ELECTRON MICROSCOPE STUDIES OF THE INTRACYTOPLASMIC MEMBRANE SYSTEM IN CLOSTRIDIUM TETANI AND CLOSTRIDIUM BOTULINUM

Abstract

The existence of an intracytoplasmic membrane system produced by the invagination and extension of the cytoplasmic membrane into the cytoplasm, and which appears as lamellar, concentric, or vesicular in configuration, has been established in a number of bacterial species(3). This membrane system is sometimes referred to membranous organelles, or mesosomes. Reports of the intracytoplasmic membrane system in anaerobic bacteria are encountered only rarely in the literature. FitzJames(6) working with Clostridium pectinovorum and the authors'laboratory(15,16) working with Fusobacterium polymorphum, report what appears to be the membrane system in these organisms. The aim of the present investigation is to learn the intracytoplasmic membrane system in Clostridium tetani and Clostridium botulinum, and if possible, to elucidate the role of this system in the sporulation process and in cell division. MATERIALS AND METHODSStrains . A Cl. tetani strain (Riku-3) and a Cl. botulinum type E strain (Saroma ) were used .Media . Liver broth fortified with 1% glucose was used to grow vegetative cells. To preserve spores and to observe the sporulation process, the same medium without glucose was used .Culture conditions. The cells from stock cultures in liver broth were transferred to the same medium, cultured for 72 hours at 37 C, and then kept for 4 days at room temperature to obtain sufficient numbers of spores. These cultures were heated for 15 minutes at 70 C to kill vegetative cells, and resulting spore preparation were inoculated into the same medium, and cultured at 37 C for 10 to 48 hours or more according to the purpose of experiments .Ultrathin sectioning. Procedures were performed largely according to the method of Kellenberger, Ryter, and Sechaud(10). After specified periods of incubation, the broth culture were cooled in ice water and spun down. The sedimented cells were fixed in 1% osmium tetroxide for 18 hours at 4 C. After embedding in 2% agar, agar blocks were treated with 0.5% uranyl acetate for 2 to 3 hours at room temperature.Fixation in 2% potassium permanganate in tap water for 4 hours at 0 C

Ultrastructure of the cell walls of two closely related clostridia that possess different regular arrays of surface subunits

“…Tetragonal arrays of subunits similar to those on the surface of the cell wall of C. thermosaccharolyticum have been observed on a range of gram-positive bacteria, including Bacillus polymyxa (2,8,22), B. cereus (7), B. fastidiosus (17), B. sphaericus (1,15,23), Clostridium tetani and C. botulinum (30), C. (Desulfotomaculum) nigrificans (26), and C. tartarivorum (12,27). In the majority of these arrays, the center-to-center distance between the subunits is in the range 9 to 12 nm (11,31).…”

Regular Array in the Cell Wall of Lactobacillus fermenti as Revealed by Freeze‐Etching and Negative Staining1

“…Since the presence of a macromolecular array of the cell walls of gram-positive bacteria was first reported in an unidentified bacterium by Labaw and Mosley [15] and then in Bacillus polymyxa [2], similarly ordered structures on bacterial surfaces have been found in a number of gram-positive bacteria using shadowing, replicas or negative staining [7,18,22,26,29,30]. The ordered struc tures so far described mostly consist of a tetragonal array of subunits as summarized by Glauert and Thornley [8].…”

Rhapidosomes in Clostridium Botulinum

“…There are seen several rhapidosomes BT sectioned longitudinally or transversely in an extramembranous space between the two daughter cells. It is a well-known fact that the mesosomes are frequently observed around the septum, relating to cell division (23).…”