diplococcus as the causal virus of epidemic cerebro-spinal meningitis. Jaeger’s defective technique and faulty chain of reasoning were disclosed in one of his subsequent communications (22nd December 1902), and Albrecht and Ghon (1903) are fully justified in believing that although Jaeger admittedly observed Micrococcus meninnitidis cwebrospinalis in the exudates of some of his cases, he failed to cultivate and subcultivate this micrococcus.

The position taken up by Jaeger in 1902 being so obviously untenable, it is surprising to find that in so accurate and sound a work as the fourth edition of Lehmann and Neumann’s <‘ Atlas und Grundriss der Bakteriologie und Lehrbuch der speziellen bakteriologischen Diagnostik,” published in 1907, the “ Jaeger-Henbner type” of coccus is still described in the section on Micrococcus intmcellularis. Lehmann and Neumann (1 907), moreover, state that they have isolated strains of Micrococcus inlracellularis which, although originally Gram-negative, eventually became Gram-positive, in the same fashion as the I‘ Meningococci” studied by Lepierre (1903), Kob (1905), Castellani (1905), and Weyl (1905). Vansteenberghe and Grysez (1906) also record a Gram-positive meningococcus, while Stuart hPDonald (1907), in a study of over fifty strains of meningococci, found a Gram-positive reaction in three instances, in one of which the coccus sometimes appeared in short chains.

In recent epidemics of cerebro-spinal meningitis Micrococcus meninyitii‘is cerebrospinalis has been found in a great proportion of the cases. Faber (1900) detected this micrococcus in 87 per cent. of his examinations, Coohez and Lemaire (1902) isolated it in 88 per cent., Schottmuller (1905) in 87.7 per cent., and von Lingelsheim (1906) in 64.6 per cent. ; while Bettencourt and Franqa (1904), examining 271 cases, isolated the micrococcus in every instance.

It remains to add that prior to 1901 there was much perplexity regarding the identity of many of the Gram-negative diplococci found in the throat and in the respiratory passages. I n that year Ghon, Pfeiffer, and Sederl published their researches upon over forty strains of Micrococcus catarrhalis. The diagnosis of the meningococcus, Micrococcus catarrhalis, and other Gram- negative cocci from one another and from Diplococcus crassus-by which name Jaeger’s Gram-positive diplococcus is now known-has recently been greatly facilitated by methods of differentiation based upon fermentation of carbo-hydrate media. But even upon these useful diagnostic methods the opinions of all writers are not at one.

EPIDEMIC CZRERR 0-SPINAL MEfl1NGITI.S. 459

The majority of the cases on which we base the observations recorded in this paper occurred during the earlier months of 1907. They were met with during the course of an epidemic of cerebro- spinal meningitis which reached its height daring the months of March and April.

1907

CHAR.^ 1. A, Deaths from epidemic cerebro-spinal meningitis. E, Deaths froin meningitis other than epidemic cerebro-spinal. C, Deaths from convulsions.

The accompanying chart shows the actual mortality froni the disease in the eight largest towns in Scotland during each of the first eight months of the year. The regularity of the curve which can be plotted completes the proof of the epidemic character of the visitation.

We have included several other cases which have occurred outside the period stated.

460 TflEODORE SHZNNAN AND l% T. BITCHIE.

The cases, which occurred during the prevalence of the epidemic in 1907, were distributed as follows: two in January, seven in February, six in March, four in April, one in Ma.y, one in June, one in August, and two in September.

Most of t he patients lived in Edinburgh, b u t some lived in the more or less adjacent towns of Haddington, Musselburgh, Hawick, Grangemouth, and Kirkcaldy. The spinal fluid was in t h e majority of cases taken by one of ourselves, and in the remainder it was sent to Edinburgh very shortly after being withdrawn. In other cases we examined exudate removed froni t he cerebral or spinal meninges after death. In niost of the cases the illness was of very acute onset and short duration. Death took place in every case with two exceptions, Nos. 5 and 27.

The short histories which follow give the salient features of each case, and permit the formation of a judgment, froni t he clinical stand- point, of t he nature of the cases which came under our observation. We gladly take this opportunity of acknowledging our indebtedness to the physicians who have kindly furnished us with these references, and have permitted us to make use of t he cases.

DESCRIPTION OF CASES.

CASE 1.-Young man, from whom Dr. Alexander James removed spinal fluid which was sent to us for investigation on 17th January 1907. The patient’s symptoms left no doubt that the case was one of acute leptomeningitis. The spinal fluid contained numerous polymorphonuclear leucocytes, in many of which Gram-negative diplococci were observed, and yielded a pure culture of similar organisms, which we identified as Micrococcus meningitidis eerebro- spinalis (Weichselbaum).

CASE 2.-Female, zet. 6 years, Haddington. The father was a tramp. She was seen 6th February 1907, within twenty-four hours of the onset of the disease. She was unconscious, with marked head retraction. A hemorrhagic rash was scattered pretty generally over the body. The lips were covered with herpes, which as time went on became hsmorrhagic and scabbed. At the first visit lumbar puncture was employed to withdraw some spinal fluid. The first drops escaping consisted of pure, thick pus, but the fluid rapidly became thinner and less opaque, the last escaping being opalescent only. Blood serum, blood agar, and plain agar were inoculated directly with the fluid, and films were made from the deposit which quickly formed in the fluid. These contained very numerous diplococci of gonococcus type, chiefly contained within the polymorphonuclear leucocytes. These cocci were invariably Gram-negative. Pure cultures, corresponding in all respects with the meningococcus of Weichselbaum, were obtained from the first on all media. Subcultivations on agar from the primary agar cultures failed to develop after one transference.

With careful nursing the child lived for over thirteen weeks (ninety-three days). From the cerebro-spinal fluid at the post-mortem examination- 10bh May-we easily recovered cocci in films and on cultivation (Strain 2, u). These corresponded in almost every respect with those obtained from the same case at the commencement of the illness, except that in films they were more exclusively contained within the leucocytes ; moreover, the proportion

EPIDEMIC CEREBR 0-SPINA L MENINGITIS. 461

of mononuclear cells had greatly increased. were identical.

The cultural characters, however,

CASE 3.-Female, at. 4 years. This case was one of three which occurred in one family. Of the three, this case and another-a boy of sixteen-died.

On admission to hospital (Kirkcaldy) her head was held rigidly on the neck. There was orthotonos. The pupils were irregular, unequal, reacting slightly to light. The abdomen was scaphoid and was covered with petechial spots. The lips and gums were covered with sordes. She was semi-conscious, resisted movement of the limbs, could be wakened, complained of severe headache, and vomited all food. She continued thus for four days, after which slight improvement took place, but on the sixth day she relapsed into her former condition and died on the seventh day.

Spinal fluid was received 11th February 1907, within a few hours of its removal, I t was opalescent. Films contained characteristic, intracellular, Gram-negative cocci, and pure cultures mere easily obtained upon ovarian fluid agar. The cocci seemed a little larger than usual, but in general cultural behaviour, staining characters, and biochemical reactions they corresponded with those separated from other cases.

CASE 4.-Female, at. 4 years, Haddington. She was seen within twenty-four hours of the onset of the symptoms. Nutrition was good. She was very irritable; conscious; spoke a little; was very sensitive to touch. She complained of pain in the head and in the back of the neck. Head retraction soon developed, with Kernig's sign, and the chin became covered with herpes. Only 2 C.C. of opalescent spinal fluid was obtained by lumbar puncture, 14th February 1907. Films contained numerous intra- and extra- cellular, Gram-negative diplococci. They held to the stain longer than usual, but were distinctly Gram-negative when compared with a film of Staphylococcus albus. Pure cultures, typically Gram-negative, were secured from the first.

This case survived for ten weeks, the head symptoms becoming very marked towards the close of life. A t the post-mortem examination the spinal dura mater was firmly adherent to the arachnoid, and cultures were not obtained.

CASE 5.-Male, zt . 17 years; an athlete, Musselburgh. Four days previously he had visited Edinburgh, apparently in perfect health. On the following day he complained of gastro-intestinal symptoms, which were ascribed to his having eaten a pork pie. When seen, 13th February 1907, he was semi-conscious, restless, and struggled while the spinal fluid was being withdrawn. Only about 1.5 c.mm. of slightly opalescent, watery fluid was obtained. Films contained Gram-negative diplococci, both within the cell and extracellular, similar to those found in previous cases. Numerous colonies of Weichselbauni's diplococcus developed on ovarian fluid agar, along with two colonies of Staphylococcus albus (epidernzidis). Pure cultures were separated with the greatest ease, and maintained on ascitic fluid agar. The patient recovered, but unfortunately with complete loss of hearing.

He was at work, apparently in good health, on 5th February. On the 6th he felt indisposed, complained of headache, and stayed at home. On the 7th at 5 a.m. the headache became extremely severe, and he soon lapsed into unconsciousness. At 8 a.m. his temperature was 104" F., and pulse about 80 per minute. The pupils were unequal, the left dilated, reacting badly to light. He remained very restless, and could not be kept quiet. The spinal fluid was sent in for investigation. Films showed scanty Gram-negative intracellular diplococci. Inoculated agar remained sterile, but on glucose

Photophobia was marked.

She died emaciated on 22nd April.

CASE 6.-Male, at. 18 years, Grangemouth.

He died about four days later.

462 THEODORE SNENNAN AND K T. RITCHIE.

agar and ascitic fluid agar, pure cultures of Weichselbaum's coccus developed.

He was in good health until 27th February 1907. On that day he came in from play complaining of pain in the back of the neck. He remained restless and cross all night, and in the morning appeared not to know his parents. When visited in the afternoon he was unconscious, crying out at intervals. He held his head stiffly, and gave indication of pain when it was moved. There was internal strabismus of the right eye ; Kernig's sign was present; the temperature was 103" F. ; no spots of any kind could be seen on the skin surface. There was marked head retraction in the evening. I n films and cultures made from fluid taken by lumbar puncture on the evening of the 28th characteristic nieningococci, without other contaminating organisms, were found.

CASE &-Male, zt. 20 years. Illnesg began on 12th March 1907, with headache and pain in the abdomen. He became more or less delirious in a short time. On admission to the Royal Edinburgh Infirmary, on the 15th, he was noisy and troublesome, with tenderness and fulness over the upper part of the abdomen. The urine contained blood and casts. He died 18th March. At the sectio, which took place on the same day, purulent exudate was found most thickly at the base of the brain, less plentifully over the convexity and along the cord. The lungs were intensely congested. I n the upper part of the jejunum a congested, cedematous patch was seen, with numerous petechial hemorrhages scattered over it. The remainder of the intestine showed no pathological change. Intense hemorrhagic nephritis was present. Films from the exudate contained typical Gram-negative diplococci, chiefly intracellular, and pure cultures of the meningococcus were obtained upon ovarian fluid agar from the first.

He was taken ill on 20th March 1907, with pains all over the body and frontal headache. He felt too ill to work. There was no sore throat and no vomiting. During the following night he became unconscious. Slight improvement took place on the morning of the 21st, but he relapsed into unconsciousness a t 1 p.m., jerking his arms about and heaving his body from side to side. He was admitted to the Royal Infirmary on the same day, and died a t 6 p.m.

At the sectio, conducted next day, very marked suppurative lepto- meningitis was found over both base and convexity. Films contained typical Gram-negative diplococci, and in the primary inoculations on ovarian fluid agar meningococci in pure culture were isolated. Apart from the cerebral condition, we found that the mouth and pharynx were very foul and many teeth were carious. Nothing worthy of note was found in stomach or intestines.

On 6th March 1907 he was a t school, apparently in good health. Next day he complained of slight shivering, and had a frontal headache all day. At night he became delirious. On admission to the Royal Infirmary Edinburgh, on 12th March, the nostrils were red and tender ; swallowing caused pain ; the tonsils were swollen and congested ; the lymphatic glands behind the angle of the jaw were enlarged and tender. There was no abdominal pain or tenderness, and no cutaneous eruption. Patellar reflex was increased ; Kernig's sign was present, but there was no ankle clonus, and Babinski's sign was not elicited. The sight was good, and the pupils were of equal size. The head was held stiffly, and movement of it caused pain. He was intelligent, answering questions readily. H e had slight delirium a t nights. After 19th March he began to be restless

CASE 7.-Male, zt. 29 years.

He was very irritable, and would not take any food.

He died next morning.

CASE 9.-Male, set. 29 years.

CASE 10.-Male, at. 9 years.

EPIDEJfIC CEREBRO-SPIiVd L MXNINGITIS. 463

during the day, and at times drowsy. On the 21st convulsive seizures were noticed. He was removed to the City Fever Hospital on the 25th March, and died there on 5th April. At the sectio typical lesions were found in brain and spinal cord. On two separate occasions during life almost clear, watery fluid was withdrawn by lumbar puncture, and from the second quantity pure cultures were obtained with the usual ease on ovarian fluid agar. Films from both lots of fluid showed intracellular Gram-negative cocci of typical shape.

CASE 11.--Female, st. 19 years. On 8th March 1907, when about to go out, she shivered and felt very ill. She was sick in the evening, and complained of headache. Later she became excited and delirious. On admission to the Royal Infirmary on the 10th she was stupid, restless, moaning, and complained of coldness and headache. The left upper eyelid drooped. Next day herpes developed over the chin and the right side of the upper lip. There was marked polyniorphonuclear leucocytosis. The urine reduced Fehling's solution. There were no tremors or jerks, and no exagger- ation of the tendon reflexes. A mottled petechial eruption, which faded slightly on pressure, was seen over the buttocks and over both knees. On 13th March there was severe headache, but no retraction. On the 15th watery fluid, almost transparent, was removed by lumbar puncture. It escaped under moderate pressure. Films made from this showed polyniorphs containing cocci of typical shape, position, and staining reactions. On inoculating upon ovarian fluid agar only one colony, but that of the nieningococcus, developed. From this time onwards her condition varied from day to day, consciousness and relative comfort alternating with semi- consciousness and distressing pains in the head, neck, abdomen, and lower extremities. On 19th March more spinal fluid, similar to that obtained formerly, was withdrawn, but inoculated media remained sterile in all cases. She was tra,nsferred to the City Fever Hospital on 26th March, and died a few days later. Swabs of the nasal cavities, taken on 18th March, contained principally Staphylococcus aureus and Micrococcus catarrhalis. Meningococci were not found.

No sectio was allowed.

CASE 12.-Lunibar puncture fluid was received (14th March 1907) from Dr. Stuart M'Donald, who had just previously removed it from an infant in the Royal Edinburgh Hospital for Sick Children. On inoculated ovarian fluid agar a pure culture developed; the colonies were numerous, small in size (about 1 mm. in diameter), and had little tendency to coalesce. The cocci did not decolorise so easily as usual when treated by Gram's method, but were distinctly Gram-negative when compared with a film of Stap7~~Zococcus albus placed on the same slide and stained simultaneously. Anilin oil dis- charged the colour a t once.

CASE 13.-Female, set. 45 years, a housekeeper. She was brought to the Royal Infirmary with a history of a week's illness-" Influenza." On the day before admission she had been delirious and unmanageable. On admission (9th April 1907) she was delirious, could not answer questions, and would not remain in bed. There was diffuse redness with puiictiform hsmorrhages over the buttocks, and to a lesser extent over the elbows and on the inner aspect of the knees. Ankle clonus and Babinski's sign were present on both sides. There was retention of urine, which was albuminous and reduced Fehling's solution. A blood examination revealed a leucocytosis of 36,000 per c.mm., principally polymorphonuclear. On lumbar puncture next day, turbid fluid escaped under moderate pressure. I n films a few Gram-negative intracellular diplococci were discovered after a very prolonged search. Three ascitic fluid

The knee-jerks were exaggerated. Kernig's sign was also present.

464 TBEODORE SHELVNAN AND W. T. RITCNIE.

agar tubes were inoculated, and four colonies of meningococcus alone developed. The patient died on 9th May in the City Fever Hospital.

Was temperate, and had had no previous illnesses. On 15th April a t 10 a.m. he complained of headache and shivering. The head pain was frontaI in position; there was no pain in the back of the neck. At 1.30 p.m. he was unconscious, jerking about his hands and feet, and calling out loudly. He vomited at 2.15 o’clock. On admission in the evening to the Royal Infirmary he was unconscious. Kernig’s sign was present. The skin was livid, with circumscribed scattered red areas. The leucocytes numbered 28,000 per c.mm., and were chiefly polymorphonuclear neutrophiles. Lumbar puncture fluid was turbid, containing Gram-negative cocci with the characters of the meningococcus. These were readily obtained in pure culture on ovarian fluid agar from the first. On the following day he was removed to the Fever Hospital, where he died at 9 p.m.

CASE 15.-Mrs. W., aet. 57 years. At night on 13th April 1907 she had a severe headache, and felt indisposed. Next morning she got up as usual, and, though feeling no better, went about her household duties throughout the day. On 15th April she rose at 6.15 a.m., but rapidly became unconscious, and died at 7 a.m. At the sectio, performed a t 11 o’clock in the forenoon of the same day, no visible exudation was found in the meninges, but these were much Congested. Pure cultures of the meningococcus were obtained from the first on ovarian fluid agar, in tubes inoculated from the surface of the pons and spinal cord. The glomeruli of the kidneys were extremely congested and enlarged, standing out very distinctly on the pale grey background produced by the accompanying acute parenchymatous changes.

CASE 16.-MaIe, aet. 18 years; admitted to RoyaI Infirmary, 22nd June 1907, with a history of one day’s illness with vomiting and headache. He was very restless and violent. A leucocytosis of 22,600 per c.mm. was present. His urine was loaded with albumin. Death took place next day. At the sectio the sulci of the cerebrum were filled with greyish-green turbid fluid. There was a consider- able amount of purulent lymph in the interpeduncular space, over the pons, and on the under surface of cerebellum. The foramen of Majendie was closed by this exudation. Similar exudation was present along the whole length of the spinal cord. The lungs were markedly congested, with small subpleural haemorrhages. The kidneys showed marked recent parenchymatous change, and congestion of the glomeruli. Filnis made from the purulent exudate contained typical Gram-negative diplococci, lying principally within polymorphs. Pure cultures of the meningococcus were obtained from the start.

She was brought into the Royal Infirmary unconscious, on the night of 10th September 1907, with indications of cerebral, renal, and pulmonary disease. She did not regain consciousness, and died a t 8 p.ni. on the following day. On the 11th at 1.30 p.m. we removed 10 C.C. of spinal fluid by lumbar puncture. The fluid was turbid and escaped under low tension. This was preserved a t 37” C. for two hours, until we were free to examine it. By that time a delicate fibrinous coaguluni had formed. This was deposited by centrifngalising, and ascitic fluid agar plates were inoculated. Films contained numerous cells, principally polymorphonuclears, but also very numerous Iarge and small nononuclear cells and fibrin. Only Gram-negative cocci were found, usually within the polyrnorphs, but also free. Some of those within the polymorphs were

CASE 14.-Male, aet. 19 years.

Half an hour later he went to bed, but soon rose again.

At night she felt very ill.

Answers to questions were unintelligible.

CASE 17.-Mrs. H,, aet. 29 years.

EPIDEMIC CEREBK 0-SPINAL MZNINGITIS. 465

swollen and degenerating. Films from the plates when stained by Gram’s method displayed, to the naked eye, resistance to decolorisation, but on microscopic examination this appearance was found to be due to thc retention of the violet stain by remains of the fibrin. The patient died six hours after the lumbar puncture, and sixteen hours later a sectio was performed. The case mas remarkable for the number of complications present. Thick fibrino-purulent exudation covered the interpeduncular space, and did not extend on to the pons beyond the roots of the fifth nerves. It was less plentiful and more fluid over the medulla, in the posterior arachnoid cistern, and along the spinal cord. The whole surface was deeply congested, and the sulci over the convexity contained turbid serous fluid. The sphenoidal, ethmoidal, and frontal sinuses were apparently absolutely healthy. Acute myelitis with softening was found in the lower dorsal cord. There was recent acute pericarditis. The kidneys were acutely congested, but there was no distinct tubular change. Tho inner surface of the stomach was covered with thick tenacious mucus, and its mall was congested, with some petechial hzmorrhages scattered over it. I n the upper part of the jejunum the mucous membrane was thickened and congested. There was some patchy congestion in the ileum, but none of the Peyer’s patches were thickened. There was 110 distinct enlargement of the mesenteric or retro-peritoneal glands. Films made from the exudate at the base of the brain contained characteristic Gram- negative cocci. Pure cultures of the meningococcus developed from pus taken from the same situation, but media inoculated from the spleen and from the pericardium remained sterile.

CASE 18.--Male, zt. 12 years, admitted to Royal Infirmary with a history of ten days’ illness, and complaining of sickness and vomiting, severe frontal headache, severe pains in back and body generally. Before admission he had been drowsy, with headache and vomiting, apparently unaccompanied by nausea. Five years previously he had suffered from double middle ear disease, resulting in perforation of the tympana. On admission he lay in a semi- conscious condition, occasionally starting up with a sharp cry. Forehead was contracted in a continual frown. There was ptosis of left upper eyelid and marked external squint of left eye; tongue was thick and furred, breath foul. He was removed to the Fever Hospital, where he died a few days later. Fluid removed by lumbar puncture contained Gram-negative intracellular diplococci of characteristic shape, and inoculated media produced pure cultures of an organism correspond- ing in every way to the meningococcus. At the sectio the lesions on the brain and cord were typical.

CASE 19.--Female, zt. 13 years. She was said to have received a blow upon the head when at work on a Wednesday morning. She was brought into the Royal Infirmary next day complaining of severe frontal headache. There were some indications pointing to lobar pneumonia on the right side. Later she developed meningeal symptoms. Lumbar puncture fluid contained numerous cocci. The white blood corpuscles numbered 32,000 per c.mm. She died early on the Sunday morning. The post-mortem examination was made thirty hours later. On opening the head, yellowish opalescent serous fluid was found in the sulci over the convexity. Thick fibrino-purulent lymph covered the interpeduncular space, and a small amount also lay posteriorly between the lobes of the cerebellum. The spinal cord was covered with thin milky exudate. The lungs showed subpleural haemorrhage, partial collapse, and acute bronchitis. The kidneys were acutely congested, and showed recent acute parenchymatous change. I n the films numerous intracellular Gram- negative diplocci mere found. On parovarian fluid agar pure cultures of meningococcus developed from inoculations from the base, and almost pure

Purc cultures were obtained on the plates.

The cocci were completely decolorised.

Both superficial and deep reflexes were increased.

The spinal exudate contained meningococci.

466 THEODORE’ SHENNANAND W. T. RITCHIE.

from the cord,-three colonies of Staphyloeoecits aureus appeared as well in one tube. No pneuinococci were found. Two mice inoculated intra-peri- toneally died within twenty-four hours. A guinea-pig remained well.

CASE ZO.--Male, Zt. 24 years. Previously he had been quite healthy. He fell out of his perambulator in the evening of 26th June 1906. When brought home the same night he looked very white, and he was said to have had some convulsive attacks. On admission to the Royal Infirmary he had continuous green, fe t id diarrhea, and occasionally, convulsive seizures with stiffening, particularly of the arms and neck, with head retraction. At the sectio, 29th June 1906, acute leptonieningitis, recent pericarditis, acute suppurative arthritis of both knee-joints were found. From the pus in the knee-joints, as well as from the cerebral exudation, pure cultures of the meningococcus developed. In films from the same situations characteristic Gram-negative diplococci were found lying within the polymorphs.

CASE 21.-Female, et. 58 years. She was found leaning against a wall in a dazed condition. She was seini-conscious, and her whole body seemed to be stiff. On admission, 25th January 1907, the sensory functions were a little exalted ; she occasionally was able to return monosyllabic answers. Kernig’s sign was elicited next day, but Babinski’s reflex was not present. Convulsive seizures were frequent towards death. On inoculating ascitic fluid agar with the fluid removed by lumbar puncture a scanty growth of Diplococcus meningiticlis cerebrospinalis developed. The culture was not maintained. Gram-negative intracellular diplococci were found in the subarachnoid fluid post-mortem.

CASE 22.-Male, zt. 2 years, 8 months. H e had been ailing about a week. He complained of pain in the upper part of the abdomen. After being hot and restless during the night he vomited early in the morning of 10th April 1907. This seemed to relieve him for the time, but about 10.30 a.m. he suddenly became unconscious, with marked head retraction, his attitude indicating irritation of the middle lobe of the cerebellum. Kernig’s and Babinski’s signs were present. The pupils were large, and reacted to light. Lumbar puncture resulted in a gush of clear fluid under considerable pressure. He died next day. I n films made from the fluid, scanty Gram-negative intracellular diplococci were found, but pure cultures were obtained from the first. These were not maintained.

CASE 23.-A young woman was admitted, September 1907, to the Royal Infirmary displaying the classical symptoms of the disease, and died in a few days. Spinal fluid was obtained during life, and pus was obtained from the meninges after death. On both occasions inoculated media remained sterile, but on the latter occasion this was to be ascribed to the lapse of time (two days) between the death and the post-mortem examination. Films made from the exudate at the base of the brain, and in the posterior arachnoid cistern showed many intracellular and free Gram-negative diplococci of typical shape.

CASE 24.-Female, zt. 82 years. She was admitted to the Royal Infirmary in a comatose condition. The pupils were contracted and unequal ; the right pupil reacted to light. Albumin and tube casts were found in the urine. Fluid obtained by lumbar puncture during life, 24th May 1907, contained Gram-negative diplococci within the leucocytes, and these also developed on hydrocele fluid agar, along with a contaminating bacillus. The patient died two days after admission. At the post-mortem examination, unavoidably delayed until two days after death, acute leptomeningitis, with a small amount of grey opaque exudate, greenish in parts, was found. Films

He objected to being moved or handled.

EPIDEMIC CEREBRO-SPINAL MENINGITIS. 467

made from this contained diplococci of typical position, shape, and staining reactions.

CASE 25.-A boy, the youngest of three children, aged 10 years, 3 years, and 13 months respectively, members of the same family. They all died within seven days with symptoms characteristic of epidemic cerebro-spinal meningitis, the two youngest each after only thirteen hours’ illness. We received a considerable quantity of fluid, which appeared, macroscopically and in microscopic films, to be pure blood. We could find no bacteria of any kind, and nothing developed on cultivation, even after prolonged incubation. W e learned later that post-mortem examination had confirmed the clinical diagnosis, but as no blood was found within the spinal meninges we concluded that the fluid sent us had not been derived from that situation.

CASE 26.-Female, Et. 34 years, Hawick. KO other cases ha3 been known to occur in the district. Her brother-a soldier-visited home a week before, coming from Maryhill barracks (Glasgow), where the disease was still prevalent. She took ill a t midnight on 6th August. She had rigidity of the back, great headache, and a hydrocephalic cry. No cutaneous eruption was detected. She died in nineteen hours with a temperature of 106” I?. A sectio was conducted next day (8th August). The spinal dura mater contained a large quantity of turbid sero-purulent fluid, and covering the cerebrum there was a slight amount of fibrino-purulent exudate. Films contained numerous pus cells, and numerous typical, chiefly intracellular, Gram-negative diplococci. Scanty colonies of the nieningococcus developed on inoculated media, but were not maintained. The history of the possible method of infection in this ciise is of interest in view of the recent observations by Jehle (1906-1907), and of Fraser and Cotnrie (1907), as to conveyance of the infective virus by intermediaries.

I n the evening he was playing with his companions. It was said that he had fallen and struck the back of his head on the ground. After a short time he resumed his play and then went home to bed. At 2 o’clock next morning his father found him un- conscious. On being visited shortly afterwards meningeal symptoms were present. He screamed occasionally ; the head was retracted ; Kernig’s sign was present, most marked on the right side. Fluid obtained by lumbar puncture was slightly turbid, and contained numerous polymorphonuclear neutrophiles, but few other cells. Some of the polymorphs were vacuolated and degenerating. Only one or two diplococci-Gram-negative and intra- cellular-were discovered after prolonged and repeated search. The principal organism developing on cultivation was the Staphylococcus albus. After an illness lasting for five weeks the patient recovered completely.

During the progress of the epidemic we had occasion to investigate other cases displaying close clinical reseinblances to epidemic cerebro- spinal meningitis, but these were found to result from infection either with the tubercle bacillus or with the pneumococcus.

Nothing developed upon inoculated media.

She was previously in good health.

CASE 27.-Boy, let. 6 years, Haddington.

This rdsunii: of the clinical histories of the twenty-seven cases which came under our observation indicates that all of them manifested those symptoms which are usually associated with acute non-tuber- culous meningitis affecting especially the base of the brain and spinal cord.

Excluding case No. 25, in which the fluid sent to us was not derived from the meninges at all, but was most probably pure blood,

468 THEODORE SHENNANAND W. Z RITCHIE.

we were successful in demonstrating typical intracellular diplococci in films, made either from the spinal fluid during life or directly from the meningeal exudate post-mortem, in every case (100 per cent.).

I n all the cases, with the exception of Nos. 8, 9, and 15, the clinical diagnosis was substantiated during life by the bacteriological examination of fluid withdrawn by lumbar puncture. I n these three cases the course of the illness was so rapid that no opportunity was afforded of using this diagnostic method, but the ineningococcus was obtained from the meninges a t the post-morteni examination. I n thirteen other cases of our series a post-mortem examination was like- wise permitted, and thns in sixteen out of our twenty-seven cases the clinical and bacteriological cliagnosis was confiriiiecl after death.

I n twenty-three cases we isolated iMicrococczis nzeningitidis ccrebro- spinalis upon artificial media, either from spinal fluid removed by lumbar puncture during life or from the meningeal exudate, post- mortem.

From one of these cases we separated two identical strains (2 and 2a) : one during life, the other thirteen weeks later a t the post-mortem examination. Fliigge (1 90 6) isolated the meningococcus from the lumbar meninges at a sectio seventy-five days after the commencement of the illness, and Rautenberg (1 9 0 5) records a similar success at so late a date as nine weeks. Kutscher (1907) comments upon the extreme rarity of such results so late in the course of the disease. From this aspect, therefore, our case would appear to be one of exceptional interest. These twenty-three cases thus afforded us twenty-four strains of the meningococcus, and the nineteen of these which were maintained gave us a basis for further study of the organism.

I n the four remaining cases, although diplococci of characteristic forn, position, and staining reactions were detected in films, we failed to cultivate the meningococcus from fluid removed during life, or from meningeal exudate obtained a t the post-mortem examination. Such failure must be closely correlated with the low degree of vitality possessed by the micrococcus under certain conditions. Thus all the niicro-organisms may have died-( I ) on account of undue delay in transniission of the lumbar puncture fluid, or (2) by the lapse of too long a period between death and the post-niorteni examination. Even under more favourable conditions thg micro-organism is one of such low vitality that culture media must be inoculated with a large amount of the exudate in order to obtain a successful result. Many of the micro-organisms visible on microscopic examination of such fluid are undoubtedly dead-it may be in virtue of a solvent power of the cerebro-spinal fluid or of an autolytic action of the micro- organisms referred to by Flexner (1 906)-and the failure to obtain growths in certain cases may be ascribed to the scanty numbers of viable organisms present.

We examined, then, twenty-eight specimens of cerebro-spinal

EPIDEMIC CEREBR 0-SPINAL MENINGITIS. 469

fluid, and isolated the meningococcus twenty-four times = 8 5.7 per cent. On only three occasions did we encounter contaminating organisms, these being in two instances Staplzylococcus albus. On no occasion did we find the meningococcus associated with pneumococcus, streptococcus, Diplococcus crassus, tubercle or diphtheroid bacilli.

We examined blood removed during life from three of our cases, but were unsuccessful in demonstrating meningococci either in films or on inoculated media.

XXAMINATION OF SPINAL FLUID REMOVED BY LUMBAR TUNCTURE DURING LIFE.

The fluid was in all cases received directly into sterilised test-tubes or centrifuge-tubes, and conveyed to the laboratory, where we either investigated it at once or placed it in the incubator at 37” C. until we were at liberty to do so.

I n a few cases pure creamy pus escaped at first, but as the flow continued the fluid became thinner and more milky. I n other cases the material in suspension was less in amount, producing a varying degree of turbidity, or merely causing an opalescent appearance. Frequently the fluid was transparent, and limpid like water. Speaking generally, the denser the fluid the more luxuriant the growth obtained on culture media, but this was not invariable. Again, the proportion of cells had apparently no special relationship to the severity of the illness. Many of the more acute cases, in which the disease ran a very rapid course, yielded fluid with very slight turbidity, from which, however, even though few cells and organisms were detected in stained films of the deposit, numerous colonies developed.

I n the majority of our cases the fluid was watery, opalescent, or moderately turbid, hence, as a rule, it was centrifugalised, and films were made and media inoculated from the deposit thus obtained.

We employed the former method, as recommended by its originator (1904). Gram’s method was always performed in the following manner: The film, after fixation, was stained in anilin-water-gentian-violet, or in carbolic-acid- gentian-violet for three to five minutes at the room temperature. Lugol’s solution was then applied to remove the stain, and allowed to remain on the’ film for two minutes. The film was then washed in absolute alcohol until no more stain was discharged, the average time required for this being about two minutes. I t was then counterstained in weak carbol-fuchsin (one part in fifteen of distilled water) for five to ten seconds ; washed in distilled water ; blotted and allowed to dry in the air.

W e consider the following points in the technique of Gram’s method to be of some importance. The films should not be allowed to dry at any intermediate stage of the staining process, and should not be washed in water until after the counter-stain has been applied. They should not be allowed to lie in the fuchsin solution, but watched carefully until they have taken up the stain, and no longer. This is to avoid the risk of discharging any considerable amount of gentian-violet remaining in the bacteria.

The appearance of the fluid varied in different cases.

The films were stained by Leishman’s and by Gram’s methods.

Appearances in Films.

Cells.-The great majority of the cells present in the films proved to be polymorphonuclear neutrophiles, some of them degenerating.

470 THEODORE SHENNANAND W. T. RITCHIE.

Few lymphocytes were seen as a rule, but in some films, particularly in those from very acute recent cases, many large mononuclear cells, mostly of endothelial origin, mere seen.

Cocci-The meningococci occurred as uniformly Gram-negative cocci, either single and oval or rounded in shape, or more frequently in pairs, each being reniform, with the flattened or concave sides apposed. Most of them lay within the cytoplasm of the polymorph, sometimes with a narrow clear zone separating them from its substance. We looked on this appearance as evidence of the presence of a vacuole within which the cocci lay. We failed to detect any appear- ance representing a true capsule, and we have never seen cocci actually within the nucleus. The number of cocci contained in different cells in the same film varied between wide limits. I n most filnis extracellular cocci were also seen possessing the same general characters as those within the cells. As a rule the intracellular cocci greatly predominated, but in one case, No. 7, the great majority of cocci appeared t o be free. The cocci are indistinguishable in shape and size from goEococci.

I n none of the films could we detect any other contaminating organisms, either Gram-negative or Gram-positive, although in two cases these developed on cultivation, and in none could we find any diplococci within niononuclear cells.

The number of cocci found in the films bore no apparent relation- ship to the severity of the case. I n some of the more acute cases cocci were scanty, requiring prolonged search before any were found. From such cases, however, cultures were always obtained, colonies developing usually in excess of the numbers which examination of the films would have led us t o expect. The greatest number of cocci within individual polymorphs were observed in the films made post-mortem from Case No. 2, thirteen weelis after the commencement of the illness.

I n a few of the films made from eerebro-spinal fluid or meningeal exudate the cocci held the violet stain in Gram’s method more strongly than usual, but when compared with an average staphylococcus on the same slide they were always Gram-negative. Anilin oil invariably discharged the violet with great rapidity.

CULTIVATION OF THE MENINGOCOCCUS.

During 1906 one of us had been employing sloped ovarian fluid agar (ovarian fluid, 1 part; peptone agar, 4 to 6 parts) for the cultivation of the gonococcus, on the recommendation of Dr. Bulloch of the London Hospital. The ease with which the gonococcus was isolated and maintained on this medium suggested it as a suitable one to use in the case of the meningococcus. We found that colonies developed rapidly and grew to a larger size-up to 3 to 5 mm.-than on other media containing raw serum, although aacitic fluid agar and hydrocele fluid agar proved almost as useful for the isolation and maintenance of the organism. We found parovarian fluid agar a less

EPIDEMIC CEKBBR 0-SPINAL MENINGITIS. 471

suitable mcdium, doubtless because of its low albumen content. Agar made with parovarian fluid instead of bouillon gave results very similar to those on nasgar (Gordon, 18. ii., 1907).

The fluid, whether ovarian, parovarian, ascitic, or hydrocele, was transferred with aseptic precautions to small sterile flasks, and, to ensure its sterility, was shaken up with a small quantity of chloroform, or exposed to 58" to 60" C. for one hour on four successive days. This chloroformed fluid after a few days was distributed, for convenience in handling, into sterilised test- tubes. For use the fluid, in the proportions already indicated, was added to melted agar a t 45" to 50" C. in test-tubes, and the mixture either poured into Petri's capsules or sloped and allowed to set. Tubes and plates were placed i n the incubator for a t least twenty-four hours, to drive off all traces of chloroform and to test sterility. On only three occasions during the past year has stock medium made u p in this manner proved to be contaminated. Such media are referred to later in this paper as "serum" agars.

A considerable amount of the deposit obtained by centrifugalising spinal fluid, or of the pus taken directly from the meninges after death, was smeared over t h e surface of such media. Only a small number of the cocci present seem able to grow and develop, the organism in this respect closely resembling the gonococcus.

I n taking pus from the meninges a t the post-mortem examination we were careful to choose thin or creamy fluid pus rather than fibrinous or inspissated pus, as the latter did not afford as good results.

Appearance of Colonies in Cultures.

Slight variations in detail were observed in different strains, but the following description gives the characters more or less common to all those separated by us:-

I. OVARIAN, PAROVARIAN, ASCITIC, OR HYDROCELE AGAR. - 1. After ticenty-four hours at 37" (2.-To the unaided eye the colonies of meningo- coccus appear as rounded discs, with a diameter of 1 to 2 mm.; they are colourless and translucent, with a moist, glistening surface, only slightly raised above the surface of the medium; their margins are thin a n d regular. Viewed with a low power ( x 50) with transmitted light, colonies have a slightly yellow colour a t the central parts, and here they are also finely granular. Occasionally there is a small darker central spot or '' nucleus." They become thinner towards the peripheral part, and near their margins are thin, transparent, homogeneous and almost colourless. The borders are uniform, usually regular or presenting very slight irregularities.

Primary inoculations, as a rule, result in the development of numerous isolated colonies. On continued subcultivation the colonies developing become more numerous still, and tend a t times to be a little denser than young strains, usually coalescing towards the lower end of the streak. This depends to some extent on the heaviness of the inoculation. Even i n late generations, however, isolated colonies always develop a t the top and a t t h e margins of the streak.

2. A.fter thi~ty-s iz hours' incubation.-The colonies are now larger, on ovarian fluid agar plates occasionally reaching 4 mm. or even 5 mm. in diameter ; the central part of each colony is now rather more distinctly raised, while the thin marginal part is relatively more extended. Viewed under a low magnification, the central parts now appear of a darker yellow colour, and display a few scattered refractile granules of larger size than those seen a t

31-~~. OF PATH.-POL. XII.

472 THEODORE: SHENA’ilN AND W. I: RITCHIE.

earlier stages. These granules are not uniformly prominent in all strains, and, as we have frequently seen somewhat similar appearances i n colonies of other micro-organisms, e.g. gonococcus, other Gram-negative cocci, and some staphylococci, we do not regard central coarse granulation as a wholly essential characteristic of the colonies of meningococcus. W h e n granules are present in colonies of meningococcus they become still more prominent as the colonies grow older. Sometimes radiating streaks or ridges are seen on the surface of the colonies.

3. After forty-Pight to seventy-tzco hours’ incubation.-The size of the colonies reniains almost stationary, and the appearance of their central parts undergoes little change. The extreme edge of the homogeneous peripheral part is now more distinct, and even as seen with the unaided eye appears to be thickened, forming as i t were a “ halo” round the colony. I n late stages small wedge- shaped knobs or rounded elevations sonietinies develop on this ‘’ halo,” and can be seen under a low magnification. Occasionally a second raised margin or ‘( halo” develops outside the first after five to seven days’ incubation. Young colonies are slightly viscid, and they cannot be moved about as a whole over the surface of the medium. Old colonies tend to become denser a n d friable, particularly if the inediuni have become partly inspissated.

Growth i n the Condensation Water.-The growth in the condensation watw in the case of such media is practically identical with that i n serum broth. Turbidity is slight, and most distinct in comparatively young cultures. A surface pellicle develops a t an early stage, and as successive pellicles form and are shaken down a white deposit of cocci forms a t the bottoni of the fluid. Coarse pellicles, which cannot be shaken down or readily broken up, and dense, perniaiient turbidity of the condensation water generally indicate t h e presence of contaminating micro-organisms.

After continuous subcultivation of ouia strains, in some cases for more than twelve months, they have, as a rule, tended to become somewhat denser, bu t the essential characters, as described above, have remained constant throughout. For successful subcultivation it 1s necessary to transfer il relatively large amount of a previous culture. W e transfer newly isolated strains on to fresh media a t least every third or fourth day, but in the case of old strains a longer period-a week to ten days or even more-is allowed t o elapse.

Vitality.

On testing the vitality of our strains we obtained in two cases a n active growth froni serum agar cultures sealed with paraffin, anu‘ preserved a t 37” C. for seventy-eight and a hundred days respectively, while growth was likewise obtained from three other strains sealed and preserved in the same manner for eighty-five days. On the other hand, certain strains, i n spite of long liabituation to artificial media, cannot now live for three weeks or even a fortnight.

11. BLOOD AGAR (PFEIFFER’S).-~eVelOplilent is almost as strong on this medium as on ascitic 01’ liydrocele fluid agtr. The characters of the colonies are siniilar to those already described. Hamolysis takes place after a time, and in old cultures the medium becomes turbid superficially.

111. LOFFLER’S GILKID SRRUM.-Although this medium does not prore quite so useful as serum agar in the isolation of the ~neningococcus, me find it extremely useful in subcultivation of that organism. The colonies are appreciably moister, denser, more opaque and usually smaller than those 011

serum agar. They also tend more to coalesce on this medium. The growth has occasionally a faint yellowish tinge. The appearances of the groIvth in

EPIDEMIC CEREBRO-SPINAL MENINGITIS. 473

the condensation water correspond with those already described i n t h e case of the special ‘‘ serum ” agar media. It was noticeable tha t the vitality of cultures upon this medium was, on the average, decidedly less than tha t of cultures on media containing raw serum.

IV. PEPTONE AGAR.-If colonies develop upon agar they have in general the same characters as have those grown upon special “serum” agar, but the growth is less luxuriant and consists almost entirely of isolated colonies, which are usually of simller size than others of the same strain inoculated simultaneously upon the more favourable media. In some of our strailis we have been unable to obtain growth upon agar, even after such straiix have been long subcultivated upon other media.

I n Case No. 2 we obtained a plentiful primary growth upon simple agar, but the spinal fluid contained a large amount of pus, and a few drops were passed directly into the tubes a t the bedside, and not allowed t o cool before being placed in the incubator. On first subcultivating on to similar iiiediuni a scanty development took place, but subsequent subcultivation upon agar failed.

I n Case No. 20 agar was inoculated from the knee-joints, which mere the seat of secondary suppurative arthrit.is. A plentiful pure growth of nieningococcns developed, hut after three days’ subcultivation on all media failed, the organism having, even i n that short time, died out, although preserved a t 37” C.

After one t o four months’ cultivation of our stock strains on the “ serum ” agar media, subcultures were made upon agar. Growth took place in the case of Nos. 2, 4, 6, 7 , 8, 9, 10, 14, 15, and 19, but in the remainder it was difficult t o determine whether any development had really taken place. In the cases i n which growth had occurred this was possibly assisted by the transference of some of the serum medium along with the culture to the surface of the agar.

The capacity for growth upon agar was again tested after subcultivation for seven months longer, and now in inany of oiir strains several trans- ferences from agar to agar in series were successfully carried out. Even now, however, as we have already noted, some strains may fail to grow upon this medium.

V. ON NASGAR (Gordon, 1907) and upon a similar agar medium we made from parovarian fluid we found that all our strains grew well, but not .quite so actively as upon the serum agars.

VI. OVARIAN-FLUID hoTH.-Bouillon to which was added sterile ovariaii fluid. All our strains grew upon this mediuni. A faint turbidity develops, which varies in degree in the different strains, but is always distinct by the third or fourth day. About the second day a th in grey marginal ring develops on the inner wall of the tube a t the level of the upper surface of the fluid. A day or two later a delicate pellicle has formed upon the surface of the medium. To detect such pellicles one must avoid disturbing the cultures, and our procedure was as follows: The racks holding the tubes were placed close to the front of the incubator, so that they could be examined without being moved. A light, for example, of a taper, was held to one side of each tube, rather below the surface level of. the broth, so as to illuminate specially tha t part of the medium. I n this way i t was easy to observe even delicate surface pellicles.

Gentle shaking breaks up the pellicle, and i t falls to the bottom of the broth and lies there in a white granular layer. After a time the general turbidity diminishes.

This we take as a proof of the aerobic character of the organism.

W e noticed that the turbidity cleveloped from above downwards.

474 THEODORE SHENNAN AND W. I: RITCHIE.

VII. PEPTONE BRoiw.-ordinary '' bouillon." Growth in all cases takes place in broth. The appearances resemble those presented by the cultures in ovarian-fluid broth, though the growth is not so vigorous ; moreover, the pellicles are less obvious and more easily broken up.

We find that the addition of glucose to broth produced a much stronger growth of meningococcus, and in addition cultures retain their vitality for a longer time than when grown on plain broth.

VIII. POTATO.--GrOwth is uncertain, and in any case scanty, a hardly visible layer developing and causing a slight localised " varnishing " of the surface.

IX. GELATIN MEDIA. - Peptone gelatin, ascitic - fluid gelatin and ovarian-fluid gelatin. Even after the repeated subcultivation of our strains upon appropriate culture media during periods varying from nine to twelve months, transference to gelatin at 20" to 22" C. always fails to give any growth.

X. LITNUS MILK.-when tested after being maintained for a time upon artificial media all our strains grow in this medium, but produce no distinct alteration in the reaction, and no coagulation. On first subcultivating on this medium from the primary culture in strain No. 2 we noticed a very slight reddening of the colour, bnt this has not occurred again on any subsequent testing of that strain.

XI. PEPTOWE WATER.-NO visible development takes place.

XII. NEUTRAL RED AGAR. - On this medium surface colonies of meningococci are colourless. Bettencourt and Franpa (1 904) report that cultures on glucose neutral red agar develop a light rose colonr.

XIII. INDOL PRODUCTIOh-.-%ZiinS 3 and 10 were not tested. The strains were cultivated in ovarian-fluid broth for ten days at 37" C. The test was carried out following the usual method of adding potassium nitrite and pure sulphuric acid to the broth cultures, these being kept warm or gently heated.

After fifteen minutes a positive result was given by Nos. 1, 4, 6, 8, 9, 11, 12, 13, 16, 17, 18; after thirty minutes the reaction appeared in No. 7. Three drops more of the nitrite solution and two more of the acid were now added to those which did not show any reaction. I n five minutes more, that is, thirty-five minutes altogether, No. 2 showed the reaction distinctly, and after one hour from the commencement of the experiment Kos. 5 , 14, and 15 showed a faint reaction. Thus all of the strains tested gave the indol reaction.

On another earlier occasion forty-eight days old ovarian-broth stock cultures were tested, the nitrite and acid being added as before. The first sixteen strains were available at that time. The tubes were first kept a t 15" C. for one hour. At the end of that time Nos. I, 3, 6, 7, 10, 11, 13, and 14 gave the reaction. They were then placed for two hours longer in the incubator at 37" C. On examination at the end of that time Nos. 4, 6, 7, and 10 gave a very strong reaction ; KOS. 1, 3, 5, 9, 11, 13, 14, and 15 gave a distinct reaction; Nos. 12 and 16 gave no evident reaction, and Nos. 2 and 8 were not examined on this occasion.

Behaviour on Media containing Yayious Carbohydrates. The carbohydrates employed were those used for similar purposes by

von Lingelsheim, namely, glucose, kevulose, galactose, cane sugar, maltose, lactose, dulcite, and mannite. In some of the later isolated strains

B P I D E M I C CEREBR 0-SPINAL M E N I N G I T I S . 475

mffinose, dextrin, and inulin were used in addition. The media were prepared by a method similar to that described by v. Lingelsheiin (1906). W e found, however, great difficulty in obtaining sterile media in Petri’s capsules when prepared according to his instructions, and as we considered that slight alterations in reaction would be more easily detected in relation to colonies localised upon solid media we devised the following modification of his method, using tubes instead of the Petri’s dishes.

To unneutralised 2 per cent. agar we added 1.2 per cent. of a given carbohydrate, tubed the mixture, and sterilised a t 100” C. for a quarter of an hour on two occasions at an interval of thirteen hours. After cooling to 50” C. sterile ovarian or ascitic fluid, in approximately the usual proportion, and sterile 1 per cent. litmus solution, the latter rendered distinctly alkaline by the addition of hydrate of soda (10 per cent. solution), were added, so that the resulting medium was of a faint but distinct violet colour. The tubes were now sloped, allowed to set, and incubated for at least twenty-four hours t o test their sterility before being inoculated. We have also employed litmus ovarian broth containing 1 per cent. of the carbohydrate and also carbohydrate media prepared according to the method of Hiss, but for the reason stated above we prefer to use solid media.

We are fully alive to the difficulties and risks which arise in the sterilisation of these carbohydrates by heat. We realise that chemical changes take place in them, particularly in the presence of alkali, even with the short exposure to high temperatures indicated above, but after all our primary aim has been to compare our results with those obtained by other workers, for example, von Lingelsheim (1906), Gordon (1907), Rundle, Mottram, and Williams (1907), Goodwin and von Sholly (1907), and Symmers (1907), who employed “sugar ” media prepared after a somewhat similar method. In niany instances the reaction obtained must be one, not upon the actual substance dissolved, but upon some derivative of it. For example, one of us, working a t this’ subject, found that inulin suspended in distilled water was profoundly altered by exposure to a temperature of 70” to SO” C. Our carbohydrate media, prepared as above described, gave characteristic reactions with B. typliosus. B. coli conanzunis, B. xeroszs, Hoffniaiin’s pseudo-diphtheria bacillus, and B. diphtheria.

In repeated trials upon solid carbohydrate media, prepared according to the above method, our results with meningococcus correspond with those obtained by von Lingelsheim and Symmers, and differ in some particulars from those of Gordon and the earlier conclusions of Goodwin and von Sholly. No gas was ever produced in these media. We found-like Ghon (1907)-that the reactions vary in intensity and in time of development, but they are, with perhaps one exception, quite characteristic of the nieniagococcus, and show that we are in no instance dealing with Diplococcus c7’assus or any of the Grain-negative cocci described and named by von Lingelsheim (.Micrococcus pharyngis flavus, Micrococcus cinereus, etc.). Our meningeal cocci were also differentiated by these reactions from Micrococcus catarrhnlis, Micrococcus gonorrhoea, and from several other Grani-negative diplococci which were isolated by Dr. Williamson and Mr. Lindsay in the course of routine bacteriological reporting in the Pathological Department of the Edinburgh Royal Infirmary.

When examining the tubes for reactions we preferably stand with our back to the window and place a sheet of white paper

476 THEODORE SHENNAN AND W. Z RITCHIE.

behind the tubes, which receive the daylight directly and not by transniission. We found i t easier to detect slight alterations in the colour of the litmus thus than when we held up the tubes between the eye and the source of light.

It is convenient to present liere in tabular form the reactions we obtained in nineteen of our strains, and also to compare our results with those obtained by certain other observers. All cultures were exaniined claily for a week.

-

1 . 2 . 20. . 3 . 4 . 5 . 6 . 7 .

9 . 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 .

a .

Strains.

. . .

. . .

. . .

. . . . . .

. . . . . .

. . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . .

. . . J f e ~ t i ~ ~ g o e o c c ~ ~ s (Gordon) . .

dGcrococe?cs catarrhaliu (Gardoll) ilficrococcus p~norrhoea: (Gordon) Mic~ococc~as gonorrhoea! (Royal

Infirmary, Edinburgh) . . DipZococc~~s c m s m s (von Lingels-

iieim) . . . . . - . Throat COCCIIS (Royal Infirm-

ary, Edinburgh, No. 1) . . Throat COCCIIS' (Royal Infirm-

ary, Edinburgh, No. 2) . .

+ + + + + + + + .t -t + + + + + + + + + + 0 +

+ t

+ +

~ -.

0 0 0 0 0 0 0 0 0 0 0 0 0

...

...

...

0

+ + +

0 0 0 0 0 0 0 0 0 0 + 0 + +

+ + +

2 F

5 n 0 c

-

0 0 0 0 0 0 0 0 0 0. 0 0 0 0 0 0 0 0 0 0 0 0

0

+ + +

p; 0 c.'

d 4

__ - .~ ~

+ + + + + + + + + + + + + + + - t - + + + + 0 0

0

+ + +

a: 0 I ., 4

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 (r 0

d 42

0 .- - 6 -

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

. . . . . . .

. . . . . .

... i ...

+ i o

0' - .d - - - -.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ... ... ... 0

0

0

0 ~~ ~~

- - .3 r-.

I Y

o 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ... ... ... 0

0

0

0 ~ ~~

...

...

...

...

...

...

...

...

... 0 0 0 ... ... ... IJ

0 0 0 ... ... ... ... ... ... ...

t..

... + ... ... ... ... ... ... ... + + .,. ... + ... ... ... + ... ... ... ... ...

Separated in Course of routiiie reporting in the Pathological Departnient, Edinburgh Royal Infirmary; tho throat cocci, by Dr. G. S. Williamson and Mr. M. A. Lindsay, from swabs. Thesc were both Gram-negative diplococri, resembling in niany respects illicrococezts pharyagis siccras of van Lingelsheim.

It will be noticed that we have never succeeded in obtaining any acid reaction on galactose agar medium. All our strains grew well on this medium. I n m e experiment we incubated the tubes for a week, and in another for four wecks, without any alteration of reaction occurring. Control tubes were preserved on both occasions, for the purpose of comparison of the tints. These results difler froin those of Gordon (1907), whose cultures in fluid media produced an acid

EPIDEMIC CEXEBRO-SPINAL MENIflGITIS. 477

reaction after twenty-four hours' incubation, and of Rundle, Mottram, and Williams, who found the maximum acid reaction on the sixth day of incubation, also in fluid media. We have never, in spite of repeated trials, found any of our strains producing acid in lactose media, except on one occasion, when strain No. 6 produced a very slight reaction. A previous test of the same strain and all subsequent tests were quite negative. This strain was also slightly variable in its action on glucose media, chiefly in the degree of acidity produced. Our results with lactose iiiedia differ from these which Goodwin and von Sholly report in their earlier communication (ii., 1 9 0 6).

Discordant results may be explained by variations in the methods of preparing the media, one probably causing greater preliminary alteration in the carbohydrates than another. Possibly an important difference in the media used is that some are fluid and others solid. We have already given our principal reasons for preferring the agar media, but our experience with several of our cases shows that the same organisni in the same hands ma.y give discordant results. We recently observed an acid reaction in parovarian galactose broth on the fourth day in strains 7 , 9, 10, 11, 16 , 18, and 19, whereas the same strains on parovarian galactose agar showed no acid reaction. Similar anomalies were met with in the case of dextrin media. Plates inoculated from these fluid media invariably gave pure typical growths. We were not able a t the time to complete this line of investigation.

We have also noticed that on adding identical quantities of litmus to measured quantities of the different carbohydrate agars or broths the resulting media differ in colour, some being distinctly blue or violet, and others, 0.9. galactose media, having a magenta colour which, if seen in other media, similarly made up, e.g., with dextrin, cane sugar, maltose, etc., might indicate an acid reaction.

The alkalinity of these media should be of the slightest, and we found it easy to prevent the appearance of the acid reaction by increasing only slightly the preliminary alkaline reaction. The pro- duction of acid by the meningococcus is never so great as in the case of such organisms as L'. coli eommzinis and some streptococci, so that if the alkali be in too great amount an acid action may not be elicited even in glucose and l~ialtose, although the cultures develop as luxuri- antly as usual. If, after an experience of this kind, the tests be repeated with media presenting very siight, though distinct alkalinity, the same strains of meningococci readily cause an alteration of the reaction in the case of these two sugars. Strain No. 8 gave a some- what weak reaction with glucose. The failure of No. 6 to produce distinct acid in glucose has already been noted. The distinct reactions produced by these two strains in maltose media, however, differentiate them from Aficrococeus cntardzalis and Mic~ococcus yonord~oem.

478 THEODORE SHENNANAND W. I: RITCHIE.

Microscopical Appearances of Meningococci f r o m Cdtwes . Films made in the customary manner were stained usually by

the same methods as were employed in the examination of filnis inade from exudate or spinal fluid. Our usual procedure, when critically testing the behaviour of a given strain under Gram’s method, was t o place with i t upon the same slide a spot of a staphylococcus culture which had been found t o present an average degree of resistance to decolorisation, thus acting both as a macroscopic and a niicroscopic indicator. All our strains are, and have been from the first, Gram- negative. A few show a slightly greater resistance to decolorisntioii with the alcohol, but all give up the stain, usually within two minutes at the very most. This resistance to decolorisation varies from time t o time in subcultures of individual strains, and also varies with the nature of the media employed. For example, we find that subcultures made upon our “ serum ” agars or Loffler’s blood seruin from previous cultures in liquid media are as a rule easily decolorised, whereas subcultures maintained upon solid media frequently display a greater resistance to decolorisation. This is not invariable. The violet is invariably very rapidly discharged by anilin oil froni all films, whatever the media upon which cultures have developed. This result does not agree with that reported by some authors, and we are driven t o the conclusion that they were not working with the true meningococcus.

I n those cases recorded in the literature, in which the cocci have been Gram-positive, we believe that either they were not Micrococcus meningitidis cerebrospinalis or the method of applying Gram’s stain was at fault, or alternatively, the true meningococci were accompanied by some other coccus which retained the violet with greater tenacity, the former predominating in the exudate, but only the latter develop- ing on cultivation, an apparent “ change in type ” being thereby simulated. It is interesting to note, in this connection, that Jaeger, recording in 1905 the results of some recent investigations, admits that the meningococcus decolorises in films made direct from exudates or aspirated fluids, but states that in some instances the strains decolorise slowly and unequally.

Morphology.

The meningococcus has a very close family resemblance t o Micrococcus gonorrhoem. In cultures it occurs as single rounded cocci, varying a little in size, or in gonococcus-like pairs with flattened or .concave apposed surfaces. Tetrad forms may be looked on as quite characteristic. We have never observed true chains, though soinetimes one may find four or six cocci-two or three pairs-forming an irregular short row. We look on this as an accidental arrangement. Chains wera never seen in films of tbe exudate or spinal fluid.

EPIDEMIC CEREBR 0-SPINAL MENINGITIS. 479

Involution forms are frequently seen, but in twenty to twenty- four hours old cultures they are not common. At a later stage- twenty-four to forty-eight hours-they are prominent features in the field, either in the form of single, deeply stained large cocci, or similar cocci may be arranged in pairs or tetrads. Both pairs of a tetrad rarely correspond in size.

After forty-eight to seventy-two hours' incubation, involution and degenerating forms predominate. The inost of the organisms present stain feebly, many are represented as rings from two to five times the size of the young coccus, but most as granular material which scarcely takes up the stain. Along with these degenerate forms even in cultures incubated for fourteen clays, one can detect sinall diplo-forms of normal size which stain in a normal manner, and on which most probably the vitality of the culture depends.

AGGLUTINATION TESTS.

The agglutinative power of serum, whether from patients suffering from the disease or from animals that have been frequently inoculated with meningococci, has been repeatedly tested by many investigators within the past three years. As the literature upon this subject has been so fully considered in a recent review by Kutscher (1907) we do not think it necessary to comment on it in any detail, but need only refer to the observations of Flugge (1906), Jochinann (1906), Kolle and Wassermann (1 9 0 6), von Lingelsheirn (1 9 0 6), Goodwin and von Sholly (1 9 0 6)) and Ceradini (1 9 0 7). Their observations indicate that the serum of artificially immunised animals agglutinates meningococci even in low dilution, the effect being most marked upon the homologous strain, whilst the blood serum of patients may not .agglutinate meningococci until the illness has lasted for one or two weeks.

Our tests were performed with the serum of two cases of epidemic cerebro-spinal meningitis, and with one of the commercial anti- meningococcal sera. The patients' sera were obtained on the seventh .and on the thirtieth day of the illness respectively. The results of .our tests are shown in tabular form. They show that a heterologous strain may be distinctly agglutinated in one hour, or rarely in from fifteen to thirty minutes, but that complete agglutination of a strain, whether homologous or heterologous, was never observed in less than twelve hours. The number of positive results would doubtless have been larger had the tests been performed a t 37" C. or a t 55" C., as recommended by Kutscher. Our observations on the agglutinative power of therapeutic serum are admittedly few in number, yet they illustrate the feeble agglutinative effect of at least one of the thera- peutic sera on the market, a fact to which attention has already been directed by Houston and Rankin (1 9 0 7).

480 THEODORE SHENNANAiVD Pi? T. RITCHIE.

Table of A:pjhti?tation Tests.

Serum of Case No. 8 tested on the '1 f i 1 : 20 se~en th day of the ilhiess / I 1; 1 : 5 0

Serum of Case No. 4 tested on the thirtieth day o t the illness

Anti-meningococcal serum (B. W. & C O . )

I ,

I , I ' 1 : 2 0 I 1 :50

I- 1, 1 : 2 0

( 1 l : e \ , 1 : 2 0

I :50

1 : 2 0 1 : 50

1 : 20 1 : 50

1 : 20 1: 50

I :20 1 : 50

1 : 20 1 : 50

1 : 2 0 1 : 5 0

1 : 2 0 1 : 5 0

1 : 2 0 1 : 50

1 : 2

1 : 5 { 1 : 2 0

1 : 20 I I

Result.

} + i n 30 minutes.

t , I

I 1 ,

+ in 1 hour; * in 12 } hours.

+ in 1 hour. x in 1 hour ; + in 12 hours

\ x iu 1 hour and in 12 hours.

}x i i l l hour ; +inl2honrs

\ +in 1 liour ; * in 12 j honrs.

\ + in 15 niiiintes ; + in 14 j hour.

1- x in 16 hour.

10 in 14 hour.

+ in 2 hours. Oin 2 hours.

1 + in 1 hoar.

x in 1 hour. 0 in 14 Iiour.

0 in 2 hours.

*=complete agglutination ; + =distinct agglutination ; x =slight agglutination ; 0 =no agglutination.

EXPERIMENTAL OESERVATIONS.~ We do not wish at the present time to detail fully the results of

our experimental observations, and shall only give the general results and conclusions.

Cultures which had been maintained upon the " serum " agars were rubbed up in 0.85 per cent. saline and inoculated both intraperitoneally

lThese observations were carried out in the Research Laboratory of the Royal College o f Physicians, Edinburgh.

EPIDEMIC CERBB R 0-SPINA L MENINGITlS. 481

death resulted rapidlyfrom intra- peritoneal inoculation usually within twenty-four hours. The result mas thus a little variable. The orgnnisiii was recovered after death i n most of tho cases.

Differential counts of the cells present in the peii- toneal exudate after inocula- tion gave some interesting results, which, however, were not altogether satisfactory unless taken as additional proof of the low virulence of the nieningococcus. Control animals were inoculated with a corresponding arliount of 0.85 saline, and their peri- toneal cells were exaniined concurrently. In all cases the cells present in the peritonea’ fluid at the

and subcutaneously into a series of mice. I n the few cases in which we inoculated guinea-pigs of 250 to 3 00 grms. weight intraperitoiieally the animals showed so little disturbance that we abandoned their use.

Weichselbaum (1887) had a similar experience, and as a conse- quence stated that meningococci were feebly pathogenetic to guinea-pigs. One of our latest experiences, however, supports the statement of von Lingelsheini and Leuchs (1 9 0 S), and of Kolle and Wassermann (1 9 0 S ) , who found that young guinea-pigs were more susceptible than mice.

Portions of the second subcultures of strain 18 were inoculated into four mice, in two subcutaneously and in the other two intraperitoneally. They were all alive and well a fortnight later. Half of a subculture of the same strain, on,pnrovarian fluid agar of a similar agd, was inoculated intraperitoneally i n a guinea-pig weighing 117 grms. It was fount1 dead fifteen hours later, and the nieningococcus was recovered in pure culture from the scanty peritoneal exudate.

The amount inoculated into mice varied from one-tenth to one-half of a 24-hours’ culture upon serum agar. A11 the strains which were maintained were inoculated with the exception of Nos. 6 and 8. I n four cases death followed suhcutaneous inoculation, but in all cases except 2, 13, 14, and 16

CHAW 2.-Mouse inoculated intraperitoneally with

482 THEODORE SHEA'NAN AND W. T. RITCHIE.

On examination every hour thereafter the proportion of mono- nuclears uniformly decreased in direct proportion to the increase of

polyniorphs. For example, in one case the small niono- nuclears decreased in two hours from 50 per cent. t o 41 per cent. of the nucleated cells present, whilst in the same time the polymorphs increased from 27 per cent. to 43 per cent. In another case the niononuclears de- creased in an hour ancl a half from practically 1 0 0 per cent. to 68 per cent., while the polymorphs rose during the same period to 18 per cent. This went on until five and a half hours after inoculation the polyinorphs numberecl 8 2 per cent. and the mononuclears only 18 per cent. (Chart 2). This

nuclears was apparently not solely a relative one. I n the control animals, however,

76 instead of 66 as shown. practically identical resulis were obtained. In these

animads, in which a t the coinmencement of the experiment only mononuclear cells were present, in five and a half hours the polyniorphs represented 7 6 per cent. of the nucleated cells, and mononuclears 24 per cent. (Chart 3).

The only difference was that in the control animals, on the day following the date of inoculation, the polymorphs had greatly decreased, and in thirty-six hours had alinost altogether disappeared ; whereas in those inoculated with meningococci the reaction continued till death, or if the animal displayed more resistance, numerous proliferated endothelial cells, frequently in clusters, appeared on the second or third day.

The results obtained on inoculating saline solution confirm those reported by von Mikulicz-Radecki (1 9 04), who produced a hyper- leucocytosis by injecting physiological saline into the peritoneal cavity.

The films made from peritoneal exudate during life and after death showed usually active phagocytosis, both by polymorplis and by endothelial cells. I n one film the polyinorphs in filins from the heart

cHAI:T 3.-~ontrol mouse inoculated intraperi- dilninution of the nlono- toileally with 0'85 per cent. NaCI.

A, Mononuclear cells. B, Polyniorphs.

I n the last column the polymorphs should number

EPIDEMIC CEREBRO-SPINAL MENINGITIS. 453

blood contained well-defined Qram-negative diplococci, and in another they c,ontained rounded pale spots whose size corresponded to that of swollen degenerated cocci.

CONCLUSIONS.

1. Twenty-eight specimens of cerebro-spinal fluid were examined from cases of epidemic cerebro-spinal meningitis. The duration of the illness varied from two to ninety-three days. The age of the patients varied from 1 3 months to 5 2 years, and in nineteen cases was under 20 years.

2. In every instance typical Gram-negative diplococci were detected in films made either froin the spinal fluid during life or directly from the meningeal exudate, post-mortem.

3. In twenty-four specimens of cerebro-spinal fluid (from twenty- three cases), Micrococcus meningitidis cerebrospinalis was isolated (8 5.7 per cent.). I n one case we isolated two identical strains: one during life, the other thirteen weeks later a t the post-mortem examination.

4. On only three occasions was the meningococcus accompanied by other bacteria, these being in two instances (Cases 5 and 1 9 ) Staphylococcus albus, and on the remaining occasion (Case 24) a bacillus.

5. Our strains were readily isolated and maintained upon special serum agar media. The characters of the growth upon these and upon other media, as described in the foregoing pages, prove that the organism we isolated and studied was the Grani-negative Micrococcus ineningitidis cembrospinalis of Weichselbaum, and not Diplococcus crassus.

6. The viability of the micrococcus varies greatly within wide limits. Strains recently isolated may, if maintained upon agar, require to be subcultured every second day ; but in serum media which were preserved at 37" C., and were prevented from drying, strains were found to be viable after seventy-eight, eighty-five, and one hundred days respectively.

7. The micrococcus was found to produce indol after ten days growth in ovarian-fluid broth.

8. All our strains produced acid without gas in glucose and maltose media, but there was a certain degree of variability in the time of appearance and in the intensity of the reaction. I n galactose agar media, no matter how faint its alkalinity, no strain gave an acid reaction even after four weeks' incubation.

9. The agglutinative power of one of the commercial anti- meningococcal sera, and also of the sera of two patients suffering from the disease, was tested upon several of our strains of nieningococci, and it was found to present a considerable degree of variability-some strains reacting, whereas others failed to do so.

10. The pathogenicity of our strains was tested experimentally. The conclusion is drawn that the intraperitoneal inoculation of mice and of young guinea-pigs is usually attended by a fatal issue.

484 THEODORE SHENNAN AND ?% T. RITCNIE.

ALBRECHTUND GHON . .

BETTEXCOURT ET F R A N ~ . Boxom . . . . . .

BORDONI-UFFREDUZZI . .

CASTELLANI . . . . . CERADINI . . . . . .

COCHEZ ET LEMAIRE. . . COUNCILMAN, MALLORT,

EBERTII . . . . . . -4ND \VRIGHT

ELDER A S D IE\’ERS . .

FABER . . . . . . . FRAENKEL . . . . . .

F o i . . . , . . . .

FLEXNER . . . .

FLUGGE . . . . . . . FRASER AND COIIRIE . .

,GHON . . . . . . . GHOX, PFEIFFER, UND

,GOODWIN AND VON SHOLLI’ SEDERL

GOLDSCIIMIDT . . . . .

GORDON . . . . . .

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EPIDEMIC CERRBR 0-SPINAL MENINGITIS. 485

HEUBNER . . . .

HOUSTON AND RANKIN . . HUNTER AND NUTHALL . . JAEGER . . . . . . .

JEHLE . . . , . . .

JOCHMAKN. . . . . . K AJIEN . . . . . . .

KIEFER . . . . . . . KISCHENSKY . . . . . KISTER . . . . . . . KLEBS . . . . . KOB . . . . . . . KOLLE UND WASSERMAXK KUTSCHER . . . . .

LAZARUS-CARLOW . . . LEICHTENSTERN . . . . LEPIERRE . . . . . .

VON LEYDEN . . . . .

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486 EPIDEMIC CEREBRO-SPINAL MRNIA’GITIS.

RAUTENBERG . . . . .

RUNDLE, MOTTRAM, AND

SCHOTTMULLER . . . .

SENGER . . . . . . .

WILLIAMS

STILL . . . . . . . SYMMERS AND WILSOS . URBAN . . . . . . VANSTEENBERGHE ET GRY-

VANZECTI . . . . . . WEICHSELBAUM . . . .

SEZ

WEYL . . . . .

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69. Gior. d. r. Accad. di naed. d i Torino, 1901,

anno lxiv. 11. 653. Metl. Jahrb. d. 76. k. Gsterr. Staates., Wien, 1886,

N.F. Ed. i. S. 483 ; Fortsclzr. d. Med., Berlin, 1887, Ud. v. SS. 573, 620; Wien. Idin. Wchnsclir., 1888, Ed. i. SS. 573, 595, 620, 659 ; Centralbl. .f. Bakteriol. u. Parasitenk., Jena, 1903, Abth. 1, Orig., Bd. xxxiii. S. 510; and in “Handbuch d. path. Mikroorgan- ismen ” von Kolle u. Wassermann, Jena, 1903, Bd. iii. S. 356.

Jali rb. f. IGnderh. u. phys. Erziehung, Berlin, 1905, Bd. Ixi. S. 385.

Heft 31, cited by Kutscher.

1617.

1886, Bd. xx. S. 389.

1898, vol. v. p. 147.

DESCRIPTION OF PLATE XLIX.

FIG. 1.-Film preparation made from the lunlbar puncture fluid removed during life from Case 4. The characteristic micro- scopic appearance of the purulent exudate is seen. The cells are chiefly polymorphonuclear neutrophiles, and the cocci, for the most part in pairs, are contained within the cytoplasm of these cells. A portion of a large mono- nuclear cell is seen at one part of the margin of the field. ( x 900.)

FIG. 2. -Microscopic appearance of a culture of Micrococcus meni?qilidis cerebro-spinalis, incubated a t 37” C., and examined on the third day. In addition to single and paired elements, tetrads are present, both of normal size and swollen. Some swollen pairs are also seen. Degenerated cocci are all faintly stained and small in size or, as seen particularly near the top and bottom of the field, show the ring form.

FIG. 3.-Photograph of colonies of Afierococcus mizingitidis cerebro-spinalis, after twenty- four t o thirty hours’ incubation a t 37” C. In some of the colonies the very earliest stages of development were shown. ( x 20.)

FIG. 4.-Similar colonies after thirty-six to forty-eight hours’ incubation. They show a finely granular centre, with a very few coarser granules, and the thin, extended, homogeneous periphery described in the text. ( x 20.)

FIG. 5.-Colonies after incubation for forty-eight to seventy-two hours. The central parts show more coarse granules, and the granularity spreads outwards. The clear margins of the colonies are slightly thickened.

Here the general description corresponds with that given of the last figure, but in addition the thickening of the margins is more marked, and little knob-like elevations are begirlni~~g to appear in them.

It was stained by Leishman’s method.

( x 900.)

( x 20.) FIG. B.-Colonies after about seventy-two hours’ incubation.

The outlines are slightly sinuous. ( x 20.)

JOURNAL OF PATIIOLOGY.-VOL. XII. PLATE XLIX.