411 IDENTIFICATION OF THE MENINGOCOCCUS. · to 65° C. to kill the coccus and to inactivate its...

411

IDENTIFICATION OF THE MENINGOCOCCUS. By MAJOR M. H. GORDON,

Royal Army Medical Corps. AND

MR. E. G. MURRAY.

IN a previous paper, published in this Journal for May, 1915, the result was described of some preliminary experiments made for the purpose of ascertaining the extent to which the absorption of agglutinin test can be applied practically for the purpose of identifying the micro-organism of the present outbreak of cerebrospinal fever. Anti-meningococcus sera on the market having proved unsatisfactory for this purpose, a suspension of a culture· of meningococcus, recently isolated from the cerebrospinal fluid of an acute case, was injected intravenously into a young rabbit,. and a specific agglutinating serum thus prepared. Having obtained in this way a serum giving good macroscopic agglutination, meningococci isolated from the cerebrospinal fluid of three further cases of meningitis were then tested against this serum and found to agglutinate practically as well as the homologous strain. On making absorption tests, it was found that three of these meningococci absorbed the homologous agglutinin; the fourth failed to do so in the experiment described, but as this coccus failed at the, same time to absorb the agglutinin for itself the result was inconclusive. It may be added that this coccus has since been found toabsorb the homologous agglutinin in question.

Application was now made of this serum for the purpose of determining the behaviour towards it of three Gram-negative cocci isolated from the nasopharynx of contacts, and of cases of doubtfull illness suspected of being anomalous forms of cerebrospinal fever. The cocci in question were indistinguishable from the meningococcus in the morphological, staining, cultural, and fermentative properties examined. One of them altogether failed to agglutinatewith the serum. The other two agglutinated to a certain extent with it, but did not absorb the specific meningococcus agglutinin. N one of these three nasopharyngeal cocci, therefore, could be identified with the meningococcus against which the serum had been prepared.

The inference drawn from these preliminary observations was· that the absorption method promised to have a distinct value in certain cases for the purpose of differentiating the meningococcus. from micrococci closely resembling it in cultural characters, but.

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412 Identification of the Meningococcus

devoid of the same pathogenic significance. It was considered, however, that as there are possibly, and even probably, several distinct races of meningococcus concerned in the present outbreak, the value and limits of the method in this connection could not be stated until a far larger number of meningococci from the cerebrospinal fluid of cases had been collected and examined in the same way.

During the interval that has elapsed since the preliminary experiments referred to, it has been our good fortune to be able to obtain a considerable number of cultures of meningococci all isolated in the first place from the cerebrospinal fluid of cases during the present outbreak. We are greatly indebted to Dr. Arkwright, of the Lister Institute; Captain Gaskell, of the 1st Eastern General Hospital, Cambridge; Dr. O'Brien, of Brockwell Hall; Lieutenant Macmahon, of York; Lieutenant Compton, of Weymouth; Dr. Barrington White, of Nottingham; Dr. Claridge, of Norwich; and others who have placed cultures at our disposal for this purpose. As a result, we are now able to report on thirty-two specimens of meningococcus, all of which came in the first instance from the cerebrospinal fluid of cases during the recent {)Utbreak. In addition, we have been able to examine in the same way nine Gram-negative cocci, indistinguishable from the meningo,coccus in cultural and fermentative characters, isolated from the nasopharynx either of contacts or of cases of illness suspected to be instances of cerebrospinal fever, in which symptoms of meningitis were absent or suppressed.

METHOD.

Before proceeding to state the result of this further investigation, we will describe as briefly as possible the method which we have been using. In order that our results should be comparable, and in order that they should be easily checked by others, we have tried to make our tests on as simple and standard a basis as possible. Our procedure has been as follows:-

(1) Preparation oj a Suspension oj the Ooccus.-In the first place the Gram-negative coccus is plated out on a series of legumin-agar plates and a subculture made from a single colony ·on to a slope. On the following day this slope is subcultured into .a tube of trypsin broth (Douglas) neutral to phenolphthalein, to which about one-tenth of its volume of sterile serum or ascitic fluid has been added. This ascites broth culture has the great .advantage that the meningococcus will live in it for three weeks .or more.


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M. H. Gordon and E. G. Murray 413

From the legumin-agar slope culture we now proceed to inoculate six or eight Petri dishes of the same medium. These plates are spread in the usual way with a platinum or iron wire, or with a piece of capillary glass tubing bent to a right angle. After twenty-four hours' incubation these plates show, as a rule, a profuse confluent growth of the coccus. Five cubic centimetres of sterile saline is now poured into each plate, and the growth raked off the agar and distributed in the saline by means of a bent wire. If the growth sticks to the agar, it is advisable to rub it off with a small swab. The suspension is now poured off into test-tubes, a separate tube being used for each plate. Film preparations are then made from the contents of each tube, stained with Gram, and examined microscopically. Any tubes showing contaminations are at once rejected, the rest are mixed together and heated for half an hour to 65° C. to kill the coccus and to inactivate its autolysin, as recommended by Raymond Koch, applying Flexner's work on meningococcus autolysin. Heating in this way also appears to help the coccus to emulsify.

(2) Standardization of the Suspension.-The heated suspension of the coccus is standardized in the following way :-

By means of a pipette holding 0'1 C.c., this amount of the suspension is transferred to a special glass test-tube kept for the purpose. Then with a 5 c.c. pipette graduated in tenths of a cubic centimetre, tap-water is run in until the suspension is so dilute that it is only just turbid to the naked eye when compared with a control tube of tap-water per se. This end-point is taken to represent a content of 100 million per cubic centimetre. The volume of the suspension is then measured and a simple calculation gives the total number of cocci contained by it. For example, 0'1 C.c. of the suspension requires to be diluted with 5 c.c. of water to reach the end-point. It contained, therefore, 500 million cocci in 0'1 c.c., or 5,000 million per cubic centimetre.

Its strength having been in this way determined, the suspension is diluted with saline so as to make its value approximately 2,000 million per cubic centimetre, and 0'5 per cent of phenol is added. This standard phenolated suspension of the coccus is kept in a glass-stoppered bottle and placed in the cold storage. It is used for all agglutination and absorption tests, and also for injecting rabbits.

(3) Preparation of Agglutinating Serum.-We can endorse the statement of Elser and Huntoon that young rabbits are to be preferred for this purpose. Our best sera have been obtained from


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rabbits weighing between 1,000 and 1,500 grammes. The material with which they are injected is the standard suspension already described, and all of our injections have been intravenous. As we could not wait for a month or longer for an agglutinating serum, we had to use an intensive method of some kind. We began by using Fornet and Miiller's method of giving increasing doses at twenty-four hours intervals for three days in succession. We have also used the following method which occasionally gives a serum with a titre as high even as 1 in 800 within ten days. The rabbit is given t C.c. of the suspension (1,000 million cocci) and then fortyeight hours later it receives three doses of 1,000 million at hourly intervals. This method, which we call our "saturation" method, was suggested by some experiments made by one of us in conjunction with T. J. Horder some years ago. We may add that the relative merits of various modes of dosage for the purpose of producing an agglutinating serum in the shortest time is under investigation by Captain Hine.

(4) Absorption Tests. - It is very necessary in making an investigation of this kind to do all the tests in exactly the same way, and we had variable results until we standardized our mode of doing absorption tests. At first we added a number of loopfuls of living growth to a known dilution of serum, placed the tube at 37° C. or at 55° C., and centrifuged out the cocci after two or three hours. The results were uneven, and we then made the following comparative experiment. Five cocci were tested against the same serum; in the first series the serum was diluted directly with the standard suspension; in the second, the serum was diluted to the same extent but half with saline, and then half with suspension. The tubes were allowed to stand overnight at 37° C., then centrifuged next morning and titrated against the coccus of test and the coccus homologous to the serum respectively, a control being done at the same time against the unsaturated serum. The result of this experiment was to show that the latter method, i.e., dilution half with saline and half with suspension, gave the best results; and accordingly we adopted it for routine use.

Our procedure in carrying out the absorption test is briefly as follows:- .

Say that the serum has a titre of 1 in 800 against the standard emulsion of the homologous coccus in twenty-four hours at 55° C. We propose to test it in dilutions of 1 in 100, 1 in 200, 1 in 400, and 1 in 800, before and after saturation with a given coccus in order to see if this coccus does or does not absorb the homologous


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M. H. Gordon and E. G. MU1'ray 415

agglutinin. We make first of all a sufficient quantity of a 1 in 50 dilution of the serum in saline and put this into a test-tube in the rack used for absorptions. This is our specimen of the serum before saturation. We next put out a sterile centrifuge tube for each coccus that we propose to test, including one for the homologous coccus (control positive), and another for a heterologous coccus (control negative).

Into each of these tubes we put 2'5 C.c. of a 1 in 25 dilution of the serum of test. Next we add an equal amount of the standard suspension of each coccus in turn. We then place these tubes in the incubator at 37° C. overnight. Next morning we note those that have agglutinated and centrifuge all of the tubes for half an hour. 'rhe serum before saturation is then titrated against the coccus of test, and the serum after saturation is titrated against the coccus homologous to the serum and also against the test coccus. In addition, a control tube is put up of each coccus against normal rabbits' serum. Should the result of an absorption test made in this way be at all doubtful, we then saturate the same serum over again and proceed as before. The first saturation sometimes clears off " agglutinoids " very nicely from serum.

Up to the present we have done all our agglutinations at 55° C. and have read them off after twenty-four hours exposure to this temperature. We use small test-tubes three inches long by half an inch wide plugged with cotton-wool. The tubes are arranged in small racks designed for the purpose by Captain Hine. These racks hold twelve tubes abreast and have strips of xylonite nailed in front of each row for the purpose of labelling. Having arranged the tubes in the rack for the experiment, we first put t C.c. of the required dilution of serum in each of the tubes, and then add to the tubes of each group t C.c. of the standard suspension of the necessary coccus. In adding the suspension, it is advisable to add first of all the suspension of the coccus homologous to the serum of test to the batch of tubes devoted to it, and then to replace the plugs, or to cover these tubes over. In this way the possibility of adding suspension to the wrong tubes is avoided. A pipette graduated in ! c.c.'s and worked by a rubber teat is useful. When judging the result we find it advisable to use in some cases a small hand-lens giving about eight diameters. If there is any doubt about the agglutination, we give the tube a shake and look for flocculi. If these are absent we enter the result as negative.

The arrangement of the tubes is seen from the following experiment ;-


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No.

--I 2 3 4

416 Identifioation of the Meningooooous

SERUM JONBS (RABBIT 19)

Normal rabbit Before saturation After saturation serum (con-trot)

v. Test coccus v. Jones coccus v. Test coccus -- --

Coccus 1: 50 1: 100 1: 200 1: 300 1: 400 1: 100 1: 200 1 : 300 1: 40C 1: 100 1: 200

-----. ---------- ------------Jones .. - + + + + - - - - - -Smith .. - - - - - + + + + - -Robinson - + (+) - - + + + + - -Jenkins .. - + + - - - - - - - -

I + = agglutination well marked; (+) = slight agglutination.

. In this experiment, J ones, the control homologous coccus, has by saturation absorbed its own agglutinin in all of the four dilutions. Smith, the control heterologous coccus, neither agglutinates with J ones serum nor absorbs the J ones agglutinin. Robinson agglutinates somewhat, but does not absorb. Jenkins agglutinates to the same degree as Robinson, and absorbs Jones agglutinin. Clearly, then, of the above cocci J ones and J enkins alone combine with the specific agglutinin in this serum and remove it, and the Robinson coccus was only affected by a group agglutinin.

RESULTS.

(1) Meningococci isolated from the Cerebrospinal Fluid of Cases during the 'recent Outbreak.-Specimens of these cocci from thirtytwo different cases have been examined up to date. So far as morphological, staining, and cultural characters go, they all appear to be undoubted meningococci. All except three failed to grow on legumin agar at 23° C., and all except two fermented glucose in three to four days at 37° C. The two cocci negative to glucose were in all other respects typical meningococci, and one of them has been found on repeated trials fitfully to produce some feeble fermentation of glucose. None of these thirty-two meningococci fermented saccharose.

In addition, a culture of the parameningococcus of Dopter, kindly sent to us by Dr. Louis Martin, of the Pasteur Institute, Paris, has been investigated in the same way. Like the majority of the above cocci this parameningococcus failed to grow on leguminagar at 23° C., and fermented glucose, but left saccharose unchanged.


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Procedure has been as follows: In the first place a suspension was prepared of each coccus and this was standardized and phenolated in the manner described. Then one of these suspensions was injected intravenously into a young rabbit and an agglutinating serum prepared. Each of the meningococci was now titrated against this serum, and its capacity of comhining with the homologous agglutinin determined.

TABLE I.-CEREBROSPINAL FLUID MENINGOCOCCI V. SERUM OF RABBIT PREPARED AGAINST MENINGOCOCCUS No. 1.

SERUM M.I.

Before saturation After saturation

v. Test coccus v. M. 1 coccus v. Test coccus

__ Dilution ~_I~ ~l~ 100 200 400 800 IOO 200

Meningococcus 1 + + + I + 2 + + + I (+) 3 + + + -4 + + +' (+) 5 + + + + 6 + + + -7 + + + -8 + + + -9 + + + (+)

10 + + (+) -11 + + (+) -12 + + I - -13 + + - -14 + + I + + 15 + + + + 16 + + + (+) 17 + + + I + 18 + + + I (+) 19 + + + -20- -,--

~~ = = I = = ~~ +. (+) I = =: ~~ (+) =: I=:=: 27- - --28- - --~ + - - -00 - - - -31- - --32- - --

Parameningo- - - - -coccus

+ (+)

+ +

(+) +

(+)

+ + + + + + + + + + + + + +

+ + + + + + + + + + + + + +

+ + + + + + + + + +

(+) + + +

+ + + +

(+) + +

+ +

+ -+ -

(+) I _ + I _

+ I -+ I -

The results are seen in Tahle I. The readings recorded in this table show that the serum in question divided the meningococci


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into two distinct groups. The first nineteen specimens both agglutinated with the serum and absorbed the specific agglutinin. Tbe last fourteen cocci, on the other hand (specimens 20 to 33), for the most part failed to agglutinate with the serum, and they all failed to absorb the specific agglutinin.

TABLE lI.-CEREBROSPINAL FLUID MENINGOCOCCI V. SERUM PREPARED AGAINST MENINGOCOCCUS No. 20.

SERUM M. 20

Before saturation I After saturation

". Test coccus ". M. 20 coccus '11. Test coccus

Dilution 100 200 300 400

_ 1:0_1_~_ 300 400 100 200

MeningococcuS 1 - + (+) 2 + (+) - - + + + (+) - .. 3 + - - - + + + (+) - .. 4 (+) - - - + + + (+) - .. 5 + - - - + + + (+) (+) -6 + - - - + + + (+) (+) -7 - - - - + + + (+) (+) -s - - - - + + + ~+) - .. 9 - - - - + + + +) - ..

10 - - - - + + + 1+) - .. 11 (+) - - - + + + +) - .. 12 + - - - + + + (+) + -13 + + - - + + + - - .. 14 (+) - - - + + + (+) (+) .. 15 (+) - - - + + + (+) - .. 16 (+) - - - + + + - - .. 17 - - - - + + + (+) - .. 18 - - - - + + + <+) - .. 19 - - - - + + + (+) - .. 20 + + + + + - - - + -21 + + - - + - - - + -22 +

I + + + + - - - + -23 + + + + + - - - + -24 + + + - + - - - + -25 + + + (+) + - - - + -26 + + + - + I - - - + -27 + (+) - - (+)

I

- - - - -28 (+) - - - + + + - - .. 29 - - - - + + + - - .. 30 -

I

- - - + + + - - .. 31 (+) - - - +

I + + (+) - ..

32 - - - - + + + - - .. Parameningo- + I - - - + + + - - ..

coccus

The next step was to prepare a rabbit against the first of these fourteen heterologous cocci and to test the whole of the cocci in the same way with this serum. The result is shown in Table H.


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It is seen that the first nineteen meningococci showed a comparatively minor degree of agglutination with this serum, and that they all failed to combine with the agglutinin of the meningococcus (N o. 20) against which the rabbit had been prepared.

TABLE IlL-CEREBROSPINAL FLUID MENINGOCOCCI V. SERUM PREPARED AGAINST

MENINGOCOCCUS No. 28.

SERUM ll. 28

Before saturation I After saturation


Dilutiou 100 200 300 400 100 200 300 : I

400 100 200

-------------------Meningococcus 1 + + + - + + (+) ,

- - .. 2 + + + (+) + + (+) - - --3 + - - - + + - - - .. 4 + + + + + + (+) - (+) -

5 + + (+) - + + (+) I - - .. I

6 + + + - + + (+) I - - .. 7 + + + - -+- + (+) I - - .. 8 + + + - + - - - (+) -9 + + + - + + + - - ..

10 + + + (+) + + + - - .. 11 + + + (+) + + (+) - - .. 12 + + + - + + + - - .. 13 + - - - + + (+) - - .. 14 + + - - + + + - - .. 15 + + (+) - + + + (+) (+) -16 + + (+) - + + (+) - - .. 17 + + - - + + + - - .. 18 - - - - + + + (+) - .. 19 (+) - - - + + (+) - - .. 20 - - - - + + + !+) - .. 21 - - - - + + + +) - I .. 22 (+) - - - + + + - - I .. 23 - - - - + + + (+) - .. 24 - - - - + + + - - .. 25 (+) - - - + + + - - .. 26 - - - - + + + ~+) - .. 27 - - - - + + + +) - .. 28 + + + (+) - - - - - .. 29 + + + + - - - - - .. 30 + + + + - - - - - .. 31 (+) - - - (+) - - - - --

. 32 - - - - + + + (+) I - .. Parameningo- I

I coccus + - - - + + + - - ..

On the other hand, no fewer than eight of the fourteen meningococci negative to the first serum both agglutinated and absorbed with the present serum.


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The next step was to prepare another rabbit against the first of the remaining six cocci still unaccounted for, and to examine the whole of the meningococci in the same way against this serum. The results are shown in Table Ill. The readings given with this third serum are distinctly interesting. In the first place, no fewer than four of the six meningococci not yet accounted for combined with the specific agglutinin of this third serum. Moreover, one of these cocci absorbed the agglutinin although it agglutinated with the serum to a comparatively minor degree.

The second point of interest with regard to this serum is that several of the cocci of class 1 agglutinated quite well with it, and one of them (No. 8) that had absorbed the specific agglutinin of No. 1 serum absorbed also a certain amount of the specific agglutinin of the present serum.

These three sera, therefore, accounted for no fewer than thirtyone of the thirty-two meningococci under investigation. The last meningococcus, viz., No. 32, is so far unique. It came from the cerebrospinal fluid of a very protracted case of meningitis at York. A rabbit has been prepared against this coccus, and specimens of all the other groups put up against its serum, but they neither agglutinate with the serum nor do they combine with the agglutinin of coccus No. 32.

Coccus No. 33, the parameningococcus of Dopter, which was also examined in these tests, is apparently distinct from all the four groups into which these thirty-two specimens of meningococci from the cerebrospinal fluid of cases in the present outbreak are resolved by the absorption of the agglutinin test.

The investigation is being continued, and meningococci are being examined in the same way as they come to hand.

(2) Meningococcus-like Cocci from the Nasopharynx.-The immediate object for which this investigation in the first instance was undertaken was to obtain a specific serum whereby the microorganism of the present outbreak could be more readily and accurately identified in cultures from the nasopharynx.

When these sera were being prepared we had in stock nine nasopharyngeal cocci, all of which were practically indistinguishable from the meningococcus in morphological, cultural staining and fermentative characters. Seven of these cocci had been isolated from contacts, and two from cases of illness suspected of being instances of cerebrospinal fever without meningitis.

These nasopharyngeal cocci have been tested with the sera prepared against the four types of meningococcus referred to above.


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M. H. Gm'don and E. G. Murray 421

and also with a serum prepared in the same way against the paramenmgococcus.

As a result we have found that five of them agglutinate with the serum prepared against type 2 meningococcus, and also combine with the specific agglutinin of that strain. The reverse experiment was then maae, and a rabbit prepared against one of these nasopharyngeal cocci (D. 24), that had been identified with the second type of meningococcus. All of the thirty-three cerebrospinal fluid cocci were then put up against this (D. 24) serum, with the result that the same eight meningococci were selected by it as had been picked out by the original type 2 meningococcus serum. This result seems to us to establish the identity of these five nasopharyngeal cocci with the second strain of meningococcus beyond all reasonable doubt.

Only four of these five cocci thus identified with the meningococcus came from contacts. The fifth came from the nasopharynx of a case of pyrexia of unknown origin invaliaed home from the Front for fever. No meningeal symptoms occurred in this case. We have to thank Dr. Frank Taylor, Pathologist to the Queen Alexandra Military Hospital, for kindly giving us this culture.

It was puzzling to us to find that we had no specimen of type 1 meningococcus among our nasopharyngeal cocci. On reflection, however, it occurred to us that these nasopharyngeal cocci which we had in culture had all been isolated at a more recent stage of the outbreak than the cerebrospinal fluid cocci which we had been testing, and therefore we thought that possibly had we still possessed nasopharyngeal cultures from contacts in an earlier stage of the outbreak we might have found among them type 1 of the meningococcus. Confirmation of this view has since been afforded in a remarkable way, for having recently been asked to examine a carrier who had been in isolation for over six months, and had last been in contact with a case of cerebrospinal fever in the earlier stage of the outbreak, we obtained from his nasopharynx a Gramnegative coccus indistinguishable in cultural and fermentative characters from the meningococcus. On trial against our five sera this coccus was found to agglutinate and absorb with one only of them, namely, with the serum which had been prepared against type 1 of meningococcus.

The results given by these ten nasopharyngeal cocci with sera prepared against types 1 and 2 meningococci respectively are shown in Tables IV and V.


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TABLE IV.-NASOPHARYNGEAL COCCI V. SERUM PREPARED AGAINST MENINGOCOCCUS No. 1.

SERUM M. 1



- --600 800 200 400 I 600 800 200 400

------.--~I~ ----I 2 3 4 5 6 7 8 9

10 11 12

I 2 3 4 5 6 7 8 9

10 11 12

M.1 " + + + + - - - - - - Control positive. M.20 .. - - - - + + + + - - Control negative. D.24 " - - - - + + + + - -D.32 .. - - - - + + + + - -D.35 .. + (+) - - + + + I + - -D.40 .. - - - - + + + ! + - -H.6 .. - - - - + + + + - -B.12 .. - - - - + + + I + - -I s.

"1 - - - - + + + + - -A. "I + + - - + +

I + + - -

B.1 .. + + - - + + + + - -B.2 +

I + + - - - - - - -

TABLE V.-NASOPHARYNGEAL COCCI v. SERUM PREPARED AGAINST MENINGOCOCCUS No. 20.

SERUM M. 20


v. Test coccns v. M. 20 coccus 1J. Test coccus

100 200 I 300 400 100 200 300 400 100 200

--- -- -- ---- - --------M.1 .. - - - - + + + + - - Control negative. M.20 .. + + + + + - - - - - Control positive. D.24 .. + + + (+) - - - - - -D.32 .. -+ + + + + - - - - -D.35 .. + + + + (+) - - - - -D.40 .. - - - - + + + (+) - -H.6 .. + + (+) - - - - - - -B.12 .. - - - - + + + (+) - -S. .. + + + - - - - .- - -A. .. - - - - + + + + - -B.1 .. - - -

I - + + + + - -

B.2 .. - - - - + + + + - -


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SUMMARY.

(1) Specimens of meningococcus isolated from the cerebrospinal fluid of thirty-two cases during the present outbreak were resolved by the absorption of agglutinin test into four groups. Of the meningococci in question 19 belong to Group I; 8 to Group II; 4 to Group III; and 1 to Group IV. So far, only one specimen of Group IV has been obtained.

(2) One specimen appeared to absorb the specific agglutinin of two groups, i.e., to be amphoteric.

(3) All of these thirty-two meningococci are distinct from the parameningococcus of Dopter.

(4) Of nine specimens of Gram-negative cocci isolated from the nasopharynx of contacts and suspected cases, and closely resembling the meningococcus in morphological, cultural, staining, and fermentative characters, five have been found by the absorption of agglutinin test to be identical with Group II of the cerebrospinal fluid meningococci. A further specimen recently isolated from the nasopharynx of a chronic carrier has been identified in the same way with Group I of these same meningococci obtained from thecerebrospinaifluid of cases in the present outbreak.

31


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411 IDENTIFICATION OF THE MENINGOCOCCUS. · to 65° C. to kill the coccus and to inactivate its...

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