The Location of Chemical Components on Ultrathin Sections of Bacillus cereus Embedded in Glycol Methacrylate

Abstract

The structure of Bacillus cereua during sporulation and germination is described after aldehyde fixation and ernbedding in a water soluble resin. The resistance of ribosomes to digestion with ribonuclease has been investigated during the various phases of growth. Increasing resistance to digestion was observed a t a stage just prior to the development of the spore coat. The location of polysaccharides on similar sections corresponded with their location previously found in osmic acid fixed material. Materials and MethodsOrgan ism Bacillus cereus var. terininalis u as used exclusively in the prcsent, st,idy. Growth and processing for electron microscopyCells at various stages of sporulation and germination were prepared as previously described (Baillie, Thomson, Bttty & Walker, 1967). At appropriate stages 25 ml samples were removed from the growing culture and fixed in 2.5% wlv glutaraldehyde [4631 WALKER, P. D. & SHOIIT, J. (1969). The location of bacterial polysaocharide during various phases of growth.

Immunocytochemical Localization of Spore Specific Antigens in Ultrathin Sections

“…Cells at various stages of sporulation were prepared as described by Baillie et al (1967). Samples were removed and processed for electron microscopy using the fixation and embedding procedure described by Walker (1969). Mature spores were prepared in the medium described by Delafield et al (1968) at 37 "C with continuous shaking for 3 d. A further 3 d at room temperature allowed for autolysis of the vegetative cells.…”

(Symposium on Bmterial Spores: Paper I). Cytology of Spore Formation and Germination

“…Another method for locating chemical components on ultrathin sections consisted of observing the changes produced following digestion with specific enzymes. For example, ribonucleic acid was detected on ultrathin sections of B. cereus fixed with glutaraldehyde and embedded in glycol methacrylate, a water soluble resin, by observing the changes which occurred following digestion with ribonuclease (Granboulan & Leduc, 1967; Walker, 1969). Ribosomes were readily removed from ultrathin sections of young vegetative cells and also from the forespore during the initial stages of sporulation.…”

“…The results indicated a change in the ribosomes themselves, making them impenetrable to ribonuclease. I n germinating spores ribosomes became sensitive to digestion with ribonuclease as early as 2 min after germination of the spore (Walker, 1969).…”

Morphological and Chemical Characteristics of a Cytophaga sp. Grown under Conditions of Magnesium Excess and Magnesium Limitation

“…The silver methenamine method (Walker, 1969) was used mainly to detect whether the bacteria had a capsule or produced polysaccharide-containing slime, which is one of the properties said to characterize most species of the genus Cytophaga (Christensen, 1977), but it also gave an indication of whether different amounts of polysaccharide were produced when the bacteria were starved of Mg2+. The results suggest that more carbohydrate-containing slime is produced during Mg2+-limited growth.…”

“…The sections were stained with uranyl acetate (Stempak & Ward, 1964) for 5 min and with lead citrate (Venable & Coggeshall, 1965) for 2 min, unless stated otherwise. The silver methenamine method for localizing polysaccharide material in ultrathin sections was performed essentially as described by Walker (1969): the sections were placed on grids and stained with silver hexamethylenetetramine solution at 60 "C for 15 to 60 min and subsequently treated with 2 % (w/v) sodium thiosulphate for 5 min. Some sections were post-stained with uranyl acetate and lead citrate as described above.…”