JOURNAL OF BACTERIOLOGY, May, 1966Copyright © 1966 American Society for Microbiology

Vol. 91, No. 5Printed in U.S.A.

Morphology of the Spore of Some Strains ofClostridium botulinum Type E

W. HODGKISS AND Z. JOHN ORDAL'Ministry of Technology, Torry Research Station, Aberdeeni, Scotland

Received for publication 15 February 1966

ABSTRACTHODGKISS, W. (Torry Research Station, Aberdeen, Scotland), AND Z. JOHN

ORDAL. Morphology of the spore of some strains of Clostridium botulinum type E.J. Bacteriol. 91:2031-2036. 1966.-The spores of four strains of C. botulinum type Eshow an unusual and elaborate morphology. Numerous tubular appendages radiatefrom the surface of the spore. The spore and its appendages are enclosed in a delicateexosporium. The electron microscopic morphology of the spores, as seen in metal-shadowed and negatively stained preparations, and by the carbon-replica technique,is described.

The morphology of the bacterial endosporehas been the subject of a number of studies. Areview of the information then available on theformation and morphology of spores waspresented by Robinow (16). The spores ofdifferent species vary in size and in shape andmay exhibit an irregular surface structure. Vanden Hooff and Aninga (9), by electron micros-copy, substantiated the observations of Meyer(15) on the ribbed or starlike structure of Astasiaasterosporus (Bacillus polymyxa). This spore haslongitudinal ribs which make it appear as amulti-pointed star when examined in crosssection. Bradley and Williams (1) and Franklinand Bradley (6) introduced the use of the carbon-replica technique to study the surface char-acteristics of the spores of several Bacillus spp.and of Clostridium welchii (C. perfringens). Thesestudies demonstrated that the outer spore surfacewas not always smooth and that the spores ofcertain species exhibited a characteristic sculp-tured effect. More recently Krassil'nikov, Duda,and Sokolov (11) presented electron micrographsof the spores of 20 soil isolates of Clostridiumspp. These spores possessed varied and elaborateprotrusions which the investigators considered tobe significant enough to place the strains in sixgroups made up of 13 new species.Our studies were initiated following a routine

electron microscopic examination of spores of astrain of C. botulinum type E (NCIB 4248).Attempts were being made to produce a cleanspore preparation, one free of vegetative and

1 Visiting scientist. Permanent address: Departmentof Food Science, University of Illinois, Urbana.

sporangial debris. When dark contrast phaseoptics were employed, it was noted that thespores possessed a distinct halo and that fre-quently bits of debris appeared to cling tena-ciously to this halo. Our initial electron micro-scope observations prompted us to undertake amore detailed study of the morphology of sporesof C. botulinum type E.

MATERIALS AND METHODSThe cultures used were transfers of two well-known

strains (Beluga and Nanaimo) and two recent isolatesthat were obtained from sea bottom deposits off theScandinavian coast. These strains are from the Na-tional Collection of Industrial Bacteria, Torry Re-search Station, Aberdeen, and are identified by thefollowing NCIB numbers: 4240 (Nanaimo), 4248(Beluga), 4256 and 4275 (Torry isolates).

Spores were produced in the medium of Schmidt,Nank, and Lechowich (17) or in this medium modifiedby the addition of calcium and manganese. The sporeswere removed from the culture medium by centrifuga-tion and washed three times in 0.75% (w/v) sodiumchloride solution. As sporulation was never complete,enzymes were used to free further the sample of vege-tative debris (trypsin alone, or trypsin, pepsin, ribo-nuclease, and lysozyme). The spores were then washedagain with the saline solution, and the preparationwas examined under dark contrast phase optics. Afterthree additional washings with distilled water, thespores were suspended in distilled water for electronmicroscopic examination.

Metal-shadowed preparations were made by placingdroplets of the spore suspension on Formvar-coatedgrids, which were then shadowed with gold-palladium(40:60) at 200. Negatively stained preparations weremade on carbon-coated grids. A droplet of the sporesuspension was allowed to air-dry on the grid, which

2031

on February 3, 2019 by guest

http://jb.asm.org/

Dow

nloaded from

HODGKISS AND ORDAL

was subsequently treated for 1 min with 1% aqueousammonium molybdate or 1% aqueous phospho-tungstic acid adjusted to pH 7.0 (2). Carbon replicaswere prepared by the method of Bradley and Williams(1) and were shadowed with gold-palladium (40:60)at 20°.

Preparations were examined in a Siemens ElmiskopI with a single or double condenser, a 200-,u con-denser aperture, a 50-,u objective aperture, and anaccelerating voltage of 80 kv. Micrographs were takenat initial magnifications of 10,000 to 80,W0 X onIlford N50 plates.

RESULTS

In all, four strains of C. botulinum type E havebeen examined by the techniques described. Inprinciple, the morphology of the spores of theseveral strains is very similar; the minor differ-ences observed do not appear to be significant.For the sake of brevity, we will therefore confineour remarks to the morphology and structure ofthe Beluga strain (NCIB 4248).

The appearance of a spore lacking an exo-sporium, as seen in a shadow-cast preparation, isshown in Fig. 1. Radiating from the well-definedspore are numerous stalklike appendages whichare slightly swollen at the ends. A carbon-replicapreparation (Fig. 2) demonstrates that theappendages are randomly distributed over theentire surface of the spore. Such a preparationalso suggests that the coat of the spore is notperfectly smooth but has surface irregularitieswhich may be the result of a folding of the outerlayer. Additional detail of the structure of theappendages is brought out in a negatively stainedspore (Fig. 3). This electron micrograph suggeststhat the appendages are variable in length andhave a characteristic substructure. The finestructure of an appendage, as seen in negativelystained preparations, is represented diagram-matically in Fig. 4 and illustrated by the micro-graph in Fig. 5. The appendages are tubular instructure, and, although of varying length (up to

M-_C4I ;, .. t F 7

MN,M-1_4

FIG. 1. Trypsin-treated spore, gold-palladium shadow. The appendages are seen radiating from the surface ofthe electron-dense spore. At the distal end ofeach appendage is an enlargement (the cap). All the electron micro-graphs are ofpreparations of the Beluga strain, NCIB 4248. Figures 1 to 4 are electron micrographs of sporeslacking an exosporium. The fragile exosporia of these specimens have been lost during the cleaning of thesespore suspensions.

FIG. 2. Trypsin-treated spore; carbon replica, gold-palladium shadow. The appendages project from the surfaceof the spore.

2032 J. BACTERIOL.

on February 3, 2019 by guest

http://jb.asm.org/

Dow

nloaded from

SPORE MORPHOLOGY OF C. BOTULINUM

0.56 ,u in the Beluga strain), they are of quiteuniform diameter. The overall diameter is of theorder of 200 A, and the diameter of the lumen is80 A. The wall of the tubule is 60 A thick andappears to be composed of spherical subunits ofthat order of diameter. The distal end of eachtubule appears to be solid or to have a plug ofmaterial closing it. The lumen terminates about400 A from the distal end of the appendage, andthe tip of each tubule is surmounted by a hemi-spherical cap. The cap is not a continuousmembrane but is composed of several sphericalsubunits each approximately 40 A in diameter.Between the subunits in the cap and the distalend of the tubular appendage is a gap of theorder of 40 A which is filled with a material thattakes up the negative stain.The presence of a discernable exosporium was

variable and could not be related to any particularspore preparation technique. It was usual to findspores with and spores without an exosporiumon the same microscope grid. In many cases theexosporium was obviously disintegrating, and inothers only residual fragments of the exosporiumremained. Figure 6 illustrates the delicate nature

FiG. 3. Trypsin-treated spore, ammonium molybdatepreparation. The tubular nature of the appendages isapparent.

C

L

200A

FIG. 4. Diagrammatic representation of the finestructure of an appendage as revealed by the negative-stain procedure. C = cap; L = lumen.

of the exosporium in which the spore and itsappendages are enclosed. Additional detail onthe structure of the exosporium is presented inFig. 7. The exosporium is in the process ofdisintegration, and the micrograph demonstratesthat it is composed of macromolecular fibrilswhich are essentially parallel to each other. Thiselectron micrograph also shows some of theminute debris that characteristically clings to theexosporium.

DISCUSSIONAs far as we are aware, no description of the

gross morphology of the spore of C. botulinumtype E has yet been published. Stewart (18) madean electron microscopic study of spores of C.botulinum type A. She reported that spores of thisorganism possess an exosporium but that thespore itself does not contain any unusual struc-tures attached to its surface. Takagi, Kawata, andYamamoto (19) examined ultrathin sections ofspores of a strain of C. botulinum type E duringthe sporulation process, but did not demonstratethe presence of an exosporium or any unusualstructures attached to the spore. They did, how-ever, demonstrate a pronounced intermediatespace between the inner and outer spore coats.Krassil'nikov et al. (11) were the first to describeclostridial spores possessing a variety of structural

2033VIoL. 91,y 1966

on February 3, 2019 by guest

http://jb.asm.org/

Dow

nloaded from

5 l_S -; ;f ."

I g~~~~~~~~~~~~~~~~~~~~~~M

FIG. 5. Appendages of a trypsin-treated spore, ammonium molybdate preparation. Fine detail of the tubularappendages is revealed by the negative stain. The distal end of each tubule is surmounted ty a hemispherical capcomposed ofseveral spherical subunits.

FIG. 6. Spore and exosporium from a mixed enzyme preparation, gold-palladium shadow. The delicate natureof the exosporium is shown by the narrow shadow and the fact that the detached spore appendages are clearlyvisible within it.

FiG. 7. Exosporium from a mixed enzyme preparation, gold-palladium shadow. The fibrillar structure of theexosporium is particularly clear in the portion that is fragmenting. The intact portion demonstrates that the micro-fibrils are parallel to each other (arrow).

on February 3, 2019 by guest

http://jb.asm.org/

Dow

nloaded from

SPORE MORPHOLOGY OF C. BOTULINUM

appendages or protrusions. They considered thatthe spore morphology of the 20 soil strains whichthey examined was constant enough to be oftaxonomic value. Our results with the spores offour strains of C. botulinum type E would atfirst appear to support this suggestion. However,our investigations have not yet covered enoughstrains to confirm entirely this viewpoint.

It is well established that the spores of certainBacillus spp. have a characteristic surface struc-ture (1, 6). A parallel is to be found in the sporemorphology of Streptomyces spp. (21). A cau-tionary note should be made of the fact that thespore morphology of the Streptomyces may varywith the composition of the culture medium andthe period of incubation (8). Similar factors mayequally apply to the morphology of spores of thegenera Bacillus and Clostridium, although thisaspect has not yet been adequately examined.Of the many morphological spore types

described by Krassil'nikov et al. (11), the mor-phology of the spores of the four strains of C.botulinum type E is most similar to their strain 80,Clostridium sporospilum nov. sp. Unfortunately,their electron micrographs do not permit us tomake a detailed comparison between the sporesof their strain 80 and those of C. botulinum typeE. Our electron micrographs demonstrate adegree of detailed structure in the tubular append-ages as well as for the delicate exosporium.A variety of spores, members of the genera

Bacillus and Clostridium, have been shown topossess an exosporium. The most detailed studyso far presented is that of Gerhardt and Ribi (7)on the exosporia of B. cereus and B. anthracis.They demonstrated that the exosporium of B.cereus consists of two main layers. The outerlayer has a nap of hairlike projections and is about250 A thick. The inner basal layer is highlyorganized, with a perforate hexagonal surfacepattern of holes on 76-A centers and consistingof four lamellae. The lamellae of the inner basalmembrane can be fragmented into crystal-likefragments. The entire basal membrane has athickness of 190 A. Between the two layers, andessentially a part of the outer layer, is an inter-mediate covering which adds another 60 A to theoverall thickness of the exosporium (500 A).The exosporium of spores of B. anthracis issomewhat thicker (720 A), but the extra thicknessis largely due to its deeper hairlike nap. Althoughwe have not as yet conducted a detailed study ofthe exosporium of the spores of C. botulinumtype E, our electron micrographs do clearlyindicate that its ultrastructure is quite differentfrom that described for B. cereus. It wouldappear that this exosporium consists of a single

layer of subunits arranged in filamentous threadslying side by side. The overall thickness is of theorder of 60 A.The morphological description that we have

presented does not provide a satisfactory clue asto the function of the tubular appendages of thisunusual spore. One is tempted to speculate thatthey may serve either as attachment processes oras chemo-sensory organelles which might beadvantageous to the spore in the germinationprocess. In the vegetative cells of certain gram-negative bacteria, pili or fimbriae are consideredto act as attachment processes (3, 4, 5, 10).However, fimbriae do not possess the elaboratestructure of the tubular appendages (20). Thediameter of the tubular appendages is of the sameorder of magnitude as the diameter of theubiquitous cytotubules or microtubules of thecytoplasm of plant and animal cells. Thesecytoplasmic microtubules were noted by Manton(13, 14) and first described in detail by Ledbetterand Porter (12). The precise function of thesecytoplasmic microtubules is still a matter forconjecture. Speculation upon the function of theappendages of the spore can be substantiated ordisproved only by knowledge gained fromadditional study of these structures.

ACKNOWLEDGMENTSWe thank D. C. Cann for tranfers of the strains

used, for spore suspensions of strains NCIB 4240 and4256, for rechecking the toxicity of the strains, andfor his wholeheated cooperation and interest through-out the entire study.

This investigation was supported by Public HealthService fellowship 1-F3-AI-28, 391-01 from the Na-tional Institute of Allergy and Infectious Diseases.

LITERATURE CITED1. BRADLEY, D. E., AND D. J. WILLIAMS. 1957. An

electron microscope study of spores of somespecies of the genus Bacillus using carbonreplicas. J. Gen. Microbiol. 17:75-79.

2. BRENNER, S., AND R. W. HORNE. 1959. A negativestaining method for high resolution electronmicroscopy of viruses. Biochim. Biophys. Acta34:103-110.

3. BRINTON, C. C. 1965. The structure, function,synthesis and genetic control of bacterial piliand a molecular model for D.N.A. and R.N.A.transport in Gram-negative bacteria. Trans.N.Y. Acad. Sci. Ser II 27:1003-1053.

4. DUGUID, J. P. 1959. Fimbriae and adhesiveproperties in Klebsiella strains. J. Gen. Micro-biol. 21:271-286.

5. DUGUID, J. P., I. W. SMITH, G. DEMPSTER, ANDP. N. EDMUNDS. 1955. Non-flagellar filament-ous appendages ("Fimbriae") and haemagglu-tinating activity in Bacterium coli. J. Pathol.Bacteriol. 70:335-348.

VOL. 91,y 1966 2035

on February 3, 2019 by guest

http://jb.asm.org/

Dow

nloaded from

HODGKISS AND ORDAL

6. FRANKLIN, J. G., AND D. E. BRADLEY. 1957. Afurther study of the spores of some species ofthe genus Bacillus in the electron microscopeusing carbon replicas, and some preliminaryobservations on Clostridium welchii. J. Appl.Bacteriol. 20:467-472.

7. GERHARDT, P., AND E. RIBI. 1964. Ultrastructureof the exosporium enveloping spores of Bacilluscereus. J. Bacteriol. 88:1774-1789.

8. HODGKISS, W., AND T. G. MITCHELL. 1965. Varia-tion in spore morphology in a Streptomycete.J. Gen. Microbiol. 41:xix-xx.

9. HOOFF, A. VAN DEN, AND S. ANINGA. 1956. Anelectron microscope study on the shape of thespores of Bacillus polymyxa. Antonie vanLeeuwenhoek J. Microbiol. Serol. 22:327-330.

10. HOUWINK, A. L., AND W. VAN ITERSON. 1950.Electron microscopical observations on bac-terial cytology II. A study of flagellation.Biochim. Biophys. Acta 5:10-44.

11. KRASSIL'NIKOV, N. A., V. I. DUDA, AND A. A.SOKOLOV. 1964. Protrusions on the surface ofspores of anaerobic bacteria of the genusClostridium. Microbiology (Mikrobiologiya)33:454-458.

12. LEDBETTER, M. C., AND K. R. PORTER. 1963. A"microtubule" in plant cell fine structure. J.Cell Biol. 19:239-250.

13. MANTON, I. 1957. Observations with the electronmicroscope on the cell structure of the an-theridium and spermatozoid of Sphagnum.J. Exptl. Botany 8:382-400.

14. MANTON, I. 1959. Observations on the micro-anatomy of the spermatozoid of the bracken

fern (Pteridium aquilinum). J. Biophys. Bio-chem. Cytol. 6:413-417.

15. MEYER, A. 1897. Studien uiber die morphologieund entwickelungsgeschichte der Bacterien,ausgefuhrt an Astasia asterospora A.M. undBacillus tumescenis Zopf. Flora (Jena) 84:185-248.

16. ROBINOW, C. F. 1960. Morphology of bacterialspores, their development and germination,p. 207. In I. C. Gunsalus and R. Y. Stanier[ed.], The bacteria, vol. 1. Academic Press, Inc.,New York.

17. SCHMIDT, C. F., W. K. NANK, AND R. V. LECHO-WICH. 1962. Radiation sterilization of food. II.Some aspects of the growth, sporulation andradiation resistance of spores of Clostridiumbotulinum type E. J. Food Sci. 27:77-84.

18. STEWART, G. J. 1963. Studies on spores ofClostridium botulinum. Ph.D. Thesis, WestVirginia Univ., Morgantown.

19. TAKAGI, A., T. KAWATA, AND S. YAMAMOTO.1960. Electron microscope studies on ultrathinsections of spores of the Clostridium group,with special reference to the sporulation andgermination process. J. Bacteriol. 80:37-46.

20. THORNLEY, M. J., AND R. W. HORNE. 1962. Elec-tron microscope observations on the structureof fimbriae, with particular reference toKlebsiella strains, by the use of the negativestaining technique. J. Gen. Microbiol. 28:51-56.

21. TRESNER, H. D., M. C. DAVIES, AND E. J. BACKUS.1961. Electron microscopy of Streptomycesspore morphology and its role in species dif-ferentiation. J. Bacteriol. 81:70-80.

2036 J. BACTERIOL.

on February 3, 2019 by guest

http://jb.asm.org/

Dow

nloaded from