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tK 11401A110NAI .10VRNAL Or Lt IIRDSY^ Volume 46, Number 4Printed in the U.S.A.

Electron Microscopic Study of Colonies of

Mycobacterium lepraemurium '

S. Okada, M. Nishiura, T. Ogawa and T. Mori 2

It has already been confirmed that M. lep-raemurium can he cultivated on Ogawa's eggyolk medium by his method, and that the ba-cilli subcultured on the medium are surely

lepraemurium ( 1",13. 14 ). The coloniesof murine leprosy bacilli grown on Ogawa'segg yolk medium were observed with scan-ning electron microscopy (SEM) by the au-thors. Thus far there has been little study ofbacterial colonies by SEM L 2, 64. II. 16-22 ).

This paper deals with the relationships be-tween the gross morphology and microstruc-ture of colonies of murine leprosy bacilli bySEM, and the chemical nature of extracellu-lar material in the colonies as clarified bytransmission electron microscope (TEM)observations of ultrathin sections of the col-onies stained with ruthenium red.

MATERIALS AND METHODS

The colonies of the Hawaiian strain of M.lepraemurium Subcultured on Ogawa's eggyolk medium slants were observed withSEM. The rough type colonies of the 13thto the 14th subculture incubated for one tothree months, and the smooth type coloniesof the 14th to 15th subculture incubated forone to four months were used. Cold 2.5Ci to

Outaraldehyde in M/15 phosphate buf-fer (p11 7.2) was poured gently into the testtube containing the Ogawa's egg Yolk me-dium, and the test tube was left in the refrig-erator for 18 to 24 hours. After fixation, thefixative was removed and the test tube wascut transversely near the colonies. The siteof medium having the colonies was cut offthinly and dehydrated by means of a gradedethanol series. Thereafter, the ethanol was

Recei ed for publication 7 March 1978.'S. Okada. M.D., Associate Professor and NI. Nishi-

ura. M.D.. Professor. Leprosy Research Laboratory,K oto Unkersity School of Medicine, Sak\o-ku, Kyo-to, Japan; 'I. Ogav,a, NI.D., f o od Ilygiene Center. Kita-sato Institute, Minato-ku, ok^I. Mori,M.D.Sci., Associate Professor. Department of Leprol-op . . Research Institute lor Microbial Diseases. OsakaUnkersit■. Suita City, Osaka..lapan.

replaced with iso-amyl acetate. The colonieswere weakly adherent to the medium, andfrequently dropped off. In such an event theslice of medium from which the coloniesdropped off was removed, and the processdescribed above was carried out withoutpinching the colonies with forceps. Iso-amylacetate being replaced with liquid CO,, thecolonies were dried in a critical point dryer,HCP-1. Also when the colonies were put intothe critical point dryer, the colonies whichdropped off the medium were sucked intoa pipette with iso-amyl acetate and pouredon the filter paper. The filter paper havingthe colonies was put into a wire basket andplaced in the chamber of the dryer. Colo-nies thus dried in the critical point dryer werecemented to aluminum stubs with adhesive.This was done with care not to distort thecolonies by instrumental pinching. This wasaccomplished by lifting them by the forcepstip to which a small amount of adhesive wasattached, and then transferring them to theadhesive which was on the aluminum stub.After the adhesive dried and hardened, thecolonies were coated with iz,old in an ion-spattering apparatus (Giko 113-3). Somerough type colonies were dried by the freeze-drying method without use of organic sol-vent. and their features under SEM werecompared to those of the other rough typecolonies which were grown on the same slantand dried by the use of organic solvent, inorder to examine whether the organic solventused in the process of drying of the specimensaffected the features of the colonies. Addi-tionally, sonic rough colonies were disruptedgently with a Potter-Elvehjem type homo-genizer, stained negatively with phospho-tungustic acid solution in phosphate buffer(p11 7.4), and observed by TEM.

Ultrathin sections of the rough type col-onies stained with ruthenium red were ob-served with TEM. Following Lulls method( 12 ), the colonies were fixed in a refrigera-tor for two hours with 2.5C (" glutaraldehvdein 0.05M cacodyktte buffer (pH 7.4) in which

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4f, 4^Okada et al: Ell Study of Colonies of M. lepraemurium^365

ruthenium red was dissolved at a concentra-tion of 0.5 mg/ ml. Then they were treatedfor four hours in a refrigerator with 1Ci, solu-tion of osmium tetroxide in 0.05M caco-dvlate buffer (p1I 7.4) in which rutheniumred was dissolved in a concentration of 0.5mg/ ml. Subsequently, the specimens wererefixed in a refrigerator for three to fourhours with Icy solution of osmium tetroxidein 0.05M cacodvlate buffer (pH 7.4). Afterrefixation, they were dehydrated with thegraded ethanol series, embedded in metha-crylate resin, ultrathin-sectioned, and ob-served with TEM.

Some rough type colonies were treatedwith chloroform in the process of prepara-tion. After dehydration with ethanol, theywere first immersed in a I : I mixture of I 00(:( ,

ethanol and chloroform for 30 minutes, thenin chloroform for one hour, next in a I: I mix-ture of chloroform and iso-amyl acetate forone hour, and finally transferred to iso-amylacetate. After this they were dried in the crit-ical point drying apparatus. Other coloniesgrown on the same slant were preparedwith the ordinary method.

SEM used in this study was the Hitachifield-emission type SEM, HES-2S, whichwas operated at 25 KV, and TEM is Aka-shi's AFM-80.

RESULTS AND DISCUSSIONIn the typical rough type colonies of mu-

rine leprosy bacilli, filamentous strandswhich were 40-148 mp ( chiefly 70-120 mp )in diameter, were observed between the ba-cilli; and granules which were not spherical,but disc-like, and 60-300 mp (chiefly 100-200 mp ) in diameter, were occasionallyfound on the bacilli or filamentous strands(Fig. I). These were not observed in thesmooth type colonies, save that rarely shortbridges were found between the bacilli (Fig.2).

Among the colonies which were of roughtype macroscopically, some had many inter-bacillary filaments and granules, but somecolonies on the other slants had interbacil-lary filaments and granules only partially,and other colonies had only interbacillaryfilaments partially. Subcultivation of roughcolonies repeated many times on Ogawa'segg yolk medium caused a change of typefrom rough to smooth in many colonies. Thedisappearance of interbacillary filaments and

granules occurred prior to the macroscopicchange of colony to the smooth type. Thecolony described above which had interbac-illary filaments only partially is consideredto have been in transition from rough tosmooth type, and to be not a typical roughcolony, though it showed a rough surfacemacroscopically. Therefore, in order to com-pare some aspects of the rough type with thesmooth type, macroscopic discrimination be-tween rough and smooth type is insufficient.Discriminative determination by means ofSEM is needed.

The following studies were made to de-termine %vhether or not the interbacillary fil-aments described above are an artifact. First,the rough type colonies were dried by freeze-drying without the use of organic solvent andobserved with SEM. The interbacillary fila-ments were observed also in these specimens(Fig. 3). Then rough type colonies, gentlydisrupted with a Potter-Elvehjem type ho-mogenizer, were suspended in a smallamount of physiological saline solution neg-ative-stained with phosphotungustic acidsolution in M/ 15 phosphate buffer (pH 7.4),and observed with TEM. Filaments whichwere 25-120 nip in diameter were observed(Fig. 4). Therefore, it can be said that theinterbacillary filaments are not an artifactformed by the use of organic solvent.

Next, the rough type colonies were stainedwith ruthenium red dissolved in the fixatives.Previously, leprosy bacilli and murine lep-rosy bacilli in vivo had been stained withruthenium red by one of the authors, Okada( 15 ). The bacilli isolated from human lepromaor murine leproma were stained with ruthe-nium red during fixation, ultrathin-sectioned,and observed with TEM. A coat stained withruthenium red which surrounded the outsideof the cell wall of bacilli and having a jag-ged outer margin was observed on both M.leprae and Al. lepraemuritan. This findingindicates that both bacilli have a layer ofcomplex carbohydrate outside the cell wall.Some leprosy bacilli and murine leprosy ba-cilli in vivo do not have this coating. Theabsence of the coating on these bacilli maybe due to its removal during the isolation ofbacilli from the lesion. Murine leprosy bacil,li grown in vitro also have the same coatingas that of bacilli in vivo (Fig. 5). When thebacilli adhered to each other, the coating wasabsent at the adhering site. The margin of

1978366^ International Journal of Leprosy

FIG. I. Rough type colony of murine leprosybacilli. The filamentous strands arc present be-tween bacilli, and many granules are observed onthe bacilli or filamentous strands. X 35,000, scalebar: 0.2p .

FIG. 3. Freeze-dried rough type colony of mu-rine leprosy bacilli. The interbacillary filamentsare present also in this specimen. X 35.000, scalebar: 0.2p .

FIG. 2. Smooth type colony of murine leprosybacilli. Neither interbacillary filament nor gran-ule can he found. X 35,000, scale bar: 0.2 p .

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•

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4FIG. 4. Negative staining of rough type colony

of murine leprosy bacilli which were destroyedgently and suspended in physiological saline solu-tion. The filamentous strands are observed. M:murine leprosy bacilli: F: filamentous strand.X 14.000, scale bar:

46, 4^Okada et al: EA/ Stith of Colonies of M. lepraemurium^167

interhacillary filament was stained with ru-thenium red and presented a tubule-likeappearance (Fig. 6). Filaments sectionedtransversely presented a ring-like appear-ance (Fig. 7). In the section of a colony notstained with ruthenium red, the interhacil-lary filament could not he seen clearly be-cause of its low electron density.

Judging from these facts, it can he con-cluded that the interbacillary filament is notan artifact produced in the process of prep-aration of the specimens.

Drucker and Whittaker reported that thewell-separated, tangled filamentous bacilligrowing vertically as well as laterally, are nodoubt responsible for the rough appearanceof colonies of Nocardia ,i;raminis NCTC 4728( 7 ). They also observed that the cells werehaphazardly arranged within rough coloniesof .S'Ireptomyces scabies and Cor•nchacte-rium xerosi.s . , and that the smoother coloniesof ilerococcus ruhrum, andVibrio metchnikovi consisted of microor-ganisms which were regularly arranged ( 6 ).Takagi and Katsumoto studied colonies ofS and R type of Salmonella and Shigellagroups with SEM. They attributed the dif-ference in gross morphology between S andR type to the difference of arrangement ofbacilli in these colonies. They concluded thatthe smoothness or the roughness of coloniesis related to whether the surfaces of cell bod-ies growing within colonies are hydrophilicor hydrophobic ( 17. ). In the present studyof murine leprosy bacilli, no difference ofarrangement of bacilli in the R and S colo-nies was observed. Therefore, the cause of thedifference in gross configuration of the colo-nies of microorganisMs is not uniform amongthe different species of microorganisms.

Elmros and his colleagues ) studied col-onies of Neisseria gonorrhocae with SEM.Virulent colonies of Neisseria ,i,rotiorrhocaehave highly convex surfaces while coloniesof avirulent strains exhibit a radial extensionand flat upper surfaces, though the differenceof gross form between both strains of thismicroorganism does not relate to the smooth-ness or roughness of the surface of the colony.An abundance of intercellular strands wasfound between cells in virulent colonies, butnot in the avirulent colonies. They held thatthese strands seemed to anchor the cells toeach other and to the agar surface and thatthe presence of such structures explained

the highly convex surface of virulent colonies.Kraus and Glassman ( ) also observedsuch strands in colonies of virulent types ofgonococci.

In our ultrathin sections of colonies stainedwith ruthenium red, the homogeneous sub-stance stained with ruthenium red filled upthe space between clustered bacilli (Fig. 8).The homogeneous substance was connectedwith the coating of bacilli which stained withruthenium red, having the same density asthat of the coating. Accordingly, the homoge-neous substance also consists of complexcarbohydrate. In the colony ollerved withSEM, the space between bacilli was some-times filled with a homogeneous substance.In some cases the marked deposit of homo-geneous substance concealed the contour ofindividual bacilli (Fig. 9). The homogeneoussubstance observed with SEM seems to heidentical to the substance which stained ho-mogeneously with ruthenium red in ultra-thin sections, and accordingly it also consistsof complex carbohydrate. In papers dealingwith the SE NI studies of other bacteria,some authors have designated such homoge-neous extracellular substance as "coveringfilm" or "surface film" ( 6 . 7,2 ' ). However theterm "film" is not appropriate, judging fromthe distribution of the substance shown inFigure 8. In the colony of some species ofmvcobacteria, the amount of this substanceobserved with SEMI is considerable. It canthen he held that the amount of complexcarbohydrate included in the colony is quiteconsiderable in such a case.

In the rough type colony treated with chlo-roform after dehydration with ethanol, inter-bacillary filaments were observed with SEMin the same manner as in control specimensnot treated with chloroform (Fig. 10). Draperand Rees ( 3-5 ) observed filamentous or tape-like substances on the surface of murine lep-rosy bacilli or attached loosely to the bacilliwhich were isolated from livers or spleensof mice and thought that these composed theelectron transparent zone surrounding mu-rine leprosy bacilli when seen in host cells.They at first regarded this material as waxD s and later as mycoside C. The interbacil-lary filaments found with SEMI in the roughtype colony in our study did not disappearon treatment with chloroform. Therefore, itcan he concluded that it is not mycoside C,and is a newly found structure which is dif-

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368 International Journal of Leprosy 1978

' Tod*^'4091

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7 I 8

46, 4^Okada et al: EM Study of Colonies of M. lepraemurium 369

1 0_711•11POININIP7.^ MMOr^41.111S■

FIG. 9. Colony of marine leprosy bacilli. The^Fio. 10. Rough type colony treated with chlo-extracellular substance fills up the space or valley roform. The interhacillary filaments and granulesbetween bacilli, and conceals the contour of each are observed also in this specimen. X 35,000,bacillus. X 17,500, scale bar: 1p scale bar: 0.2p .

ferent from the substance found by Draperand Rees and interpreted as composing thetransparent zone surrounding bacilli withincells.

Rough type colonies incubated for one tothree months and smooth type colonies in-cubated for one to four months were exam-ined, but no differences were found in colo-nies incubated for different periods of time.

FIG. 5. Colony of murine leprosy bacilli stainedwith ruthenium red. The coat stained with ruthe-nium red surrounds the outside of bacilli, showingthe jagged outer margin. The density of bacilliis low, because they were stained neither withuranium nor lead. When the bacilli adhere to eachother, the coat is absent at the adhering site. M:murine leprosy bacillus; C: coat of bacillus: A:adhering site of two bacilli. X48,000, scale bar:0.2p.

FIG. 7. Rough type colony of murine leprosybacilli stained with ruthenium red. The interbac-illary filament sectioned transversely presentsthe ring-like appearance, the margin being stainedwith ruthenium red (arrow). X 57,500, scale bar:0.2 p .

FIG. 6. Rough type colony of murine leprosybacilli stained with ruthenium red. The marginof interbacillar■„' filaments are stained with ru-thenium red, and presented the tubule-like ap-pearance (arrow). M: murine leprosy bacillus.X 70,000, scale bar: 0.2p .

FIG. 8. Colony of murine leprosy bacilli stainedwith ruthenium red. .1 he homogeneous substancestained with ruthenium red fills up the space orvalley between bacilli, and is connected with thecoat of complex carbohydrate surrounding thecell wall of bacilli. NI: murine leprosy bacillus:C: coat of bacillus; II: homogeneous substance.X 70,000, scale bar: 02p .

370^ International Journal of Leprosy^ 1978

SIJMNIARY

Colonies of the Hawaiian strain of murineleprosy bacilli, grown on Ogawa's egg yolkmedium, were observed with a scanning elec-tron microscope. In the rough type colony,filamentous strands which were 40-148 mp( mostly 70-120 mp ) in diameter were foundbetween the bacilli and designated as "inter-bacillary filament." Additionally, disc-likegranules which were 60-300 mp ( mostly100-200 mp ) in diameter were occasionallyobserved on the surfaces of bacilli and inter-bacillary filaments. Interbacillary filamentand granules such as those found in the roughtype colony could not he found in the smoothtype colony. The interbacillary filament is notan artifact produced in the process of prep-aration of a specimen but is a newly foundstructure which is different from the fila-mentous or tape-like substance previouslyregarded by Draper and Rees as composingthe transparent zone surrounding murineleprosy bacilli within host cells.

The cause of differences in gross config-urations of R and S type colonies is not uni-form among the different species of microor-ganisms.

The homogeneous extracellular substancewhich fills the space between bacilli in col-onies of murine leprosy bacilli is a complexcarbohydrate and is connected with a coatingof complex carbohydrate surrounding the cellwall of bacilli.

RESUMENSe estudiaron las colonias del bacilo de Ia lep-

ra murina, cepa Hawaii, crecidos en medio deOgawa con yema de huevo, usando Ia micro-scopia electrdnica de barrido (scanning). En lacolonia del tipo rugoso, se observaron estructurasfilamentosas de 48 a 148 mp de didmetro (en sumayoria de 70 a 120 mp ) las cuales se denomina-ron como "filamentos interbacilares." Adermis,ocasionalmente se observaron grdnulos dis-coides de 60 a 300 mp de didmetro (en su ma-yorra de 100 a 200 mp ) sobre Ia superficie de losbacilos y de los filamentos interbacilares. En lascolonias del tipo liso no se observaron ni filamen-tos interbacilares ni estructuras discoides. Elfilamento interbacilar no es un artefacto produ-cido durante la preparacidn de Ia muestra sinoque es una estructura recientemente encontradaque es diferente de la substancia filamentosa oacintada considerada previamente por Draper yRees como componente de la zona transparente

que rodea a h^I.0ti hacilos^.a .epra murina denimde las cdlulas hudsped.

La causa de las diferencias en las configura-ciones gruesas de las colonias de los tipos K yS, no es uniforme entre today las diferentesespecies de microorganismos.

La substancia extracelular hornogdnea queIlena el espacio entre los bacilos en las coloniasdel .11. lepracmcrim, es un carbohidrato comple-jo que estd conectado con una capa do carhohi-drato complejo alrededor de la pardd celular delos bacilos.

RÉSUMÉ

On a dtudid, au moyen du microscope dlec-tronique it balavage, des colonies de Ia souche ha-waienne de hacilles de Ia lepre murine, cultivdesstir le milieu au jatine d'oeuf d'Ogawa. Dans Iacolonic de type "rough", des extensions filamen-teuses d'un diametre variant de 40 a 148 nip (leplus souvent entre 70-120 mp ) one dtd obser-vdes entre les bacilles; on les a nommdes "fila-ments interbacillaires". De plus des granules enforme de disque, d'un diametre de 60-300 nip(principalement 100-200 mp ), ont dtd ob-servds, a ('occasion, sur Ia surface des hacilleset des filaments interbacillares. Filaments inter-bacillaires et granules tels que ceux qui ont dtdobservds dans les colonies de type "rough" n'ontpas pu titre retrouvds dans les colonies de type"smooth". I.e filament interbacillaire n'est pas unartdfact qui serait produit an cours de la prdpa-ration de I'dchantillon, mats bien une struc-ture entierement nouvelle, diffdrente de la sub-stance filamenteuse ou en ruban que Draper etRees on considdrd jadis comme faisant partiede la zone transparente qui entoure les hacillesde Ia lepre murine a l'intdrieur des cellules quiles hdbergent.

La cause des diffdrences obscrvdes dans lesconfigurations des colonies de type R et S n'estpas uniforme pour les diffdrentes espeees de mi-croorganismes.

La substance extra-cellulaire homogene quiremplit l'espace entre les bacilles dans les colo-nies de bacilles de la lepre murine, est un hydratede carbone complexe; elle est en contact avecun rev&ement d'hydrates de carbone complexesqui entoure Ia paroi cellulaire des bacilles.

Acknowledgments. We are grateful to Mr. M.Fujioka, Department of Pathology, Kyoto Univer-sity School of Medicine for his excellent techni-cal assistance in the operation of SEM. This workwas supported in part by a grant from the U.S.-Japan Cooperative Medical Science Program.

46, 4^Okada et al: EM Study (Y . Colonies of M. lepraemurium^371

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