Microstructure of Colonies of Rod-Shaped Bacteria

JOURNAL OF BACTERIOLOGY, Oct. 1971, p. 515-525 Copyright 0 1971 American Society for Microbiology Vol. 108, No. I Printed in U.S.A. Microstructure of Colonies of Rod-Shaped Bacteria D. B. DRUCKER AND D. K. WHITTAKER Department of Bacteriology and Virology, University of Manchester, Manchester, 13, England, and Department of 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, Corynebacterium hofmanni NCTC 1938, Escherichia coli, Lactobacillus acidophilus NCIB1899, Nocardia graminis NCTC4728, Pseudomonas viscosa, and Serratia marcescens were prepared for scanning electron microscopic examination by freezedrying and metal-coating. The arrangement of individual cells within colonies could be seen. Cells of Bacillus colonies tended to be longer than in liquid culture and irregular in shape and to give the appearance of branching. B. megaterium colonies frequently had a dense covering film. Colonies of gram-negative bacteria consisted of fairly short rods covered by much adherent extracellular material. L. acidophilus had colonies comprised of densely packed, well-oriented rods. C. hofmanni colonies contained coccobacilli, packed together. Correlations were observed between planoconvex colony form and densely packed cells, rough colony form and random arrangement of well-separated microorganisms, and irregular colony edge and tendency of cells to grow out from the colony in filaments. Light microscopy permits studies on the initiation of colony formation (6) but is of limited use for the examination of mature colonies. However, the advent of the scanning electron microscope has made possible the study of unsectioned material at a greater resolution (250 nm) than obtains in conventional light microscopy (2, 5). This, coupled with the 200x greater depth of field, makes the scanning electron microscope ideally suited to observation of bacterial cells. The instrument has been used by Williams and Davies (I 1) who examined Actinomycetes, by Klainer and Betsch (7) in their work on the surface morphology of liquid grown cells of Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, and Proteus vulgaris, by Klainer and Perkins (8) in their studies on antibiotic-treated cells, by Bulla et al. (4) and Murphy and Campbell (9) in their examination of bacterial spores, and by Barnes et al. (1) in their work on Candida albicans. More recently, techniques were developed (10) which made possible the preparation of whole bacterial colonies for scanning electron microscopy; intact colonies of Streptococcus mutans OMZ61, Streptococcus sp. D182, Staphylococcus aureus NCTC 6571, and Candida albicans type A MRL 3153 were examined. There appeared to be a correlation between planoconvex colony form and uniform distribution of cocci within the colony. Because the possibilities, 515 with regard to colonial "microstructure," are rather limited in the case of spherical bacteria, the previous work has been extended to include rod-shaped microorganisms in an attempt to discover other relationships between colonial microstructure and gross morphology. MATERIALS AND METHODS Bacterial strains. The following strains were used: Bacillus cereus, Bacillus megaterium, Bacillus mycoides CN 2495, Corynebacterium hofmanni, E. coli, Lactobacillus acidophilus NCIB 1899, Nocardia graminis NCTC 4728, Pseudomonas viscosa and Serratia marcescens. Growth of colonies. L. acidophilus was grown for 48 hr in 5% CO2-95% N2 on "'Oxoid" Rogosa agar. N. graminis was grown aerobically on 5% (v/v) horse blood-oxoid nutrient agar for 48 hr. The remaining organisms were grown aerobically, on Oxoid nutrient agar no. 2 for 48 hr, in the case of P. viscosa and, for 24 hr, in the case of the other organisms. All plates were incubated at 37 C. Freeze-drying of colonies. Colonies were photographed before removal from plates on agar blocks and again after drying to measure shrinkage, which was minimized by rapid freezing by immersion in a 2- methyl butane freezing mixture cooled in liquid nitrogen. Agar blocks carried on aluminum foil were held at - 159.9 C for I min and then freeze-dried for 24 hr at 10-3 Torr in a 500-ml culture vessel (Quickfit) initially cooled in dry ice-acetone. The temperature of the culture vessel was allowed to rise to ambient tem-

516 DRUCKER AND WHITTAKER J. BACTERIOL. perature over a 12-hr period. Metal-coating of colonies. Dried colonies were cemented to scanning electron microscope stubs and plated, while rotating in a vacuum of 10-5 Torr, with 15 mg of Au-Pd alloy (60-40) at a distance of 10 cm. Coated colonies were photographed in a Cambridge Instrument Stereoscan. RESULTS Samples showed little sign of distortion or shrinkage (usually <10%) and compared favorably with samples obtained earlier (8) by using a cooled planchet in a bell-jar. Colony microstructure. B. cereus exhibited some evidence of cell orientation in the center of colonies. Little extracellular material, e.g., gum or covering film, was seen (Fig. 1). Individual cells were 0.4 to 1.0 by 1.1 to 3.6,um and were frequently distorted by opaque spherical structures 1.0,Am in diameter, presumably spores. r; " Divided cells were incompletely separated and were occasionally joined by "bridges" (Fig. 2). At the edge of the colony, cells were more densely packed, and extracellular material was noted. Colonies of B. megaterium also showed localized-cell orientation (Fig. 3). In the center of colonies, 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 cells which were 0.5 to 1.0 by 1.0 to 14.8,um; sporebearing cells frequently appeared club-shaped. Separation of cells was incomplete. Cells at the edge of the colony showed more dense packing, and more extracellular material was present. Long filaments were noted growing out from the colony (Fig. 5). Occasionally, a dark covering film was seen on the surface of the colony; this obscured the underlying cells. I ra '1% - It _ e s s 'sk }s~~~~ FIG. 1. Organisms in the central area of a Bacillus cereus colony show localized orientation. There is little extracellular material. Bar represents 10,im.

VOL. 108, 1971 ROD-SHAPED BACTERIA 517 FIG. 2. Central area of a Bacillus cereus colony examined in the scanning electron microscope. Note the cellular 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 gm which showed little overall orientation (Fig. 6). Cells at the top of a ramus were occasionally flattened, and spherical intracellular bodies could be seen distending the outline of the cells (Fig. 7). B. mycoides cells were sometimes coated with extracellular material. C. hofmanni colonies were comprised of either very short cells (0.3 to 0.5 by 0.5 to 1.0 uin), which appeared to be covered by a dense film, or larger cells (0.3 to 0.7 by 0.5 to 1.5) densely, yet randomly 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) with associated extracellular material. At the periphery of the colony, cells were more oriented and more densely packed with less extracellular material. Some colonies were covered by a film which was perforated by holes, 0.2 to 1.0 gm in diameter. The film totally obscured the bacteria beneath it. Colonies of L. acidophilus were seen to consist of strongly oriented, irregular, tightly packed rods approximately 1 gm in width and 3 to 10,um in length; no extracellular material was observed (Fig. 10). A tangled mass of filaments was observed in colonies of N. graminis. Individual cells were 0.5 to 1.0 Am in width and over 20 gm in length (Fig. 11). No extracellular material was observed. P. viscosa colonies consisted of randomly arranged microorganisms (0.5 by 1.4,im) coated with an adherent extracellular material which did not obscure the outlines of the cells but appeared rather to form a supporting skeleton for them (Fig. 12).

b0- -. 44L& I FIG. 3. Scanning electron micrograph of organisms in the center of a Bacillus megaterium colony. Note the incomplete separation of cells. Bar represents 2 Am. FIG. 4. Note the localized-cell orientation in the center of this Bacillus megaterium colony. Bar represents 2,m. 518

VOL. 108, 1971 ROD-SHAPED BACTERIA 519 FIG. 5. Edge of a Bacillus megaterium colony showing covering film and long filamentous cells growing out from the colony. Bar represents 10,im.

520 DRUCKER AND WHITTAKER J. BACTERIOL. FIG. 6. Chains of B. mycoides cells in a colony "ramus" near the agar surface. Note the lack of overall orientation of bacteria. Bar represents 10,um. FIG. 7. Scanning electron micrograph of B. mycoides cells in the upper portion of a colony "ramus." Note flattened appearance ofcells. Bar represents 5 um.

VOL. 108, 1971 ROD-SHAPED BACTERIA 521 4. AV, '1".-VI," -A *}~~~~~~~~'^'w" FIG. 8. Occasionally, colonies of Corynebacterium hofmanni had no covering film. Scanning electron micrograph reveals tightly packed cells in the center of the colony without any covering film. Bar represents 2 um. Colonies of S. marcescens resembled those of E. coli except that the cells were more completely covered by extracellular material which resulted in a perforated-surface covering film. Cells were not closely packed but showed regular separation and were slightly shorter (0.5 to 1.0,um) than bacteria in an E. coli colony. At the periphery of the colony, cells were more closely packed. DISCUSSION Well-defined covering films were seen only on colonies of B. megaterium and C. hofmanni. This is in contrast to the more widespread occurrence of covering films on colonies of cocci (10). The appearance of branching forms in certain Bacillus colonies might be due to extracellular material having concealed incomplete separation of two or more rods. Thin intracellular "bridges" (7) might be either genuine cellular extensions of adherent slime; however, L. fermentis appears to show intracellular bridges (3) even after washing and centrifuging of cells, which would presumably separate cells held together only by slime. The long filaments growing out from the edge of B. megaterium colonies (Fig. 5) might be responsible for the irregular edge of such colonies and would explain the flattened appearance of B. megaterium colonies. The.Al1 rami of B. mycoides colonies appeared to consist of unorientated, fairly short rods rather than the long, parallel bundles of rods which might be expected. However, it is possible that growth of a ramus occurs by extension of parallel filaments and that later development of a ramus consists of the growth of an outer layer of shorter cells. The association of densely packed colonies with plano-convex colony form, previously shown in the case of cocci (10), holds true for C. hofmanni, whose colonies consist of coccoid forms. A similar association is found in the case of L. acidophilus, even though individual cells in the colony are distinctly rod-shaped. The orientation of Lactobacillus cells is not noted in the case of colonies of the other rod-shaped bacteria examined. This may be a reflection of the mode of division of the cells or their differing surface characteristics. The colonies of gram-negative bacteria consist of cells obscured by adherent material probably of bacterial origin. In the hydrated state, such material might contribute to the glossiness of colonies. The macroscopical appearance of Nocardia colonies differed from that of the other organisms examined. This difference was paralleled by differences in colonial microstructure. The well-separated, tangled filaments, growing verti-

522 DRUCKER AND WHITTAKER J. BACTERIOL. Akl-,-A~~' w. FIG. 9. Scanning electron micrograph showing arrangement of cells in an Escherichia coli colony. Note the extracellular material. Bar represents 5 Am.

VOL. 108, 1971 ROD-SHAPED BACTERIA 523-1 _ - - r 1a -._ FIG. 10. Colonies of Lactobacillus acidophilus consist of strongly orientated, closely packed rods. Bar represents 10 Am.

-W..w w.14 L. 4c, JWI f.,;..40 %k-i. hr. 1;1.-... I.1 4A 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 extracellular material. Bar represents 20,um. 524

VOL. 108, 1971 ROD-SHAPED BACTERIA 525 cally as well as laterally, are no doubt responsible for the rough appearance of colonies of this organism. ACKNOWLEDGMENTS We thank J. Hearle for use of his scanning electron microscope, N. Preston for the strains used in this investigation, and B. 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 Candida albicans. J. Bacteriol. 106:276-280. 2. Boyde, A., and P. J. Knight. 1969. The use of scanning electron microscopy in clinical dental research. Brit. Dent. J. 127:313-322. 3. Boyde, A., and R. A. D. Williams. 1971. Estimation of the volumes of bacterial cells by scanning electron microscopy. Arch. Oral Biol. 16:259-267. 4 Bulla, L. A., G. St. Julian, R. A. Rhodes, and C. W. Hesseltine. 1969. Scanning electron and phase-contrast mi- croscopy of bacterial spores. Appl. Microbiol. 18:490-495. 5. Crewe, A. V., and J. Wall. 1970. A scanning electron microscope with 5 A resolution. J. Mol. Biol. 48:375-393. 6. Driedger, A. A. 1970. The ordered growth pattern of microcolonies of Micrococcus radiodurans: first generation sectioning of induced lethal mutations. Can. J. Microbiol. 16:1133-1135. 7. Klainer, A. S., and C. J. Betsch. 1970. Scanning-beam microscopy of selected microorganisms. J. Infec. Dis. 121:339-343. 8. Klainer, A. S., and R. L. Perkins. 1970. Antibiotic-induced alterations in the surface morphology of bacterial cells: a scanning beam electron microscopic study. J. Infec. Dis. 122:323-328. 9. Murphy, J. A., and L. L. Campbell. 1969. Surface features of Bacillus polymyxa spores as revealed by scanning electron microscopy. J. Bacteriol. 98:737-743. 10. Whittaker, D. K., and D. B. Drucker. 1970. Scanning electron microscopy of intact colonies of microorganisms. J. Bacteriol. 104:902-909. 11. Williams, S. T., and F. L. Davies. 1967. Use of scanning electron microscope for the examination of Actinomycetes. J. Gen. Microbiol. 48:171-177.