Evaluation of freeze-substitution and conventional embedding protocols for routine electron microscopic processing of eubacteria

Journal Article · Sun Apr 01 04:00:00 EDT 1990 · Journal of Bacteriology; (USA)

OSTI ID:7192235

Graham, L L; Beveridge, T J ^[1]

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1. Univ. of Guelph, Ontario (Canada)

Freeze-substitution and more conventional embedding protocols were evaluated for their accurate preservation of eubacterial ultrastructure. Radioisotopes were specifically incorporated into the RNA, DNA, peptidoglycan, and lipopolysaccharide of two isogenic derivatives of Escherichia coli K-12 as representative gram-negative eubacteria and into the RNA and peptidoglycan of Bacillus subtilis strains 168 and W23 as representative gram-positive eubacteria. Radiolabeled bacteria were processed for electron microscopy by conventional methods with glutaraldehyde fixation, osmium tetroxide postfixation, dehydration in either a graded acetone or ethanol series, and infiltration in either Spurr or Epon 812 resin. A second set of cells were simultaneously freeze-substituted by plunge-freezing in liquid propane, substituting in anhydrous acetone containing 2% (wt/vol) osmium tetroxide, and 2% (wt/vol) uranyl acetate, and infiltrating in Epon 812. Extraction of radiolabeled cell components was monitored by liquid scintillation counting at all stages of processing to indicate retention of cell labels. Electron microscopy was also used to visually confirm ultrastructural integrity. Radiolabeled nucleic acid and wall components were extracted by both methods. In conventionally embedded specimens, dehydration was particularly damaging, with ethanol-dehydrated cells losing significantly more radiolabeled material during dehydration and subsequent infiltration than acetone-treated cells. For freeze-substituted specimens, postsubstitution washes in acetone were the most deleterious step for gram-negative cells, while infiltration was more damaging for gram-positive cells.

• OSTI ID: 7192235
• Journal Information: Journal of Bacteriology; (USA), Journal Name: Journal of Bacteriology; (USA) Vol. 172:4; ISSN 0021-9193; ISSN JOBAA
• Country of Publication: United States
• Language: English

Related Subjects

550701* -- Microbiology-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AZINES
BACILLUS
BACILLUS SUBTILIS
BACTERIA
CARBON ISOTOPES
DNA
ELECTRON MICROSCOPY
ESCHERICHIA COLI
EVALUATION
FREEZING
HETEROCYCLIC COMPOUNDS
HISTOLOGICAL TECHNIQUES
HYDROGEN COMPOUNDS
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
ISOTOPE DILUTION
ISOTOPES
MICROORGANISMS
MICROSCOPY
NUCLEIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PYRIMIDINES
RNA
TRACER TECHNIQUES
TRITIUM COMPOUNDS
URACILS