The Journal of Pathology and Bacteriology

Vol, XXV., No. I

ON n. wmcmr HBMO‘I‘OXIN AND ITS NEUTRALISATION WITH ANTITOXIN.*’

By HERBEBT HICNIW, M.D.

A R e p o r t t o the Xeetlical R e s e a r c h Cozcncil.

I. INTRODUCTIOS.

THE information that is available in regard to the capacity of B. welchii to effect intrcr. vitam h~en~olysis is partly clinical and partly experimental.

The severe anaxilia. which charactcrises certain cases of infection in man has been noted by a considerable number of investigators. For example, Klotz and Holman ( “ I ) found i t to be an outstanding feature in a series of gas bacillus infections occurring in coal mines, while Schumm (13) reported that the serum from an infected case gave the spectroscopic test for hmnoglobin.

I n wounded soldiers suffering from gas gangrene during the recent war a familiar feature of the disease proved to be the occurrence of a rapidly pro- gressing anamia. Blood examinations made a few hours before death invariably showed a serious depletion in the number of red cells, counts of less than one rnillion per cubic iiiillirnetrc being not infrequent in rapidly developing cases. In certain instances the anamia was accompanied by clinical evidence of blood destruction in thc form of R lmniolytic icterus, which might show itself as a slight yellow staining of the conjnnctiva, or might, on occasion, be represented as a germ-alised jaundice.

Further, Elliott and Henry ( 7 ) in their investigations on wounds of the chest, found that the early clevelopnicnt of hmnolysis in a h m o t h o r a x fluid provided diagnostic evidciice of infection with nnacrobes. H ~ m o l y s i s could he easily demonstrated by simply centrifuging a specimcn of puncture fluid, and not infrequently the finding of lmmolysis proved to be of particular value in that it antedated the microscopic and cultural demonstration of ana6robic organisms in the fluid.

The hzniolysis that results in aniinals which have been experimentally infected with 3. w e l c h i i has attracted the attention of numerous laboratory workers, and its exact significance has been very variously interpreted. Herter (!), Sinionds (Ii) and others were able to find a hsmolysing substance in culture filtrates. Of this substance Herter states that “treatment of the

* Received July 9, 1921. JOURN. OF PATH.-VOL. SXV. A

2 HERBERT HEiVZi Y

filtrate in an exhaustion apparatus very slightly reduced the hzemolytic action, hcating to 70" for one hour reduccd it still further ; but even boiling did not wholly destroy it." IlcCampbell (I]), on the other hand, was of opinion that the hsmolysis following the inoculation of glucose broth cultures into aniinals could be attributed to the presence of butyric acid. He found that lesions identical with those resulting from the inoculation of filtrates could be produced by butyric acid alone, and that the neutralisation of filtrates rendered them inactive.

With the appearance of Bull's work ('3 2, '3 5* "), the naturc of the hzemolysing substance could no longer rcnimn in dispute. This observer showed very clearly that filtrates prepared under certain conditions contained a true toxin which could be destroyed by heating, and which had well-defined antigenic properties, H e demonstrated also that thcrc arc: two outstanding lesions which can be obtained regularly in animals killed by this toxin, viz., hzemolysis and muscle necrosis, and, as he found that both these pathogenic effects could be inhibited by appropriate amounts of antitoxin, so he concluded that the toxin was both hsmolytic and muscle necrosing.

The following Report deals with an in vitro investigation of the hamoly sing substance present in H. .u;okhii toxin, the method employed being a modification of that originally devised by Madsen (I2) for the study of tetanolysin. Throughout the series o€ experiments herein described the hmnotoxic unitage has been standardised when iiecessary by titration against freshly prepared 1 per cent. solutions of the same specimen of a precipitatetl toxin, prepared accorcling to the method aiready outlined by us (".

Stewart and V e s t (I5) came to the same conclusion.

11. THE MEASUREMEKT OF THE HBMOLYTIC TITltE OF B. ~ E L C H I I TOXIN.

The hmiolytic titre of 6: welchii toxin has been tested on the washed red cells of a variety of animals, viz., man, horse, sheep, guinea- pig, rabbit, mouse, pigeon and fowl. The red corpuscles of mouse, pigeon and fowl would seem to be extremely susceptible; those of man, guinea-pig, rabbit and sheep are less so, while the specimens of horse corpuscles examined have provod to be comparatively resistant.

The titration tests herein recorded have been carried out with a well-washed and freshly prepared 6 per cent. saline suspension of red cells obtaincd from full-grown healthy rabbits. The toxin to be investigated is put up in gradually diminishing amounts (1.0, 0'8, 0'7, 0'6, 0'5, 0'4, 0'35, 0'3, 0'25, 0'2, 0.17, 0'13 c.c.) of a series of dilutions (1 : 1, 1 : 10, 1 : 100, 1 : 1000).

The volume of Huid in edcli tube is madc up to 1'5 C.C. with saline, and there is then added 1.0 C.C. of the corpuscIe suspension. The tubes, each now contain- ing 2'5 c.c., are immersed in a water-bath a t 38" C., the individual tubes being shaken vigorously a t intervals of twenty to thirty minutes. A t the end of two hours the tubes are rcinoved from the water-bath, shaken up for the last time, and then placed in the cold.

The degree of hzemolysis is estimated colorimetrically by using a hemoglobin scale prepared as follows. The same specimen of washed corpuscles as that used for making up the suspenbion is laked in distilled water in the proportion of six parts corpuscles to ninety-four parts of distilled water. One cubic centimetre of the resulting hzeinoglobin solution added to 1'5 C.C. of saline gives a t int which

Readings are taken a t the end of six hours.

represents 100 per cent. hwrnolysis, and fractions of 1 C.C. made up to 2'5 C.C. with saline represent equivalcnt amounts of hmnolysis. It is thus possible t o p r c ~ ~ e a hzemoglobin scale, which, if set out in tubes of the same diameter as those u,cd for the titration of toxin, can hc used for estimating the degree of htzmolysis produced by toxin iiiider test

I n working with such a scale we linre found it impossible to differentiate with certainty between the higher grades of hmnolysis. There is, however, one point on the scale, viz., 20 per cent. l~x?rnolysis, which with experience can he gauged f,iirly readily. TTe have, tllcrefore, arhitiarily fixcd on the hemolytic unit as being the sn ides t amount of toxiii which will produce a 20 per cent. hseniolysis in 1 C.C. of a 6 pcr cent. rabbit corpuscle suspension, the mixture of toxin and corpuscles being made up to a volume of 2'5 C.C. and incubated a t 38" C. for two hours. For cxaniple, if 1.0 C.C. of a 1 in 1000 dilution of toxin i s capable of producing a 20 per cent. htzmolysis under the conditions set forth, then the pa1 ticulu toxin is taken as containing 1000 hwrnolytic units per cubic centimetre.

I t is apparent that the method described above can be expected to yield measurements of h,-ernolytic titre that are satisfactory only i f coiripsrison be confined to the observations carried out on oiie day and with the same corpuscle preparation. And indeed much iiiforrriation can be obtained in regard t o B. welclbiz hsemotoxin within such a narrowed survey. On the other hand, it is often desirable to obtain resuIts that are comparable from day to day. The difficulty ill establishing a haemolytic unit is coincidont with the difficulty in obtaining a standard 6 per cent. corpuscle suspension. Such a suspension can be measured either by enumerating its cellular content with a hzmocytonieter or by estimating its liaemoglolin coiitent against standard colour tubes. It has been found, however, in testing freshly prepared 1 per cent. solutions of the same dried precipitated toxiii against a series of suspensions obtained from different rabbits, hut containing approximately the same number of cells or the samc amount of hcemoglobiu, that the resulting figures show considerabIc variation. The corpuscles of old large-sized rabbits have proved as a rule to be more resistant than those of younger animals. Also rabbits of the same age, reared uiider identical conditions, may show individual variations in corpuscle susceptibility, though these differences are not so marked as in the case (Jf age. The plan adopted, therefore, after a series of preliminary experinients, was to choose what appeared to be an average 6 per cent. s u s p i s i o n , to determine the t int produced by a 20 per cent. hamolysis of this suspension, and to make up on each day a fresh suspension of washed corpuscles with approximately the same content in hsemoglobin.

Twelve corpuscle suspensions, taken from twelve different adult rabbits, but containing approximately the same amount of hzmoglobin as determined by this method, were tested against freshly prepared 1 per cent. solutions of an alcohol precipitated toxin, viz., 383. The results, as displayed in Table I., show that, although the extreme variations in the series represent titres of 770 and 1660 units respectively, yet ten out of the twelve readings give a titre varying

4 HEREERT HENRY

Rabbit Corpuscle Suspensions.

n . . . . A, G, ,J . . . c, 12. F, I, . . B , I , K . . . H . . . .

from 1000 to 1430 units. As toxin 383 was found to retain unimpaired its toxicity when tested on animals over a period of six months, and as there was no reason to assnnie that i t had depreciated in lytic capacity during this period, it became a routine to use this toxin i n 1 per cent. solution as a control throughout the series of experiments dealt with in this Report. Where toxin 383 when tested against a corpuscle suspension has been found to give readings outside the normal range, i e . , less than 1000 or nioi-c than 1430 units, then readings with that particular suspension have not been made use of for comparative purposes except in experinients carried out on the same day.

TABLE I. Toxin 383 in 1 per cent . solut ion tfsted againpt tiuelue difevent i,abbzt co i -psc le

suspensions, each comtciinilzg appvoxinmtely the same anzomt of Iict.it,oglob?n.

Unitage. Unit Poiut, C.C.

0.0006 1660 0.0007 1430 0~0005 1250 0.001 1000 0.0013 770

111. THE EFFECT O F TEMPERATURE ON B. JVELCHII ~ ~ A 3 M O T O X I X .

The esperinients dealing with t8he effect of texnpersture on B. welchii hzniotoxin may conveniently be divided into three series.

A. Toxins stored in the Cold Room Two fresh culture filtrates, viz., toxins 389 and 38’7 and two

alcohol precipitated toxins in 1 per cent. solution, viz., 383 and 384, were kept in tightly-stoppered bottles in the cold room, without any added antiseptic. The lytic capacity of each of these toxins was tested a t weekly intervals u p to the end of the sixth week, using a freshly prepared 1 per cent. solution of toxin 383 as the control on each occasion.

The method of arriving a t the figures shown in Table 11. can be most readily explained by a brief reference to two of the experiments dealing with toxin 389. This toxin, when freslily preparcd, gave 20 per cent. lmmolysis of a, 6 per cent. corpuscle suspension in 0‘00% c.c.: i .e., it coiit:tined 400 hremolytic units. A freshly prepared 1 per cent. solution of toxin 383 tested on the same corpuscle suspension gave 20 per cent. htemolysis in 0’0007 c.c., i.e., it contained 1430 units. If, now, toxin 383 I>c taken as containing 1000 units, the unitagc of toxin 389 may bc rcprcsented as 280. After one meek in cold storage, toxin 389 gave 330 units, using another fresh suspension of corpuscles, while the control gave 1250 units nitli the same suspension. If again t.oxin 383 be represented as containing 1000 units, then the titre of toxin 389 may be expressed as 260 units. It is thus possible to rechice the titration figures given by any toxin to the samc conlparative basis, by taking the control on each occasion as representing 1000 units.

€3. WELCH11 T O X Z N A N D ANTITOXZN 5

Toxilis.

389 287 383 384

TABLE 11. The hjtic value of B. welcliii toxins stored in the cold, and examined at

weekly i.ntes.vaZs over a period of s ix weeks.

I 'Cf 'dcs. i'?, H-

0. 1. 2. 3. 4. 6 . G . -

6.7 280 260 2s0 ... 200 260 200 6.4 870 770 700 870 870 870 770 6.5 1000 870 1000 570 1000 770 870 6.0 I160 1140 1250 1160 1000 1250 1000

Toxins.

356 387 353 3S.l

If unitage variations due to experimental error be taken into consideration, it will be noted that the figures set out in Table 11. demonstrate that B. welchii toxins, either as culture filtrates or as 1 per cent. solutions of dried preparations, do not show any niarked depreciation in lytic capacity when stored in the cold rooni under aseptic precautions over a period of six weeks.

Days. I'rr cc11t.

Loss. 0. a. 4. (i. 5. 10.

-

6.C; llG0 1250 11-10 $70 ... $00 40 6.1 870 s70 770 700 610 400 54 6.6 1000 1000 870 870 770 600 40 6.0 1160 1140 S70 500 ... ... ...

The results are sot forth in Table 111. From this it will be noted that, although there is no detectable depreciation in lytic value after two days' incubation a t 38"C., yet exposure to this temperature for longer periods does result in a loss of lytic capacity, which a t the end of ten days' incubation amounts to about one half the original total. In the case of toxin 384 the material proved to be heavily con- taminated on the sixth day, so that further observations in regard to this toxin hecanie impossible.

A 2 JOORN. OF PATH.-VOL. XXV.

6

Unitage.

HERBERT HENRY

-

15. I 45. I GO.

C. Toxins heated to 60" C., 80" C. and 100" C. The effect on B. ue lch i i hmnotoxin of heating i t to higher tempera-

tures than 38" C. was investigated on the same specimens of toxin as those used for the preceding experiment. Samples of toxin were enclosed in thin-walled sealed tubvs of sriiall diameter and immersed in constant temperature water-baths for the periods of time indicated in the accompanying tables.

Each individual experiment, in so far as it concerns the titration of a toxin heated to a definite temperature for various intervals of time, was carried out with a separate corpuscle suspension, the control in

2000 1430 1250 1430

each instance being the unheated toxin tested against the

770 590 500 500 690 590 500 400 590 500 500 400 770 590 500 500

TABLE IV. The destruction of B. welchii Iicemotoxin b?y heating to 60" C.

2000 1430 1660 2000

I 1 Uiiits left after heatiiig for - minutes at 60" c.

500 330 330 330 400 330 350 280 500 ... ... 330 770 500 400 400

Toxius.

387 383 384

100 100 100 100

25 16 16 16 28 23 23 20 30 ... ... 20 38 25 20 20

PEIICENTAGES. I 386 100 38 30 25 25 387 100 41 41 35 28 383 100 47 40 40 32 384 7 00 64 4 1 35 35

TABLE V. The rlestmction of 33. welchii Iic~motoxin. b y heating to 80" C.

Toxins.

356 387 383 384

I Units left after lieatiiig for - minutes a t SO" C.

Unitage. I I I 15. I 30. 1 45. I 60.

PERCENTAGES. I I I

386 387 383 384

I I I I I'

same

B. WELCH11 T O X I N A N D A N T I T O X I N

1i:wiotoxic Units left after Percentage of Hremotoxin after Antitoxiii - miiiutes' contact. - minutes' contact.

~ Uiiits. R173, C.C.

3. 1 12. XI.

I

1660 0.0003 590 ' 330 330 1000 0~0003 330 200 200 625 0.0005 200 77 59

2000 0.0005 660 330 250 830 0.0005 770 590 600

1000 0~0005 770 770 i 9 0

7

TABLE TI. P%e destluction of B. welcliii Iitsniotozin b?j heutinq to 100" C.

Uiiits lpft after heatiiig for - minutes a t 100" C. I

PEIICENTACES. I 100 1 4 2.3 1.7

387 100 1 (i 3.3 2 '0 383 100 16 1 'G

1.4 354 100 17

suspension. The results obtained by heating to 60" C., 80" C. and 100°C. are set out in Tables IV., V. and VI. respectively, each table contaiiiing the actual experinlentel figures together with the translation of these figures into percentages.

ITr. THE NEUTRALISATION O F HBMOTOXIN WITH ANTITOXIN. A. 7% 12apicZity of tho HBmotoxin-Antitoxin C'ombination at 38" 6'.

This has been deterniined by estimating the hzernotoxic content of toxin-antitoxin mixtures after tliey have been left in contact for definite periods of time. Sriiall anionnts of antitoxin, R 1'73, as indicated in Table VII., were added to either 0.5 C.C. or 1.0 C.C. of toxin,

TABLE VII. Rapidity of E. welchii ? i ~ ~ r n ~ t o , ~ ~ i ~ - ~ i i t i t ~ x ~ ~ ~ combination. at 38" C.

Toxin.

- 1.386 9.387 ::,353 4.383 5.384 6 3 8 4

I

330 35'5 200 33.0 59 -32'0

250 33.0 500 93.0

77.0

the quantity of antitoxin added in each case being less than tha t required for complete neutralisatioii. Each mixture, made up to a volume of 1.5 c.c., was iiicitbated at 38°C. for five, fifteen, thirty and

8 HERBERT HENRY

sixty minutes respectively, and the number of hzemotoxic units present after each interval was estimated. Each of the six observations set forth i n Table VII. was carried out with a different corpuscle suspen- sion, and no attempt was made to use suspensions of the same haemoglobin content as this was unnecessary. The results tabulated show the actual number of hzemotoxic units remaining in the mixtures after each interval, and also the percentages of residual haemotoxin

FIG. 1.--Compiete saturation of Hmnotoxin 387 with Antitoxin R 173. Contact at W C. for one hour.

as calculatecl from these figures. From this it is apparent that, in the case of toxins 386 and 387, which are culture filtrates, the bulk of the neutralisable hzemotoxin has combined with the antitoxin in five minutes, and that the mixture has reached a condition of equilibrium within fifteen minutes. With toxins 383 and 384, which are alcohol precipitated toxins, there is a slight lag in the combination with antitoxin, so that stability is reached solnewhat later, viz., between fifteen and thirty minutes. This lag is clue, as will be

B. WELCH11 TOXIN A N D ANTITOXIN 9

shown later, to the presence of inert hmnotoxoicl in these precipitated toxins.

B. C'onLpletc N~utralisation of B. welchii Emmotoxin with Antitoxin.

Oue culture filtrate and three alcohol precipitated toxins were used in these experiments, the same antitoxin, R 173, being employed

FIG. Z.--Coniplete saturation of Hsernotoxin 38-3 in 1 per cent. solution with Antitoxin R 1 i 3 . Contact at 35" C. for one hour.

throughout. The exact neutralisation points have been determined as follows. One C.C. of toxin is placed in each of a iiuniher of tubes, and there is then added a gmdnated series of amounts of antitoxin, tlie whole volume in each tube being made up to 1.5 ex. The resulting toxin-antitoxin mixtures are left in contact for one hour a t 38" C., after which period 1 e.c. of a 6 per cent. rabbit corpuscle suspension is added to each tube. Thc series of tubes is thcn incubated for two

10 HERBERT HENRY

hours a t 38” C., the individual tubes being shaken vigorously every thirty minutes during this interval. The racks itre then set aside and readings are taken as described in previous experiments. The tube in which hsetnolysis is represented by one unit or by less than one unit is taken as containing the aniount of antitoxin requisite to effect full neutralisation of 1 C.C. of the toxin under test (“ the 1 ex. neutralising dose ”).

After this preliminary experiment, fractions of 1 C.C. of toxin are

4-43

FIG. 3.-Complete saturation of Hzemotoxin 384 in 1 per cent. solution with Antitoxin R 173. Contact a t 38°C. for one hour.

put up, each fraction with a series of amounts of antitoxin embracing and ranging on either side of the equivalent quantity of serum as calculated from I‘ the 1 C.C. neutralising dose.”

The figures obtained by titration according to this method are set out in Table VIII. They are also represented graphically in Figs. 1, 2, 3, 4, in which the amounts of toxin are shown on the ordinates and the quantities of antitoxin effecting full saturation of these amounts are plotted on the abscissse. I n the case of Figs. 1, 2, 3, the amounts of

B. WELCH11 T O X Z N A N D ANTITOXIN

387 389 10;

11

0~0002 0~0003.5 0.00045 0.00055 0fl007 0.0016 0~002.5 0.003

TABLE VIII.

To show the quantities of antitoxin R 173 required to give conp2ete neutralisation of the amounts of B. welchii toxin indicated.

I I

l o x l n 0 8.

354 1s: 0.0012 329 1 : 10 0 .000.'i.i 329 1 : l 0,003.7

I c. c I 0-4. 1 0 G . 0 3 .

0.0025 0'004 0'0055 0.0006 0.0011 0.0016 0.007 0.012 0.016

0.00075 0.004 0.007 0.002

FIG. 4.-Complete saturation of Hemotoxin 329 with Antitoxin R 173. Contact at 38" C . for thirty minutes.

12 HERBERT HENRY

serum are expressed in units, the unit being one-tenth of ‘(the 1 C . C .

neutralising dose.” I t will be noted from an examination of these curves that the actual titration figures for fractioncl of 1 C.C. of toxin

FIG. 5.-Fractional saturation of Toxins 3% and 387 with Antitoxin R 173.

agree very closely with the calculated amounts of serum as deterniined from “ the 1 C . C . neutralising dose.” In other words, the neutralisation by antitoxin of B. welchii hwmotoxin in the amounts indicated follows the law of inultiple proportions.

B. WELCH11 T O X I N A N D A N T Z T O X l N 13

As the highest concentration of toxiii used in the above experi- ments, viz., 1 C.C. of a 1 per cent. solution of toxin 384, represents no more than 12.5 mouse rnininial lethal doses, it was decided to determine the full saturation points over a higher range of toxicity. For this

Fur. 6. -Fractional saturation of Toxin 267 with Ailtitoxin It 173.

purpose a solution of an alcohol precipitated toxin, 329, was chosen. The minimal lethal dose for mice of this solution was found to be 0,013 c.c., so that 1 C.C. represented 77 iiiouse iiiininial doses. A glance at Fig. 4, in which the serum unit for the sake of convenience is taken as 0.0001 c.c., will show that here again the iieutralisation of haemotoxin by antitoxin follows the law of multiple proportions.

14 HERBERT H E N R Y

C. Pmetionul Saturation.

I n all thirty-two fractional saturation experiments have been carried out witti different specimens of A'. toelchii toxin, the neutralising serum in each instance boing antitoxin R 173.

FIG. 7.-Fractional saturation of Toxins 383 and 354 with Antitoxin R 173.

Each experiment is divided into three stages: (1) The hmiotoxin content of the toxin under test is first estimated. (2) The amount of antitoxin R 173 which will give complete

neutralisation of 1.0 C.C. of the toxin is then determined. (3) Equal amounts of toxin, usually 1.0 c.c., are put up with

gradually increasing fractions of the 1.0 C.C. neutralising dose of

13. WELCH11 T O X I N AND ANTZTOXZN 15

antitoxin. The mixtures are left in contact for one hour a t 38" C. and are then titrated out, so as to determine the amount of unneutralised hwmotoxin contained in each.

It is necessary to put up these toxin-antitoxin mixtures a t fifteen-minute intervals, in order to allow sufficient time for the titration of each.

Typical examples of the results obtained by this method of investigation are set forth in Tables IX., X. and XI. (Figs. 5, 6, 7).

TABLE IS. Fractional s a t i m t t i o n of toxins 386 and 387 with antitoxin R 173.

Tosin, C.C.

1 .o 1 .o 1 .o 1 .o 1 .o 1 .o 1 .o 387

1 .o 1 .o 1 .o I .o 1 .o 1 .o 1 .o

Serum, C.C.

... 0.0011

0.0001 0.0002 0.0003 0~0001 0.0005

... 0.00065

0 ~ 0 0 0 1 0.0002 0 ~ 0 0 0 3 0.0004 0~0005

Not Keutralised

1660

... 1000 500 330 200 100

1250

... 770 400 200

77 20

Hwmotoric Units.

Smtralised.

Total.

...

... 660

1160 1330 l4tiO 1560

...

... 4SO sso

1050 1173 1230

Per ccnt.

.,.

... 39.7 69.8 80-1

93.9 57.9

...

... 38.4 68.0 84.0 93.8 9s '4

lieutralissd by eacii successive Serum Unit.

Total.

...

... 660 500 170 130 100

...

... 480 370 200 123

57

Per cent.

...

... 39.7 30.1 10.3 7.8 6 .O

...

... 38.4 29% 16.0 9.8 4 '6

It is found that in the case of fresh culture filtrates (Table IX.), the first few fractions of antitoxin neutralise the bulk of the hzmotoxin, so :hat the first part of the fractional saturation curve is precipitous (Fig. 5). On the other hand, i f the toxin under test be one that has been stored over several months, then it is found that the first fraction of antitoxin neutralises little or no hEmotoxin (Table X.), a result which can be explained on the assumption that the specimen contairis inactive hwmotoxoid. With precipitated toxins (Table XI.) the initial lag in neutralisation beconies still more marked, so that i t is possible to demonstrate the existence of a definite plateau a t the beginning of the curve (Fig. 7) . Also the remainder of the curve is less steep than that which is found in the case of fresh filtrates.

16 HERBERT HENRY

TABLE X.

Fvactional satiwatzon 01- toxin 267 with ant i tox in R 173.

S O .

-

1.

2.

3.

1.

9 .

3.

~

Toxin

1 '0

1 .o 1 .0 1 .o 1 .0 1 .o 1 .o

' 1.0

1 .0

1 .o 1 .o 7 .0 1 .o 1 .o

Stwil l .

...

0.0016

0~0001 0~0003 O f I O O d 0~0005 0.001

... 0.0016

0.0003 0.0001 0.oooti 0~0003 0.001

1X;cmotoric Units.

A-ot Srntrelisrd.

1430

... 1430 1250

770 330

25

1430

... 19.30

.7 90 400 'LOO

50

iYeiitralisod.

...

... 0

180 660

1100 1405

...

...

180 840

7 030 12.30 1380

r p r cent.

...

... 0

12.6 4 6 ' 1 76.9 95.2

...

... 12'6 58.7 72 '0 86 '0 96.5

Con.cr'nsroNs.

(1) B. welchiz produces in cultures a hzmotoxin the potency of which can be measured iz vitl.0. The same substance is present in the precipitates obtained from filtrates hy treatment with ammonium sulphate or alcohol.

It can persist iininipciired for considerable periods of time i f stored in the cold. At higher teniperatures it becomes altered so as to lose its lytic capacity. The bulk of the hzmotoxin which is rendered inactive a t 60" C. disappears after iive minutes' heating, and the remaining unaltered hamotoxin loses i ts lytic value but slowly on longer heating a t 60" C. The tests carried out 011 toxins heated to 80" C. and 100" C. give a similar result in that iiiost of the hzniotoxin which is rendered inert at either of these temperatures disappears within the first five minutes of heating, tlie residual active substatice being comparatively resistant to longer heating. It is noteworthy that after boiling for fifteen niinutes hwniotoxin is still present in riieasurable quantities.

(3) The kizeniotoxin can be neutralised by B. welchii antitoxin. Combination takes place rapidly a t 38" C. Thus it more closely resembles the toxin of diphtheria than that of tetanus. The slight lag that occurs in the case of precipitated specimens of hziiiotoxin is

( 2 ) The hemotoxin is not so labile as is tetanolysin.

B. WELCH11 T O X I N AND ANTITOXZN 17

attributable to the mass of hamotoxoid which t.hese preparations contain.

Complete neutralisation experiments confined within the limits of the observations recorded in this Report, show that the neutralisation of this hamotoxin follows the law of multiple proportions.

The results of fractional saturation experiments when represented graphically, give curves which are not unlike those obtained by Madsen for tetanolysin.

Tosiii.

383

1 .o 0.’ 0.5

354

1 .o 0 5 0.5

384

1 .o 0 c7 0.5

Serum.

Hzrmotoxic Units.

4000 2000

2000 1430 1000

FFO 590 400 200 l i 0

...

5000 2500

2500 1660 13S0 1000 770

...

5880 29-10

2910 29-10 9000 1660 1430 1250 1000 530 625

...

Nrutralised.

Total.

...

...

... 0

570 1000 1230 1410 1600 IS00 1830

...

...

... 0

840 1170 1500 1730

...

...

... 0 0

940 1280 1510 1690 1940 2110 2315

Per cent.

...

...

... 0

28 ..5 50.0 61.5 70.5 80.0 90 .o 91.5

...

...

... 0

33% 46.8 60.0 69.2

...

...

... 0 0

31.9 43.5 51.3 57.5 $6 .O 11.7 78.7

JOURW. OF PATH.-VOL. XSV. B

18 HEREERT H E N R Y

REFERENCES.

1. BULL AND PRITCHETT . . “Toxin and Antitoxin of, and Protective In- oculation against, Ilacillus zuelchii,“ Joui~c. Expi , . Me& New York, 1917, vol. xxvi. p. 119.

2. BULL AND PRITCHETT . . “Identity of the Toxins of Different Strains of Hacillzts ?relchi i and factors influencing their production in vitro,” Ibid., 1917, vol. xxvi. p. 867.

3. BULL . . . . . . . . “Tlic Prophylactic and Therapeutic Properties of the Antitoxin for Bacillus welcl~ii,” Ibid., 1917, vol. xxvi. p. 603.

4. BULL . . . . . . . . “ Gangrhe Gazcnsc, Toxine sp6cifique et Antitoxine,” Conzpt. rend. Soc. de Biol., Paris, 1917, tonic lxxx. p. 06;.

5. BULL . . . . . . . . “Antitoxin for Gaseous Gangrene,” X e i u I‘ork X e d . Jotiriz., 1917, vol. cvi. p. 821.

6. BULL . . . . . . . . “ILCI‘sunid des rdsultats d’expkriences faites avec des bacillcs dc Wclch,” Arch. de med. et tleplmmz. mil., Paris, 1918, vol. Ixx. p. 198.

7. ELLIOTT AND HENRY . . “Infection of Hzemotliorax by Anaerobic Gas- producing Bacilli,” &it. Med. ,7ourr~., London, 1917, vol. i. p. 448.

8. HENRY AND LACEY . . . “Tlic Precipitation of Bacillus zvelcli i i Toxin,” .Jozi.rn. I’ath. an(! Bactevicl., Cambridge, 1920, vol. xxiii. p. 273.

9. HERTER . . . . . . . “ O n Bacterial Processes in the Intestinal Tract in sonic cases of Advanced Anzemia, with espccial reference to Infection with 1:. mi-oyeiLes capsidatus (11. welchii),” J o u m . Ilid. CJieiii., New Yorlc, 100G-7, vol. ii. p. 1.

10. KLOTZ AND HOLMAN . . “Infectioii by the Gas Bacillus in Coal-Mines,” ,TXU-IL. Infect. Dis., Chicago, 1911, vol. ix. p. 251.

11. MCCAMPBELL . . . . . “The Toxic and Antigcnic Properties of Eact&?im ?oeEchii,” ibicl., Chicago, 1909, vol. vi. p. 637.

12. $LADSEN . . . . . . . ‘‘ lieher Tetanolysin,” ZeLtschr f. Hyg., 1899, Bd. xxxii. S. 214.

13. SCEIUMM . . . . . . . L L Spdlrtrophotogranimc cles Scrums, einiger Fiille von Bacillus empliysematosus - Baktcriamie;’ B e r l . klin. Wchnschr., 1913, Bd. L. 617.

14. SIMONDS . . . . . . . “Studies in Bacillus welclbii, with special reference to Classification and to its relation to 1)iarrhcea.” Xonographs of the Rockefeller Institute for Afedical Research, No. 5, New York, 1916.

16. STEWART AND WEST . . “Stiidy of a Strain of Bacillus iueleiiii isolated in France, together with some notes on Gastric Ulcers,” .~O?L?’.l&. Inamunol., Baltimore, and Canibridge, England, 1916, vol. i. p. 18‘3.

.