OBSERVATIONS ON THE GRAM-POSITIVE GRAM-POSITIVE AN ACID-FAST PROPERTIES BACTERIA.' By T.
C. BENIANS,unior unior Assistant, Assistant, Inoculation Department Department London Hospital. From the
Lm&m Hospital Baeteridogieal Labomtory.
INTRODUCTION. AN attempt
is made in this paper to show that the bacterial substance
in itself does does not exhibit, ex hibit, in any a ny marked mar ked degree, degree, the th e property of of Gramof acid-fast positive staining, acid-fast staining, staining, but that tha t both of these properties are almost entirely dependent on the integrity integrity of the th e cell. cell. show this the writer has broken up, and reduced to an amorphous condition, various various Gram-positive Gram-positive and acid-fast bacteria. By breaking up an organism, and so destroying its character as an entity, the physical properties dependent on its integrity are done away with. The chemical chemical properties inherent inheren t in the bacteria should, however, however, persist persist in the amorphous state of the bacterial substance substance.. The experiments experime nts carried out have been collected into int o two main groups groups certain experiments in which which the crushed bacteria were were examined with regard to their capacity for retaining retaini ng Gram's Gram's stain experiments in which crushed bacteria were examined in regard to the acidfast property. In this second group only the tubercle bacillus is dealt with owing owing to the th e overlapping overlapp ing of e staining st aining reactions re actions it has been considered advisable, this case, case, to include the t he acid and Gram-fast properties both in the same section. Before detailing these experiments, and the th e deductions that tha t ma be drawn from them, it seems seems desirable to make a preliminary statesta tement on the th e one hand, to show show the t he present state sta te of opinion, which is divided betwe between en a physical and a chemical interpr int erpretat etation ion f th phenomena, and, on the other hand, introduce into the discussion certain relevant facts and considerations. the same time it must be understood that complete explanation of the Gram-positive acid-fast acid-fas t properties properti es is offe offered red.. Other factors fa ctors are yet ye t wanting want ing before this can be fully full y accomplished. The writer, however, claims to have demonstrated in this paper that th at these properties are ar e both, to very Received June
1912.
20
BENIANS.
large extent-in fact almost entirely-dependent attributes of the intact bacterial cell. TH
HISTORICALBSUME
on th e physical
GRAM-STAIN.
THEORIES GRAM-STIN
NATURE
1884 Gram (v
introduced his metho the differential staining f b acteria. Since th t time bacteria hav e been dividedwith a tolerably sh ar p dividing line-into two main classes, according to whether th ey retained or failed to retain the stain. The phenomenon, obviously of wide significance, has, since it as first observed, furnished material for a good deal of experimental and speculative research, although Gram himself offered no explanation. It been explained entirely chemical, and entirely on physical grounds, and various modifications throwing light on the matter have been introduced. It remains still, however, a question as to whether the differentiation depend8 on definite properties on t h e p a r t of bacterial substance, or on physical characteristics of the bacteria, which may, may no t indicate, and co-exist w ith chemical differences. Chemical Explanation. The principal work on the chemical conditions present in Gram-staining was done by Unna in 1887(14). Briefly, he showed th at the test could only be carried out with the pararosanilin class of stains, such victoria blue, methyl and crystal violets, or the mixture of th e two latter found in gentianviolet. The rosanilin stains, such as fuchsin, though so closely allied, were useless in this respect, He found that the iodine entered into combination with the dyes, th e compound so formed being bound the tissues by the iodine component. n th e cas of the rosanilin dyes this compound was readily broken up, and th us easily dissolved away in the alcohol that thes staius could not, in any case, be fixed by th e addition of iodine. n the casc f t e pararosanilin dyes th e compound was much more stable, and thus held firmly to those bacterial substances which it had an affinity. These latter were therefore Gram-positive. From th e others, for which t had no affinity th e compound was completely removed. These, therefore, were Gram-negative. Dreyer, Kriegler, and Walker (1911 have recently suggested that the substance in bacteria, which is responsible for e retention of Gram-stain, is clear evidence in this respect is however, forthconiiiig of a lipoid nature. t present
Physical Explanation. Following work of Fischer in 1889 the phenomenon of the retention of the stain has been explained on a purely physical basis. The stain, having ponetrated granules and masses of protoplasm, was relatively slowly washed out again in th e alcoholic solvent. n a recent paper by Bruclny (1908 4) this aspect is fully reviewed. It further claimed by him th at the difference between th e Gram-positiveand Gram-negative propcrties of bacteria is readily explained by t he easy permeability f th e Gram-positive bacteria by th e iodine solution. This penetration f the iodine results, e says, in the formation of
GRAM-POSITIVE
ACID-FAST PROPERTIES.
20
heavy deposit of stain inside the organism, which deposit is then n ot ver surmises that the Gram-positive readily washed by the alcohol. bacteria are of loose texture, in the interstitial spaces of which the granules of stains lie. Th e ram nega tive bacteria impermeable by regards
th e iodine solution. Th e precipitated stain is thu s only formed on the surface in solvent m edium. of these, and readily washes pa rt from th e conflicting theories, which have been offere as complete explanations, various fa cts bearing on the ma tter have been from tim e to time brought forward. These can no t be fully considere here, bu t some may be mentioned. Pe rha ps the most important ar th e exp erim ents of N eide (190412) th e action f t e iodine solution. e showed th t the potency of th e stain was dependent largely strength th e iodine solution, an the length of time of its application. By using the Lugol's solution for longer time, or by using solutiou con taining do uble th e am oun t f iodine present in Lugol solution, he was able to make some Gram-negative organisms retain th stain. e showed, likewise, th t th e tem perature f th e iodine solution w as of th e greate st impo rtance, because, wh en he used a he ated Lugol's solu tion, he was able again to make the Gram-negative bacteria retain the sta in with considerable tenacity. This was most mark ed in coli typhosza. This las t observation h as been confirmed by some experiments carried out by th e present writer, which are added in a appendix to th is paper (infra, 210). But, in is opinion, it demands a different explanation from th at offered by Brud ny, who has regarde t merely as due to th e increased act ivity in penetrative power f th heated iodine solution. MODIFICATIONS
GRAM'SMETHOD.
Of th e numerous modification Gram's method of staining, two only-those an d lau diu s 7 d be of U n n a ( 1 8 8 8 mentioned, as these have been made use of in the experiments that follow. both f the se methy l-violet ma y be used as th e staining reagent. tha t worked out by Claudius in 7 t h e p la c Lugol's solution of iodine taken by a half-saturated watery solution of picric acid. further difference lies in the fact that alcohol cannot be used s th e decolorising agent. Th e best medium for this purpos is chloroform, advised by Claudius. ith th is method certai bacteria, which are negative or doubtful by Gram's method, become positive by the method Claudius. The modification f nn a was introduced by him with th e object f providing t he m aximum oppo rtunity for chemical action to ta ke place during th e staining process. e used victoria blue, and, in place of the Lugol's solution, he used a freshly made mixture of equal parts watery potassium iodide solution, one third per cent., and hydrogen peroxide. By this mean8 nascent iodine is set free in th e fluid applied.
202
BENIANS,
U;
present
writer has
failed
find
that this method possesses ad
Gram’s original method, either in staining films f int ac bacteria, or films the crushed d6bris.
vantages over
TECENIQUE
AUTHOR’SETHOD.
TH
The present writer atte mpted to disintegrate th e bacteria in tw
ways--(a)
as films fixed
Disintegrafing
as bacteria
slides,
bulk.
fi1nza-A moderately thin but definite film spread along almost the whole length f the slide, and fixed over the flame in th e usual wa . The crushing or mutilating process is most readily carried out after he film has been stained, and treated with alcohol, and when i is almost, but not absolutely dry. cover-slip is laid over one end f the film on the slide, which lies evenly on a firm table. The cover-slip is pressed very firinly down on to the slide, under the tip of the thumb, and a screwing movement backwards and forwards is carried out for minute or so is then seen that th e film is scratched and smeared, and that some part o it is adherent to the now removed. It may e taken ha t the two glass cover-slip. The latter surfaces have come into actual apposition at some poinb, and that here th film bacteria in e film will have become crushed and broken up. Such examined under he microscope shows masses f amorphous material mingled with intact bacteria. Another method is put fine dusting of precipitated chalk under the cover-slip, assist in grinding up he bacterial film. The chalk can then removed by treatment with dilute acid. This, however, introduces an undesirable factor in the presence f e acid. (b) Crushing bacteria in bulk.-This was carried out with small agate pestle and mortar. It was not found that the use of any sort of menstruum was essential. The bacteria, having been washe off the medium in distilled water, were thrown down in the centrifuge, and th e deposit transferred to the mortar. After grinding for half an hour it found that even such small organisms as staphylococci could be, to large extent, completely broken up. n the experiments to be detailed shortly, the larger organisms such yeast and sarcins were chosen, as a rule, by reaso of their greater size. Some experiments were also carried out with staphylococci and a diphtheroid organism.
Extent
bacterial
to which the
bacteria
are
Teally cwcshed.
The res ults obtained by e method used for cr ushin g th e fixed films are necessarily very partial, and imperfect most cases. case f e dipht heria bacillus a nd t e tubercle bacillus it is niuch simpler obtain satisfactory result. h thes e e cover-slip moves very freely on the film as though
waxy surface, even when great pressure is being brought to bear on it. Almost the whole formed elements number
re crushed. ou t in to thi n waxlike masses.
Sometimes
small oval plaques are found together, as though each one
had been formed from
bacillus.
The act ual ext ent t o which these matter of some doubt.
bacilli are really crushed is naturally thing , however, can be gathe red from dipht heria bacilli ar e ground
th e observation
under a cover-slip into
th at thin
Somewhen
filmy
smear, stai ning by Neisser’s method will st ill elicit numerous int act blue granules scattered throughout
field of structureless yellow sub-
stance. The Gram-stained granules the tubercle bacillus may also frequently be found scattered thickly in the amorphous muses on the slides, and resisting decolorisation more strongly than the rest of th substance.
Immediate
the experimental procedures.
The objects f the following experiments on Gram-positive bacteria were to enquire--(a) whether and to what extent the staining reagents actually penetrate the cell, and whether the stain is firmly bound in the substance of the cell, whether it is.merely retained there by the protection afforded by the outer coverings the cell. Experiment 1.-To show that cmlied bacterial f l n a fails to retain &aszStaphylococcus aureus were stain.-Films f baker’s yeast cells, sarcinae, taken and fixed over the flame in the umal way ne end of the preparation s treated by the crushing process described above. The films were then stained by Gram’s method (methyl-violet pe cent. two minutes Lugol’s solution, two minutes bsolute alcohol, five minutes). After decolorisation counter-stain f neutral red was applied for half a minute. Microscopic examination showed that the intact part of the film s deeply staine Gram’s method, and none of the red counter-stain appeared. In the crushed part of e film, however, were masses f Gram-negative, amorphous substance lso a certain number of cellrr, which,although apparently intact, had failed retain the stain. No part f the formless substance retained the Gram stain. Experiment 2.-To show that the stain has permeated the w ole substance the ceZZ.-Films of yeast cells and sarcinae mere made, and fixed over flameas before. They were then stained with methyl-violet two minutes, and washed in water. The films were now a bright purple colour Lugol’s iodine solution was next applied two minutefi, and the colour t once changed to a deep purple black. After the application a neutral red counter-stain the films were examined under e microscope red-stained parts were seen. The films were then crushed, described above, at one end of th e slide, with e object of exposing the interior of some of the injured cells to the counterstain. Neutral red was again applied for half a minute. Examination showed now only mangled masses of cell debris mixed with uncrushed cells, all of deep blackish purple colour. There was no red-stained portion. There was, moreover, no sign of the original violet stain. The cells were evidently completely saturated throughout with th e methyl-violet iodine compound. Experiment 3.-To show stages deco1orbation.-The films, stained and crushed as in t he above experiment, were now treated with alcohol one minute. The colour was at once freely discharged from th e crushed area On examination the undamaged portion of the film was deeply stained by Gram. n the crushed area, however, were amorphous ame8 f very faintl purplish tinged substance, constituting the great bulk f the elements in th field. addition, cells were present n every stage f partial decolorisation. In the case f the yeast, many of the cells showed large granules f blackis stained substance lying in a colourless or faintly stained matrix. These granules of stained substance evidently resisted decolorisation more th an th sm ou nd in g tissues. After continuing the application f the alcohol some minutes longer the granules were also decolorised. faint purplish tinge, however, persisted the tissues, especially the thicker masses in the field. On the application f neutral red other counter-stain these amorphous tissues were immediately and completely overstained by it. The crushed yesst celle held the stain more strongly than did the crushed sarcinae. This experiment was also carried out with a large rapidly-growing organism f
BEAJIANS.
These bacteria showed a very marked granular appearance in the earlier stages of decolorisation, giving the appearance f a chain of rough and irregular granules lying in a thin-walled tube. This experiment shows that the cells were completely saturated with the Gram-stain. The stain was gradually removed from the protoplasm, remaining longer in th denser parts. Experiment 4.-To show staining reactions hacterin disintegrated in bulk.-A quantity f baker’s yeast was taken and dried in the incubator 37” . Films made from the dried substance showed a very large proportioll of Gram-positivecells. The mass was then ground up in the mortar for half hour. Films made from the powder, stained by Gram, showed a few intact cells, which retained the hi n, and in addition large quantities of pale purplish coloured amorphous material. After further pounding up in the mortar, films showed practically nothing but quantities f amorphous Gram-negative substance, which readily took a counter-stain of neutral red. This experiment p repented with sarcine twenty-four hours’ growth on agar, dried in the incubator at . was used. Practically the whole f this was crushed and rendered incapable f retaining the stain. With staphylococcus th results were the same, but the crushing was less complete. These staining reactions were confirmed by using the methods of Uniia and Claudius with similar results. Experiment 5.-To show the nature the residual stain.-In the procedures described above it was noticed that the bacterial detritus retained faint purplish colour after five minutes’ treatment with alcohol. This, it will be remembered, is not present where Gram-negative bacteria have been stained by Gram’s method, and decolorised again by alcohol. Tho importance f th iodine factor in the retention f this residual stain was tested as follows. Films f the ground-up sarcins and staphylococciwere shined with methylviolet. One-half of each film was now immersed n Lugol’s solution for two minutes, and the whole film then decolorised with alcohol It was not found that the iodine treated part retained the stain any more strongly than the other part of the film. From this we may conclude that the substance of Gram-positive bacteria has a definite affinity for th e methyl-violet dye. Th affinity not increased by t e action of iodine. B.
sulitilis group.
Conclusions to be drawn from the above experiments. The bacterial cell is completely permeated by th aniline gentianviolet th e methyl-violet stain. The cell is also permeated through out by th e iodine solution, which forms deep purplish black compound with the violet. The compound stain has no greater affinity for th e bacterial tissues than the original dye. The compound stain, present in the substance the bacterial cell, is freely soluble in alcohol, an d dissolves p int purple violet solution. Whilst the Gram-stained cell is intact, the alcohol is unable to remove the sta in; but so soon as th e cell is injured an broken p, the stai is, in great part, at once dissolved out. An injury far short of complete disintegration of e cell suffice set the stain free. The amorphous ddbris obtained from crushed Gram-positive bacteria will not retain Gram’s stain.
GRAM-POSITIVE
ACID-FAST PROPERTIES.
APPLICATION BBOVE CONCLUSIONS GRAM’SSTAIN.
HE
20
PHENOMENA
n attemp ting to explain th e phenomena f Gram-staining we h av e a t e as t t h re e d is ti nc t q ue s o ns t o ) h y is it t h a t only certain stains can function in this respect h y d o so m e bacteria retain th e s tain whilst oth ers fail to do By what means does the cell retain the stain no way concerned, bnt the With the first question this paper is explanation given by nn a h as been quoted. He showed that the pararosanilin dyes could function by virtue of a sta bl e compound which the y formed with iodine, an d which w as no t formed in th e c the rosanilin dyes. n answer to th e second question, th e work Brudny has been quoted. His explanation was that the Gram-negative bacteria are impermeable the iodine solution. It is doubtful, however, wh ether this represents the whole tru th f th e matter It is with tbe third question th at the experiments carried out in th e above research mostly deal. Two main points stand out clearlyth at th e bulk of th e sta in is not firmly bound to th e bacterial tissues against its removal. The conclusion to be drawn is that the chief factor, in th e retention f Gram’s sta in , is to be fou nd in a defiuite bacterial envelope. This exte rn al coat, by th e action f iodine solution, and to a less exte nt by picric acid, is rendered impermeable to alcohol stated above, explain clearly th e whole question of Gram positive staining with the factors now before us, is, in the writer’s opinion, t possible. It can only be said that, although a stable stain compound is essential, yet the iodine and picric acid do not bind the stain to th e tissues, but exe rt the ir fixing action through th e medium of the bacterial envelope. 11. GRAM-STAININGAND AGID-FASTNESS IN THE TUBERCLE BACILLUS.
INTRODUCTORY. Gram-staining in the tubercle bacillus goes on under somewhat different conditions from those obtaining in non-acid-fast bacteria. It will be best studied in conjunction an d comparison with th e acid-fast property. It is then found that these two forms staining tak e plac along very similar lines. Indeed, in th e presence the acid-fast property it is impossible to fully demonstrate aloohol-fastness. form f tub erc le bacillus described by uc (1908 the acidfast property i s absent, bu t G ram-positiveness remains. Grim me ( 1 9 0 2 lo points out some similarities between Gram- and acid-fastness. Bienstock (1886 and Gottstein (1886 found that fat-storing
20
BENIANS.
bacilli and their spores were made increasingly acid-fast and Grampositive by grow th on fat-containing media whilst growth such media as peptone d sugar-containing fluids iminish these properties. Th e tube rcle bacillus contains a large amount-up to per f y d waxy substances. It t a k e s u p s t a i n s w i t h considerable difficulty, bu t, when once th e sta in has penetrated- th organism, its removal, destruction by another solvent, is even more difficult. th e Gram -staining process th e add ition f iodine, as mig ht be expected, is a att er f minor impo rtance from th e point view of th e fastness f th e stain. ith ou t iodine th e tubercle bacillus is ui te alcohol-fast. Acid alcohol (1 per cent. HC1) likew ise fails to remove th e stain , although it quickly decolorises other Gram-positive bacteria. For th e dem onstration f th e acid-fast property th e ZiehlIt is best Neelsen st ain of carbol-fuchsin is mo stly employed. aided by t e application of heat, to en sure its be tter penetration. rule the whole bacillus is evenly stained, bu t sometimes, as almost always with Ehrlich’s aniline gentian-violet, th e bacteria may exhibit granular coccal formation.
Explanations
the acid-fast property.
n th e early days two theories were formulated by E hrlich to exp lain th is condition f acid-fastness. Th ey both tre ate it s physical problem, an d a s d ue to th e imperm eable na ture of th bacterial envelope. This att itu de was strongly opposed by Unna, who regarded chemical explanation as more probable, although he admitted th e physical explanation as a possibility. 1904 Bulloch and McLeod isolated in purity, from qu an tity of th e bacilli, alcoholic ax which was very stron gly acid-fast. They ascribed th acid-fast property of th e bacillus to th e presence this wax. This substance, however, was only present in relatively small quantity bulk of the constituents of th e bacteria being non-acid-fast. Certain ot he r snbstancea f a fa tt y natu re, such as lanoline and beeswax, have been found to possess th e prop erty f acid-fastness in a m arke degree. Baumgarten (1 11 regards the acid-fastness as presence f u nsa turated fa tt y acids. According to Helbig, quoted by Bechhol (1 2), the acid-fast property is to be ascribed to the presence of keratin, which, as is well known, in comm on wit.h similar substances, possesses t hi s char c eris ic. AUTHOR’S OW
EXPERIMENTS.
The exp eriments given below aim to show th at acid-fastness is mainly a physical phenomenon, th at th e stain is retained by the im-
GRAM-POSITIVE AND
CID-FAST PROPERTIES.
20
permeable character of the bacterial envelope, which thus prevents solvents from reaching the contained stain. in the experiments already described, e same course of procedure has been adopted to demonstrate to what extent the stain penetrates the cell and whether it is firmly bound in the substance the cell, or whethe it is merely retained there by the protection afforded by the outer covering the cell TRE GRAM-STAINING EACTION THE TUBERCLEACILLUS
penetration of the nietiryl-violet Experiment I.-To show the degree aniline gentian-violet and of the iodine solution.-Films f tubercle bacilli were stained with warm methyl-violet and aniline gentian-violet, respectively, for five minutes; treated with alcohol for five minutes; and dried. Under th microscope th e bacilli in the methyl-violet film showed slightly more marked granular staining than th e other. The films were then crushed under the cover-glass, and a counter-stain f neutral red applied for half a minute, with the object of demonstrating any unstained portions of the crushed bacteria. Examination showed very little colourless or red-stained material in the crushed area, despite th e granular staining noted in the bacilli. Similar film were stained with warm aniline gentian-violet for five minutes and then with Lugol’s iodine solution for three minutes. crushing it was found that the no trace of blackish purple iodine compound existed throughout. There the original purple stain. We see from this experiment that both the com ponents of Gram-stain have penetrated throughout the greater part of th e cell substance. Experiment 2.-Showing that bacilli whkh had been stained with methylviolet alone, by &am’s method, could be decolorised after crwhing.-l?ilms of tubercle bacilli were stained in the previous experiment with methylviolet and by Gram’s method respectively. They were then treated with alcohol for five minutes, and counter-stain of neutral red applied for half minute. Examination showed practically no Gram-negative substance. Th Gram-stained bacilli were purplish-black. Those stained with methyl-violet alone were a bright violet colour. Portions of the film were no treated by the crushing method above described, and the whole film then immersed in alcohol for one minute, when a good deal of violet colour was given from e darnaged part. Microscopic examination f a small part of the injured area showed amorphous masses of very pale purplish substance in both films. colour was slightly more marked in t e Gram-stained film. The films were then again immersed n alcohol for a further five minutes. Examination now still showed a faint purple colour the crushed substance of the Gram-stained film. This substance n e other film was almost colourless. neutral red counter-stain was next applied for half a minute. It was now found on both films that the crushed substance had completely taken up the red stain, and it appeared as amorphous, semi-translucent masses f homogeneous substance the thickes masses this substance s o m e small granules purple-stained matter showed occasionally. The intact bacilli in both films had retained th methyl-violet, and Gram-stain completely unaltered throughout. The stai thus appear slightly more strongly bound by the addition of iodine than without, but evidently the alcohol fastness of Gram-stain does not depend, t any great extent, on the iodine factor, n the case f the acid-fast tubercle bacillus. This experiment was then varied by crushing the bacterial film in one part before staining with methyl-violet, and by Gram’s method. the previous experiment the crushed portion failed to retain the stain when treated with alcohol.
H. ROPERTY ACID-FAST
BENIANS. TUBERCLE BACILLI;&
Experiment &--To demonstrate the penetration the stain.-pilms fixed over the flame were stained three minutes with hot carbol fuchsin decolorised with per cent. sulphuric acid for two minutcs, washed, and crushed at one end. The whole film was now restained with per cent. borax methyleneblue for one minute. Examination showed that the whole f the bacterial substance as still deeply stained with the fuchsin. There was no blue stained substance to be seen. Experiment 4.--To show deeolorisation the crushed bacilli.-Three films were prepared and stained as above with carbol fuchsin. These wer then treated with per cent. sulphuric acid for two minutes, and the counter stained with methylene blue. Microscopic examination showed that practically no part of the film had taken up the blue stain. portion f each film was now crushed, and the filme were placed in solutions 25 per cent. and per cent. sulphuric acid in water, and in absolute alcohol respectively for one minute. Examination under t microscope showed that in the two films exposed to acid of various strengths, almost the whole of the stain had been removed froin the injurcd parts. diffuse pink tin t remained only n the larger amorphous masses. The film alcohol was also to a very large extent decolorised n the injured area, although not so completely as the two films in acid. On adding methylene blue for one minute the whole of e amorphous crushed material was completely overstained-a deep blue in the thicker parts, and a pale sky-blue in the thinner parts the film. The uncrushed bacilli in each case retained the stain intact. The amorphous material showed in places faintly stained granules otherwise it was completely homogeneous. No sign of a bacterial capsule was be seen. Experiment 5.-On daining reactions tubercle bacilli crushed i n bulk.The experiments on Gram and fuchsin staining were repeated with bacterial dkbris obtained by crushing the bacteria in a small agate mortar. very large proportion of th e bacteria were disintegrated by this method. in the preceding experiments, th e bacterial detritus failed to retain either Gram’s stain or the fuchsin It was neither alcohol- nor acid-fast to any appreciabladegree. Occasionally one noticed smell pieces, apparently derived from bacilli, broken across. Some f these had retained both the Gram and fuschin stains; others had become decolorised. Conclusions dra.tunfront the experiments on tubercle bacilli.
The above experiments bring out certain points which may be detailed here. The crushed substance of th e tubercle bacillus, in marked contrast to th e intact organism is readily stained. The stained tubercle bacillus, whether Ehrlich’s, Gram’s, or Ziehl’s its method be used, is permeated throughout th e greater pa rt substance. This is especially the case when Ziehl’s method is used. The stains are retained only in the intact organism. The crushed bacillary substance exhibits neither he Gram-positive n or th e acidfast property to appreciable extent. The acid-fast property overshadows that of alcohol (Gram) fastness, th at Gram-positive staining cannot be demonstrated in presence of e acid-fast property.
GRAM-POSITIVE AND ACID-FAST PROPERTIES.
PROBLEM ABOVE CONCLUSIONS APPLICATION NATURE ACID-FAST ROPERTY. t present we have direct evidence on two ma in points-(1) Th difficult perm eability of th e bacterial coat by even the most pow erful stains; (2 Th e imperm eability f th e bacterial co at during long periods by alcohol and by acid. The conclusions drawn from the above experiments lend support and substance to the theory of E hrlich, which supposed t e outer laye rs f th e organism to form an imperv ious coat. The work Bulloch and ML eod likewise finds application here. These observe rs, by analysis, located the site of the acid-fastmss in one particular con stituen t f th e bacterial substance. This con stituen is wax, and therefore is presumably by natu re impermeable by acids, and to a lesser exte nt by alcohol. e have, however, ac tual proof f tru e n atu re f the im perm eable layer. The acid-fast wax may possibly have been synthesised d uring th e process f extraction. we assume that it exists as such in the bacilliis, it most probably ente rs largely into th e composition f th e envelope. the other hand, it may e disseminated through out the bacillus, and some other impermeable substance, such keratin, may form a bacterial covering. Against this last suggestion, however, is the fact t rem nan t of an y such d efinite, acid-fast she ll is to be found in the crushed bacterial substance. t th e sam e time, the wax is probably not th e most exte rna l laye r of th e bacillus, as we find th t wa solvents such toluol and benzol do not readily remove th e acidfast prope rty from the bacteria. For th e present the evidence points to th e existence of a waxy laye r enclosing protoplasmic and fatty cell substance, and conferring on the organism the property of resisting the penetration acid and alcohol. conclusion, wish express my thanks to r. Bulloch advice and assistance in connection with this paper also to Dr. Paul Fildes and other workers this laboratory. have likewise to express thanks to the London Hospital Research Committee for financial assistance received in connection with the carrying out of this research. APPENDIX.
EXPERIMENTS TH SOLUTION
NEGATIVE.
USING HEATED UGOL'R O D I N E SUBSTANCES WHICH ARE NORMALLYBAM-
EFFECT
STAINING
The work of Neide was referred to by the present writer in the earlier part th e preceding paper. Neide showed th at many Gram negative bacteria could retain Gram's etain if a heated Lugol's
BENIANS.
solution was used in place of th e usual cold solution. Hisob servation is confirmed in the following experiments, and it is applied to the Gram-negative de tritu s obtained from crushed Gram-positive bacteria. Experiment 1.-To
show th at the me thyl-v iolet iodine compound after heating in iodine solution still remains soluble in alcohol.-A few drops of per cent. methyl-violet stain were added to excess of Lugol’s solution, wit
the result that a heavy purplish black precipitate was thrown down. This precipitate was freely soluble n alcohol. The precipitate was then treate on a slide with excess of Lugol’s solution, which was kept gently steaming for five minutes At th e end f this time the precipitate remained still freely soluble in alcohol. Experiment 2.---To show that coliform bacilli ret ain the Gr am compound when a heated iodine solution is used.-A film of coliform bacilli, negative by Gram’s method, was stained with methyl-violet, two minutes. t s then treated with Lugol’s solution, kept gently steaming for five minutes. Th film was then treated with absolute alcohol five minutes. The stain wa quite fast, but was of a more brownish coloiu tha n Gram’s stai is normally.
Experiment
how that n Gra mp ositive bu cferia compound s Jxed throughout the whol j’ the cell substance.-Films
Gram
of yeast and of staphylococcus were stained with methyl-violet and with heated iodine cover-slip, and the slides placed in alcohol for five minutes. None f th stain was discharged. counter-stain of neutral red was applied xamination now showed that the amorphous bncterial substance remained saturated throughout with the compound (Gram’s) stain. There was no red stained part.
Experiment k-To
Gram-positive bmt+
show that tAe amorphous de’bris obtained from crushed retains the stain heated iodine solution used.-
Films were made from th e crushed substance of e following bacteria :-yeast, sarcinae, staphylococcus, tubercle, which had been ground in a mortar as in previous experiments. These films were stained with methyl-violet an heated Lugol’s solution as described above. r five minutes’ treatment with alcohol it wm found th at practically the whole f each film retained th e stain. n each case a control film using a cold Lugol’s solution for th e same time wa made, and these th e great bulk f every film was decolorised in alcohol.
These experiments show that the methyl-violet-iodine compound does t become insoluble in alcohol heating. the presence of the bacterial substance, however, it becomes firmly bound, and then ceases to be soluble in alcohol. It is evident that the effect of heating the iodine solution is not merely make it penetrate more deeply, but to alter the whole nature of the reaction, and change a physical into chemical process. REFERENCES. BAUMGARTEX. 2. BECHHOLD
. .
3. BIENSTOCK 4. BRUDNY
.
“Lehrbruch der pathogenen Mikro-organis641. men,” 1911, “Die Kolloide in Biologie und Medizin, 1912, S. 408. Fortschr. Med., Berlin, 1886, Abth. I. Ed. iv. 193. Centralbl. Bakteriol. u. Parasitenk., Jena, 1908, Abth. II., Bd. xxi. S. 62.
G R A M - P O S I T I V E AND ACID-FAST PROPERTIES. BULLOCH 6. CLAUDIUS .
.
M'LEOD
DREYER,KRIEQLER, AIXLEYW A L K E R
.
8. GOTTSTEIN
9. GRAM 10 GRIMME
MUCH NEIDE 13 U X N A 14
.
. .
. . . . . .
.
15--1~. OF rmi.-voL
.
Hy3., Cambridge, 1904, vol. iv. p. Ann. l'lnst. Pmteur, Paris, 1897, tome xi. p. 332. Journ. Path. and Bacteriol., Cambridge, 1911, vol. xv. p. 134. &fed., BerIin, 1886, Abth. I. Fortsclw. Bd iv S. 252. Ibid., 1884, Abth. I., Bd ii. S. 185. Centralbl. Bakteriol. Parasitenk., Jena, 1902, Abth. I. Orig. Bd xxxii. S. 172. xlv. 691. Berl. klin. Wchnsclw., 1908, Centralbl. Bakteriol. Parasitenk., Jena, 1904, Abth. I., Orig. Bd xxxv. S. 518. Ibid., 1888, d. xxxv. S. 218. Nomtsh. f: prahf. Dermatol., Hamburg, Erganzungsheft, 1887. Journ.
