Effect of chemical fixatives on accurate preservation of Escherichia coli and Bacillus subtilis structure in cells prepared by freeze-substitution

Journal Article · Sun Apr 01 00:00:00 EST 1990 · Journal of Bacteriology; (USA)

OSTI ID:7008097

Graham, L L; Beveridge, T J ^[1]

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1. Univ. of Guelph, Ontario (Canada)

Five chemical fixatives were evaluated for their ability to accurately preserve bacterial ultrastructure during freeze-substitution of select Escherichia coli and Bacillus subtilis strains. Radioisotopes were specifically incorporated into the peptidoglycan, lipopolysaccharide, and nucleic acids of E. coli SFK11 and W7 and into the peptidoglycan and RNA of B. subtilis 168 and W23. The ease of extraction of radiolabels, as assessed by liquid scintillation counting during all stages of processing for freeze-substitution, was used as an indicator of cell structural integrity and retention of cellular chemical composition. Subsequent visual examination by electron microscopy was used to confirm ultrastructural conformation. The fixatives used were: 2% (wt/vol) osmium tetroxide and 2% (wt/vol) uranyl acetate; 2% (vol/vol) glutaraldehyde and 2% (wt/vol) uranyl acetate; 2% (vol/vol) acrolein and 2% (wt/vol) uranyl acetate; 2% (wt/vol) gallic acid; and 2% (wt/vol) uranyl acetate. All fixatives were prepared in a substitution solvent of anhydrous acetone. Extraction of cellular constituents depended on the chemical fixative used. A combination of 2% osmium tetroxide-2% uranyl acetate or 2% gallic acid alone resulted in optimum fixation as ascertained by least extraction of radiolabels. In both gram-positive and gram-negative organisms, high levels of radiolabel were detected in the processing fluids in which 2% acrolein-2% uranyl acetate, 2% glutaraldehyde-2% uranyl acetate, or 2% uranyl acetate alone were used as fixatives. Ultrastructural variations were observed in cells freeze-substituted in the presence of different chemical fixatives. We recommend the use of osmium tetroxide and uranyl acetate in acetone for routine freeze-substitution of eubacteria, while gallic acid is recommended for use when microanalytical processing necessitates the omission of osmium.

• OSTI ID: 7008097
• Journal Information: Journal of Bacteriology; (USA), Vol. 172:4; ISSN 0021-9193
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
BACILLUS SUBTILIS
HISTOLOGICAL TECHNIQUES
ESCHERICHIA COLI
CARBON ISOTOPES
CHEMICAL COMPOSITION
ELECTRON MICROSCOPY
FREEZING
ISOTOPE DILUTION
RECOMMENDATIONS
SCINTILLATION COUNTING
TRITIUM COMPOUNDS
URACILS
AZINES
BACILLUS
BACTERIA
COUNTING TECHNIQUES
HETEROCYCLIC COMPOUNDS
HYDROGEN COMPOUNDS
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
ISOTOPES
MICROORGANISMS
MICROSCOPY
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PYRIMIDINES
TRACER TECHNIQUES
550701* - Microbiology- Tracer Techniques