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Abstract:
Broth cultures of Acholeplasma laidlawii were fixed with various concentrations of cacodylate-buffered glutaraldehyde. The shape and ultrastructure of the organisms varied with the osmolar concentration of the fixative. When the fixation mixture was hypertonic to the culture medium, ultrathin sections suggested that the cells had shrunk. Phosphate buffer, sodium chloride, or sucrose at comparable osmolaities had the same effect as sodium cacodylate. Glutaraldehyde itself also contributed to the osmotic effects of the fixation mixture but to a lesser extent than salts or sucrose, to which the cell membrane is impermeable. The osmolar concentration of the fixation mixture seemed of greater importance than pH in determining morphology. The mycoplasma was still susceptible to damage by high concentrations of cacodylate after fixation with 2.5% glutaraldehyde. The best procedure was to fix and wash the organism under conditions isotonic with the growth medium. These conditions were also satisfactory for a filamentous mycoplasma, Mycoplasma orale.
Insights
Fixing Acholeplasma laidlawii with hypertonic solutions caused cell shrinkage. Isotonic fixation is best for preserving mycoplasma morphology and ultrastructure, crucial for microscopy studies.
Area of Science:
- Microbiology
- Cell Biology
- Microscopy Techniques
Background:
- Accurate visualization of mycoplasma ultrastructure is essential for understanding their biology.
- Fixation methods can significantly impact cell morphology, potentially leading to artifacts.
- Acholeplasma laidlawii and Mycoplasma orale are representative mycoplasma species.
Purpose of the Study:
- To investigate the effect of fixative osmolarity on the morphology and ultrastructure of Acholeplasma laidlawii.
- To determine the optimal fixation conditions for preserving mycoplasma integrity.
- To compare the effects of different osmotic agents and glutaraldehyde concentration.
Main Methods:
- Broth cultures of Acholeplasma laidlawii were fixed using various concentrations of cacodylate-buffered glutaraldehyde.
- Osmolar concentrations of fixatives were varied using sodium cacodylate, phosphate buffer, sodium chloride, or sucrose.
- Ultrathin sections of fixed organisms were examined using electron microscopy.
Main Results:
- Hypertonic fixation mixtures resulted in apparent cell shrinkage, regardless of the osmotic agent used.
- Glutaraldehyde contributed to osmotic effects, but salts and sucrose had a more pronounced impact.
- Fixative osmolarity was more critical than pH in determining cell morphology.
Conclusions:
- Optimal preservation of mycoplasma morphology and ultrastructure requires fixation under isotonic conditions relative to the growth medium.
- This isotonic fixation method is effective for both Acholeplasma laidlawii and filamentous mycoplasmas like Mycoplasma orale.
- Careful control of fixative osmolarity is crucial to avoid fixation-induced artifacts in mycoplasma electron microscopy.