434 M . BARBEM

THE AhTTIBACTERIAL ACTION OF 4 : 6-DIMETHOXYTOLUQUINONE

MARY BARBER From the Department of Bacteriology, British Postgraduate Medical School

(PLATE LXII)

The antibacterial power of certain quinones related in structure t o the mould pigment fumigatin was reported by Oxford and Raistrick (1942) and by Oxford (1942~). In view of the very considerable activity of 4 : 6-dimethoxy- toluquinone against staphylococci recorded in the latter paper, it was decided to make a fuller investigation of the bacteriostatic action of this compound. Although the results indicate quite clea?ly that this substance is unsuitable for therapeutic purposes, they are thought to be of sufficient interest to be worth recording.

Antibacterial action in artificial culture rnediu

In all these experiments proflavine was tested in parallel for comparison. Bacteriostatic action in broth. Serial dilutions of the two compounds were

made in broth and then a suitable inoculum of the organism to be tested was added to each tube. The following two media were used. (1) Broth at pH 7.5 containing 1 per cent. " lab-lemco ", 1 per cent. peptone (B.D.H.), 0.5 per cent. NaCI and 0-005 per cent. p-aminobenzoic acid. Four per cent. serum had to be added to this medium for adequate growth of Strep. pyogenes. (2) Hartley broth at pH 7- 8 made from horse muscle.

Table I gives the results obtained with various bacteria when an inoculum of less than 500 viable organisms was used. 4 : 6-Dimethoxytoluquinone is

TABLE I

Bacterioatatic action in broth at 37' C. (Size of inoculum less than 500 organisms in each case)

Staph. aureu~ (M. 1) . , ,, (M. 2)*,

Strep. pyogenes (M. 1 I

9 , (Ma 2)

C . diphtheria (&I. 2) . Bact. typhosum (M. 1 )

Bact. coli (M. 1) . Pa. pyoqanea (M. 2) .

Limiting dilution of chemical causing complete inhibition of growth

4 : 6-Dimethoxytoluquinone

1 day

1 : 600,000

1 : 250,000 1 : 400,000

1 : ~00,000 .1 : 200,000 1 : 250,000 1 : 4000

1 : 8000 1 : 1000

2 days

1 : 250,000 -1 : 500,000 1 : 100,000 1 : 100,000 -1 : 200,000 1 : 32,000

-1 : 200,000 1 : 120,000 1 : 2000

1 : 8000 <1: 1000

7 days

1 : 120,000

1 : 100,000 1 : 100,000

-1 : 200,00( 1 : 120,00( I : 2000

1 : 8000 < 1 : 1000

-1 : 500,000

: 1 : 32,000

Pro&wine ~

1 day

1 : 32,000

1 : 128,00( 1 : 500,00(

1 : 500,00(

-1 : 64,000

... 1 : 32,000

1 : 16,000 1 : 4000

-1 : 64,000

7 days

1 : 32,000

1 : 128,00( 1 : 500,00(

1 : 500,000

- 1 : 64,000

... 1 : 32,000

-1 : 64,000 1 : 16,000 1 : 2000

M. 1 = " Lab-lemco " broth. M. 2 = Hertley broth. * 4 per cent. horse serum added.

A N T I B A C T E R I A L ACTION OF 4 : 6-DIMETHOXYTOLUQUINONE 435

shorn to have a powerful action against Staph. aureus, Strep. pyogenes and C. diphtherice, whereas the Gram-negative bacilli are relatively resistant. There appears to be falling off in bacteriostasis after 48 hours, although this varied considerably in degree. Oxford (1942b), working with 4-methoxytoluquinonc against Staph. aurew, found a similar phenomenon and suggested that it was due t o the peptone reacting with this compound and reducing the antibacterial power of the latter. Table I1 shows two results obtained with Stuph. aureas,

TABLE 11 Eflect of varying size of inoeulurn on antistaphylococcal action

I Limiting dilution of chemical causing complete inhibition of growth I 1

1 day 2 days

1 : 500,000

1 : 64,000

1 : 250,000

1 : 64,000

Size of inoculum of Staph. aecreu8

7 days ~ 1 day 7 days --

1 : 120,000 I : 32,000 1 : 32,000

1 : 16,000 1 : 32,000 1 : 32,000 1 410 viable organisms

82,000 ,, ,,

4 : 6-r)irnethoxytoluquinon~ I Proflavine 1

using as inoculum in one case 410 and in the other 82,000 viable organisms. As will be seen, whereas the action of proflavine is unaffected, 4 : 6-dimethoxy- toluquinone has a much less powerful inhibiting effect on the larger inoculum.

This bacteriostatic action in artificial media can be readily demonstrated by the agar cup method in nutrient agar plates. The comparative inhibition of Staph. aureus by 4 : 6-dimethoxytoluquinone, proflavine and penicillin after 18 hours' incubation is shown in fig. 1.

suspensions of staph. aureua in watery solutions of 4 : 6- dimethoxytoluquinone and proflavine respectively were made, such that the find concentration of chemical was I : 1000 and the number of organisms per mi. about 10 million. The tubes were incubated at 37" C. and viable counts were made after 6 and 24 hours. In a typical experiment where the original con- centration of organisms was 10.5 million per ml., 52 per cent. of the staphylococci were still alive after 6 hours in 1 : 1000 4 : 6-dimethoxytoluquinone, although none survived 24 hours. Proflavine killed all the organisms in 6 hours.

Agar plates.

Bactericidal tests.

Antibacterial action in blood and Berum

Action in blood or serum media in tubes. The introduction of 10 per cent. of blood or serum into artificial media approximately halves the limiting dilution causing complete inhibition of growth after 24 hours and the tendency for organisms to grow in higher concentrations of the compound after the second day is greatly although variably increased. These facts can be demonstrated by the use of the agar cup method in 10 per cent. horse blood agar plates. Figs. 2 and 3 show the comparative inhibition of hzemolytic streptococci by 4 : 6-dimethoxytoluquinone, proflavine and penicillin after 24 and 48 hours' incubation respectively. The area of inhibition produced by the quinone compound after 24 hours is seen to have a very irregular outline, and after 48 hours the effect of the weaker solution has almost disappeared, while a few streptococci have grown in the inhibitory zone of the stronger. By comparison, proflavine and penicillin produce areas of inhibition with regular outlines, which are not appreciably diminished by prolonged incubation.

If a medium consisting of 50 per cent. serum in distilled water be used the bacteriostatic activity of 4 : 6-dimethoxytoluquinone after 24 hours is reduced

436 M . BARBER

to about one-tenth of that in broth, and in this serum medium a small inoculum of staphylococci will grow after two or three days in what was originally a concentration of 1 : 5000 or less of 4 : 6-dimethoxytoluquinone.

Action in blood in slide cells. To determine whether the chemical was predominantly antileucocytic, it was tested against Strep. pyogenes in fresh defibrinated human blood. A control series using blood in 1 : 1000 liquoid was put up in parallel. Serial dilutions of 4 : 6-dimethoxytoluquinone and proflavine were made in isotonic saline or Ringer's fluid. These dilutions were mixed with equal quantities of blood and a suitable inoculum of organisms was added. The various mixtures were transferred to slide cells, which were then sealed and incubated for 24 hours. Table I11 shows the results obtained with

Dilutionof chemical *

No. ofcolonies

TABLE I11

Effect of 4 : 6-dirnethoxytoluquirzone and proJluvine on hamolytic streptococci

4 : 6 Dimethoxytoluquinone

1:4 1:s 1:16 1:32

3 1 33 I 44 1 42

Proflavine

I Blood in 1 : 1000 liquoid 1 Fresh deflbrinated blood I

Dilution $f ' 1 : 50 1 : 100 1 : 200 1 : 400 1 : 800 ' 1 : 1600 I 1 : 3200 ' None chemical

... 1 : 50 1 : 100 None

1No.ofcoloniesl 0 I 0 I 0 I 3 I 11 I 16 ~ 15 I G a n d I ... 1 0 1 34 1 4 2 I * Dilutions expressed in thousands. t Approximately : colonies had coalesced.

4 : 6-dimethoxytoluquinone and proflavine when the inoculum consisted of approximately 50 viable organisms per cell. It seems quite clear from these experiments that the addition of this compound to blood produces a medium which is very much more toxic to human leucocytes than to streptococci. Fresh defibrinated human blood by itself is much more efficient in dealing with these organisms than it is in the presence of 1 : 8000-1 : 64,000 4 : B-dimethoxytolu- quinone. Proflavine likewise appears to be toxic to leucocytes in a concentration far lower than it is bacteriostatic. This has been shown to be the case with acriflavine by Fleming (1917, 1939-40), and Abraham et ul. (1941) have shown that proflavine is highly toxic to leucocytes.

Action in vivo

Although the results already described indicated quite clearly that 4 : 6- dimethoxytoluquinone was likely to be a hindrance in combatting bacterial infections, in view of the conflicting results of in-vitro and in-vivo experiments described with other compounds, a small scale experiment was carried out with mice. The compound was found to be toxic if given by injection. A 20 g. mouse was usually killed by a single dose of 2 mg. injected subcutaneously. A dose of 0- 5 mg. or less was tolerated and could be repeated 6-hourly.

A mouse-virulent strain of streptococci having an M.L.D. of approximately 1000 organisms was injected intraperitoneally. Treatment was by means of subcutaneous injections ; 4 : 6-dimethoxytoluquinone was given a t 6-hourly intervals ; sulphanilamide was given every 4 hours for 24 hours and then twice

JOURXAL O F PATHOLOGY-VOL. L V I PLATE LXII

ANTIBACTERIAL ACTION OF 4 : 6-llIMETHOXYTOL~JQ7.TINONE

FIG. l.-Inhibition of growth of AS’taph. aureu.5 on a, nutriciit agttr plate after 18 hours’ incubation at 37’ C. produced by ( 1 ) 1 : 1500 and ( 2 ) I : 1000 4 : 6-dimethoxy- totuquinonc, (3) 1 : 500 proflavine, (4) penicillin (approximately 2 units psr c.c.).

FIG. 2.-Inhibition of growth of St7 e p . pgogenea on a 10 per cent. blood ng<ir plate after 24 Iioiirs’ inrubat,ioii by the same compounds.

Fic? 3.-The same after 48 1101~s’ inculxttion.

A N T I B A C T E R I A L ACTION O P 4 6-DIMETHOXYTOLUQUINONE 431

daily for the next 4 days. The results are given in table IV. As will be seen, there is nothing to choose between the untreated animals and 'those given

4 : 6.dimethoxytoluquinone I None . . . I

2 mg.

Sulphanilamide . .{I E:: None . . 1 ...

I

TABLE IV Treatment of experimental streptococcal infection in mice with

4 : 6-dimethoxytoluquinone und sulphniilamide

6500 10 1.6 6500 6 1.1

15,000 2 7 15,000 4 7 15,000

i

Chemical I No. of Average days 1 Totaldose organisms No. ofmice survival

injected (maximum = 7)

4 : 6-dimethoxytoluquinone, whereas all 6 treated with sulphanilamide survived I days, A heavy growth of hsmolytic streptococci was recovered after death from the heart blood of all the 10 mice treated with 4 : 6-dimethoxytoluquinone.

Discussion

From the practical point of view there is little to discuss. 4 : 6-Dimethoxy- toluquinone i s obviously contra-indicated as a therapeutic agent. This was most readily shown by adding the compound to blood in slide cells, and one point here demonstrated is the value of this method of testing a potential antiseptic. The substance appears to be generally toxic, as shown by its action on leucocytes and its lethal effects in mice. In view of this, it is interesting that it is considerably more selective than proflavine in its action on bacteria, and that even staphylococci, which appear to be the organisms most sensitive to its action, are only very slowly killed by it. The fact that the bacteriostatic action is so greatly reduced by blood and serum is perhaps not very surprising with such an unstable compound, and this loss in antibacterial power may be associated with a change in chemical structure. Whatever happens, however, to 4 : 6-dimethoxytoluquinone in blood, it is so toxic to leucocytes that it would be more profitable to use it as an adjunct to blood cultures than as an aid to the treatment of bacterial infections.

Summary

The antibacterial power of 4 : 6-dimethoxytoluquinone has been studied. It has a powerful bacteriostatic action against Xtupb. aureus and Strep. pyogenes in broth, but owing to the great reduction of this action by blood or serum and to the markedly antileucocytic properties of the compound, it is contra- indicated as a therapeutic agent.

My thanks are due to Professor H. Raistrick for the supply of 4: 6- dimethoxytoluquinone and to Professor J. H. Dible and Dr G. A. D. Haslewood for advice.

R E F E R E N C E S

ABRAHAM, E. P., CHAIN, E., FLETCHER, 1941. Lancet, ii, 177. C. M., GARDNER, A. D., I ~ A T L E Y , N. G., JENNINCS, M. A., AND FLOREY, H. W.

JOmN. OF PATE.-VOL. LVI 2 + 2

438 F. JACOBY

FLEMING, A. . . . . . . .

OXFORD, A. E. . . . . . . OXFORD, A. E., AND RAISTRICK, H.

. . . . . . ,,

. . . . . . ,,

A METHOD OF OBTAINING

1917. Lancet, ii, 341. 1939-40. Proc. Roy. SOC. Med., xxxiii, 487. 1942a. Cltern. Ind., hi , 189. 19423. Biochem. J., xxxvi, 438. 1942. Chem. Ind., Ixi, 128.

578.085. 23 : 578.68

PERMANENT PREPARATIONS FOR THE CYTOLOGICAL STUDY OF PURE CULTURES OF MACRO- PHAGES, WITH SPECIAL REFERENCE TO THEIR MODE OF DIVISION

F. JACOBY

From the Department of Physiology, The Medical School, University of Birmingham

(PLATE LXIII)

In 1933 Baker published a method for obtaining pure cultures of hen-blood monocytes * grown in dilute serum in ordinary Carrel flasks (type D). By its use, combined with Willmer’s serial photographic technique (1933), an extensive study of the rate of multiplication of these and related cells under various conditions has been made during the past few years (Jacoby, 1937, 1938, 1941-42; Jacoby, Medawar and Willmer, 1941). The ordinary Carrel flasks used in this work have two major disadvantages: (1) they limit, on account of their relatively thick and uneven glass bottom, the magnification which can be employed ; (2) permanent preparations for cytological study cannot be obtained unless, each time, a precious flask is sacrificed and the bottom cut out. It was, however, found highly desirable to have a method which, at any stage of the cultivation period, would permit permanent prepara- tions to be made. Interest was in the first place focussed on cell division. Although the photographic records (paper film negatives) of the living cell populations leave practically no doubt that the multiplication of these cells takes place by normal mitotic division, the magnification employed ( x 150) does not allow much detail to be seen and studied. Moreover, in view of the opinion still held by many workers that amitosis plays it major part as a mode of division of these cells, a clear demonstration of the mitotic figures seemed essential. These at certain times are very numerous in the cultures; under favourable conditions every single cell of the population will divide. Direct

fragmentation of nucleus and cytoplasm (amitosis) has not been seen in these film records in a single instance among several thousand divisions studied. At the same time it was necessary to make the cultural conditions in such a method as closely comparable as possible to those prevailing in ordinary Carrel flasks.

After various trials with Petri dishes, watch glasses, etc., all of which met with little success, the following method-though not yet ideal-was found to give so far the most satisfactory results. A large-type Carrel flask of 5 em. diameter is used, with a wide hole (30 mm. diameter) in the top (fig. 1). Through this hole either a single round no. 2 coverslip (27 mm.) or two smaller ones (22 mm.) are inserted and stuck to the bottom of the flask by means of a drop of hen plasma mixed with a minute amount of dilute embryo extract previously

* The cells are here referred t o as macrophages, as they have all the characteristics No consideration can here be given to the still obscure subject of their of the latter.

histogenesis.