Morphology of the Spore of Some Strains of Clostridium botulinum Type

HODGKISS, 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 E show an unusual and elaborate morphology. Numerous tubular appendages radiate from the surface of the spore. The spore and its appendages are enclosed in a delicate exosporium. The electron microscopic morphology of the spores, as seen in metalshadowed and negatively stained preparations, and by the carbon-replica technique, is described. The morphology of the bacterial endospore has been the subject of a number of studies. A review of the information then available on the formation and morphology of spores was presented by Robinow (16). The spores of different species vary in size and in shape and may exhibit an irregular surface structure. Van den Hooff and Aninga (9), by electron microscopy, substantiated the observations of Meyer (15) on the ribbed or starlike structure of Astasia asterosporus (Bacillus polymyxa). This spore has longitudinal ribs which make it appear as a multi-pointed star when examined in cross section. Bradley and Williams (1) and Franklin and Bradley (6) introduced the use of the carbonreplica technique to study the surface characteristics of the spores of several Bacillus spp. and of Clostridium welchii (C. perfringens). These studies demonstrated that the outer spore surface was not always smooth and that the spores of certain species exhibited a characteristic sculptured effect. More recently Krassil'nikov, Duda, and Sokolov (11) presented electron micrographs of the spores of 20 soil isolates of Clostridium spp. These spores possessed varied and elaborate protrusions which the investigators considered to be significant enough to place the strains in six groups made up of 13 new species. Our studies were initiated following a routine electron microscopic examination of spores of a strain of C. botulinum type E (NCIB 4248). Attempts were being made to produce a clean spore preparation, one free of vegetative and 1 Visiting scientist. Permanent address: Department of Food Science, University of Illinois, Urbana. sporangial debris. When dark contrast phase optics were employed, it was noted that the spores possessed a distinct halo and that frequently bits of debris appeared to cling tenaciously to this halo. Our initial electron microscope observations prompted us to undertake a more detailed study of the morphology of spores of C. botulinum type E. MATERIALS AND METHODS The cultures used were transfers of two well-known strains (Beluga and Nanaimo) and two recent isolates that were obtained from sea bottom deposits off the Scandinavian coast. These strains are from the National Collection of Industrial Bacteria, Torry Research Station, Aberdeen, and are identified by the following 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 modified by the addition of calcium and manganese. The spores were removed from the culture medium by centrifugation and washed three times in 0.75% (w/v) sodium chloride solution. As sporulation was never complete, enzymes were used to free further the sample of vegetative debris (trypsin alone, or trypsin, pepsin, ribonuclease, and lysozyme). The spores were then washed again with the saline solution, and the preparation was examined under dark contrast phase optics. After three additional washings with distilled water, the spores were suspended in distilled water for electron microscopic examination. Metal-shadowed preparations were made by placing droplets of the spore suspension on Formvar-coated grids, which were then shadowed with gold-palladium (40:60) at 200. Negatively stained preparations were made on carbon-coated grids. A droplet of the spore suspension was allowed to air-dry on the grid, which 2031 on S etem er 3, 2017 by gest http/jb.asm .rg/ D ow nladed fom