JOURNAL OF BACTERIOLOGY, Oct. 1971, p. 515-525Copyright 0 1971 American Society for Microbiology

Vol. 108, No. IPrinted in U.S.A.

Microstructure of Colonies of Rod-ShapedBacteria

D. B. DRUCKER AND D. K. WHITTAKER

Department of Bacteriology and Virology, University of Manchester, Manchester, 13, England, and Departmentof Oral Medicine and Oral Pathology, Welsh National School of Medicine, Cardiff, CF4 4XY, Wales

Received for publication 19 July 1971

Whole colonies of Bacillus cereus, B. megaterium, B. mycoides CN2495, Coryne-bacterium hofmanni NCTC 1938, Escherichia coli, Lactobacillus acidophilusNCIB1899, Nocardia graminis NCTC4728, Pseudomonas viscosa, and Serratiamarcescens were prepared for scanning electron microscopic examination by freeze-drying and metal-coating. The arrangement of individual cells within colonies couldbe seen. Cells of Bacillus colonies tended to be longer than in liquid culture and irreg-ular in shape and to give the appearance of branching. B. megaterium colonies fre-quently had a dense covering film. Colonies of gram-negative bacteria consisted offairly short rods covered by much adherent extracellular material. L. acidophilushad colonies comprised of densely packed, well-oriented rods. C. hofmanni coloniescontained coccobacilli, packed together. Correlations were observed between plano-convex colony form and densely packed cells, rough colony form and random ar-

rangement of well-separated microorganisms, and irregular colony edge and ten-dency of cells to grow out from the colony in filaments.

Light microscopy permits studies on the initia-tion of colony formation (6) but is of limited usefor the examination of mature colonies. How-ever, the advent of the scanning electron micro-scope has made possible the study of unsectionedmaterial at a greater resolution (250 nm) thanobtains in conventional light microscopy (2, 5).This, coupled with the 200x greater depth offield, makes the scanning electron microscopeideally suited to observation of bacterial cells.The instrument has been used by Williams andDavies (I 1) who examined Actinomycetes, byKlainer and Betsch (7) in their work on the sur-face morphology of liquid grown cells of Staphy-lococcus aureus, Streptococcus pyogenes, Esche-richia coli, and Proteus vulgaris, by Klainer andPerkins (8) in their studies on antibiotic-treatedcells, by Bulla et al. (4) and Murphy and Camp-bell (9) in their examination of bacterial spores,and by Barnes et al. (1) in their work on Can-dida albicans. More recently, techniques weredeveloped (10) which made possible the prepara-tion of whole bacterial colonies for scanning elec-tron microscopy; intact colonies of Streptococcusmutans OMZ61, Streptococcus sp. D182, Staph-ylococcus aureus NCTC 6571, and Candida albi-cans type A MRL 3153 were examined. Thereappeared to be a correlation between plano-convex colony form and uniform distribution ofcocci within the colony. Because the possibilities,

with regard to colonial "microstructure," arerather limited in the case of spherical bacteria,the previous work has been extended to includerod-shaped microorganisms in an attempt to dis-cover other relationships between colonial micro-structure and gross morphology.

MATERIALS AND METHODSBacterial strains. The following strains were used:

Bacillus cereus, Bacillus megaterium, Bacillus my-coides CN 2495, Corynebacterium hofmanni, E. coli,Lactobacillus acidophilus NCIB 1899, Nocardia gram-inis NCTC 4728, Pseudomonas viscosa and Serratiamarcescens.

Growth of colonies. L. acidophilus was grown for 48hr in 5% CO2-95% N2 on "'Oxoid" Rogosa agar. N.graminis was grown aerobically on 5% (v/v) horseblood-Oxoid nutrient agar for 48 hr. The remainingorganisms were grown aerobically, on Oxoid nutrientagar no. 2 for 48 hr, in the case of P. viscosa and, for24 hr, in the case of the other organisms. All plateswere incubated at 37 C.

Freeze-drying of colonies. Colonies were photo-graphed before removal from plates on agar blocks andagain after drying to measure shrinkage, which wasminimized by rapid freezing by immersion in a 2-methyl butane freezing mixture cooled in liquid ni-trogen. Agar blocks carried on aluminum foil wereheld at - 159.9 C for I min and then freeze-dried for24 hr at 10-3 Torr in a 500-ml culture vessel (Quickfit)initially cooled in dry ice-acetone. The temperature ofthe culture vessel was allowed to rise to ambient tem-

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perature over a 12-hr period.Metal-coating of colonies. Dried colonies were ce-

mented to scanning electron microscope stubs andplated, while rotating in a vacuum of 10-5 Torr, with15 mg of Au-Pd alloy (60-40) at a distance of 10 cm.Coated colonies were photographed in a CambridgeInstrument Stereoscan.

RESULTSSamples showed little sign of distortion or

shrinkage (usually <10%) and compared favor-ably with samples obtained earlier (8) by using acooled planchet in a bell-jar.Colony microstructure. B. cereus exhibited

some evidence of cell orientation in the center ofcolonies. Little extracellular material, e.g., gumor covering film, was seen (Fig. 1). Individualcells were 0.4 to 1.0 by 1.1 to 3.6 ,um and werefrequently distorted by opaque spherical struc-tures 1.0 ,Am in diameter, presumably spores.

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Divided cells were incompletely separated andwere occasionally joined by "bridges" (Fig. 2).At the edge of the colony, cells were moredensely packed, and extracellular material wasnoted.

Colonies of B. megaterium also showed local-ized-cell orientation (Fig. 3). In the center ofcolonies, some extracellular gum was apparent,and cells gave the appearance of true-branching,although this requires further investigation (Fig.4). Spores were occasionally seen in the cellswhich were 0.5 to 1.0 by 1.0 to 14.8 ,um; spore-bearing cells frequently appeared club-shaped.Separation of cells was incomplete. Cells at theedge of the colony showed more dense packing,and more extracellular material was present.Long filaments were noted growing out from thecolony (Fig. 5). Occasionally, a dark coveringfilm was seen on the surface of the colony; thisobscured the underlying cells.

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FIG. 1. Organisms in the central area of a Bacillus cereus colony show localized orientation. There is little ex-tracellular material. Bar represents 10 ,im.

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FIG. 2. Central area of a Bacillus cereus colony examined in the scanning electron microscope. Note the cel-lular morphology. Bar represents 2 ium.

Examination of the "rami" growing from a B.mycoides colony revealed that each "ramus"consisted of rods 0.5 to 1.0 by 1.4 to 3.6 gmwhich showed little overall orientation (Fig. 6).Cells at the top of a ramus were occasionallyflattened, and spherical intracellular bodies couldbe seen distending the outline of the cells (Fig. 7).B. mycoides cells were sometimes coated withextracellular material.

C. hofmanni colonies were comprised of eithervery short cells (0.3 to 0.5 by 0.5 to 1.0 uin),which appeared to be covered by a dense film, orlarger cells (0.3 to 0.7 by 0.5 to 1.5) densely, yetrandomly packed, without any covering film(Fig. 8).Colonies of E. coli revealed regular separation

of cells (0.3 to 0.5 by 1.0 to 2.0 ium, Fig. 9) withassociated extracellular material. At the pe-riphery of the colony, cells were more orientedand more densely packed with less extracellular

material. Some colonies were covered by a filmwhich was perforated by holes, 0.2 to 1.0 gm indiameter. The film totally obscured the bacteriabeneath it.

Colonies of L. acidophilus were seen to consistof strongly oriented, irregular, tightly packedrods approximately 1 gm in width and 3 to 10,um in length; no extracellular material was ob-served (Fig. 10).A tangled mass of filaments was observed in

colonies of N. graminis. Individual cells were 0.5to 1.0 Am in width and over 20 gm in length(Fig. 11). No extracellular material was ob-served.

P. viscosa colonies consisted of randomly ar-ranged microorganisms (0.5 by 1.4 ,im) coatedwith an adherent extracellular material which didnot obscure the outlines of the cells but appearedrather to form a supporting skeleton for them(Fig. 12).

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FIG. 3. Scanning electron micrograph of organisms in the center of a Bacillus megaterium colony. Note theincomplete separation of cells. Bar represents 2 Am.

FIG. 4. Note the localized-cell orientation in the center of this Bacillus megaterium colony. Bar represents

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FIG. 5. Edge of a Bacillus megaterium colony showing covering film and long filamentous cells growing outfrom the colony. Bar represents 10 ,im.

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FIG. 6. Chains of B. mycoides cells in a colony "ramus" near the agar surface. Note the lack of overall orien-tation of bacteria. Bar represents 10 ,um.

FIG. 7. Scanning electron micrograph of B. mycoides cells in the upper portion of a colony "ramus." Noteflattened appearance of cells. Bar represents 5 um.

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*}~~~~~~~~'̂ 'w" .Al1FIG. 8. Occasionally, colonies of Corynebacterium hofmanni had no covering film. Scanning electron micro-

graph reveals tightly packed cells in the center of the colony without any covering film. Bar represents 2 um.

Colonies of S. marcescens resembled those ofE. coli except that the cells were more com-

pletely covered by extracellular material whichresulted in a perforated-surface covering film.Cells were not closely packed but showed regularseparation and were slightly shorter (0.5 to 1.0,um) than bacteria in an E. coli colony. At theperiphery of the colony, cells were more closelypacked.

DISCUSSIONWell-defined covering films were seen only on

colonies of B. megaterium and C. hofmanni.This is in contrast to the more widespread occur-

rence of covering films on colonies of cocci (10).The appearance of branching forms in certainBacillus colonies might be due to extracellularmaterial having concealed incomplete separationof two or more rods. Thin intracellular"bridges" (7) might be either genuine cellularextensions of adherent slime; however, L. fer-mentis appears to show intracellular bridges (3)even after washing and centrifuging of cells,which would presumably separate cells held to-gether only by slime. The long filaments growingout from the edge of B. megaterium colonies(Fig. 5) might be responsible for the irregularedge of such colonies and would explain the flat-tened appearance of B. megaterium colonies. The

rami of B. mycoides colonies appeared to consistof unorientated, fairly short rods rather than thelong, parallel bundles of rods which might beexpected. However, it is possible that growth of aramus occurs by extension of parallel filamentsand that later development of a ramus consists ofthe growth of an outer layer of shorter cells.The association of densely packed colonies

with plano-convex colony form, previously shownin the case of cocci (10), holds true for C. hof-manni, whose colonies consist of coccoid forms.A similar association is found in the case of L.acidophilus, even though individual cells in thecolony are distinctly rod-shaped. The orientationof Lactobacillus cells is not noted in the case ofcolonies of the other rod-shaped bacteria exam-ined. This may be a reflection of the mode ofdivision of the cells or their differing surfacecharacteristics.The colonies of gram-negative bacteria consist

of cells obscured by adherent material probablyof bacterial origin. In the hydrated state, suchmaterial might contribute to the glossiness ofcolonies.The macroscopical appearance of Nocardia

colonies differed from that of the other orga-nisms examined. This difference was paralleledby differences in colonial microstructure. Thewell-separated, tangled filaments, growing verti-

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FIG. 9. Scanning electron micrograph showing arrangement of cells in an Escherichia coli colony. Note theextracellular material. Bar represents 5 Am.

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FIG. 10. Colonies of Lactobacillus acidophilus consist of strongly orientated, closely packed rods. Bar repre-sents 10 Am.

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FIG. 1 1. Tangled filaments of Nocardia graminis seen in the stereoscan. Bar represents 5 ,m.

FIG. 12. Scanning electron micrograph of a Pseudomonas viscosa colony. Cells are almost obscured by extra-cellular material. Bar represents 20 ,um.

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cally as well as laterally, are no doubt respon-

sible for the rough appearance of colonies of thisorganism.

ACKNOWLEDGMENTS

We thank J. Hearle for use of his scanning electron micro-scope, N. Preston for the strains used in this investigation, andB. Chapman, J. Hutton, and A. Williams for their assistance.

LITERATURE CITED

1. Barnes, W. G., A. Flesher, A. E. Berger, and J. D. Arnold.1971. Scanning electron microscopic studies of Candidaalbicans. J. Bacteriol. 106:276-280.

2. Boyde, A., and P. J. Knight. 1969. The use of scanningelectron microscopy in clinical dental research. Brit.Dent. J. 127:313-322.

3. Boyde, A., and R. A. D. Williams. 1971. Estimation of thevolumes of bacterial cells by scanning electron micros-copy. Arch. Oral Biol. 16:259-267.

4 Bulla, L. A., G. St. Julian, R. A. Rhodes, and C. W. Hes-seltine. 1969. Scanning electron and phase-contrast mi-

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croscopy of bacterial spores. Appl. Microbiol. 18:490-495.

5. Crewe, A. V., and J. Wall. 1970. A scanning electron mi-croscope with 5 A resolution. J. Mol. Biol. 48:375-393.

6. Driedger, A. A. 1970. The ordered growth pattern of mi-crocolonies of Micrococcus radiodurans: first generationsectioning of induced lethal mutations. Can. J. Micro-biol. 16:1133-1135.

7. Klainer, A. S., and C. J. Betsch. 1970. Scanning-beammicroscopy of selected microorganisms. J. Infec. Dis.121:339-343.

8. Klainer, A. S., and R. L. Perkins. 1970. Antibiotic-inducedalterations in the surface morphology of bacterial cells: ascanning beam electron microscopic study. J. Infec. Dis.122:323-328.

9. Murphy, J. A., and L. L. Campbell. 1969. Surface featuresof Bacillus polymyxa spores as revealed by scanningelectron microscopy. J. Bacteriol. 98:737-743.

10. Whittaker, D. K., and D. B. Drucker. 1970. Scanning elec-tron microscopy of intact colonies of microorganisms. J.Bacteriol. 104:902-909.

11. Williams, S. T., and F. L. Davies. 1967. Use of scanningelectron microscope for the examination of Actinomy-cetes. J. Gen. Microbiol. 48:171-177.

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