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Factors influencing the frequency of mesosomes observed in fixed and unfixed cells of Streptococcus faecalis
Abstract:
Mesosomes of Streptococcus faecalis (American Type Culture Collection 9790) were seen about 92% less frequently in freeze fractures of unfixed cells than in freeze fractures and sections of fixed cells. This difference in frequency was not related to any period of unbalanced macromolecular synthesis induced by chemical fixation. All measured synthetic processes (DNA, RNA, and protein synthesis, and glycerol incorporation) were halted with either osmium tetroxide (OS) or glutaraldehyde fixation. That fewer mesosomes were seen in freeze fractures of unfixed cells was probably due to the difficulty of observing cross-fractured mesosomes in this organism in the unfixed state. Unfortunately, mesosomes probably preferentially cross fracture in the unfixed state and therefore are usually only observed, infrequently, in those cases where the freeze fracture follows the surface layer of a mesosomal membrane. However, the addition of glycerol to unfixed cells, especially in the chilled state, greatly increased the frequency of observation of cytoplasmic mesosomes in freeze fractures. It is thought that glycerol, like chemical fixation, increases the number of surface-fractured mesosomes, which in turn increases the frequency of mesosome observation. It was also observed that cellular autolysis occurring during OS fixation seemingly reduced the number of mesosomes observed in thin sections and freeze fractures of OS-fixed cells.
Insights
Mesosomes in Streptococcus faecalis are rarely observed in unfixed cells. Glycerol addition significantly increases mesosome visibility in freeze-fracture electron microscopy of these bacteria.
Area of Science:
- Microbiology
- Cell Biology
- Electron Microscopy
Background:
- Mesosomes are intracellular membrane structures in bacteria.
- Their visualization in electron microscopy can be challenging.
- Previous studies have noted variability in mesosome observation.
Purpose of the Study:
- To investigate the factors affecting mesosome observation in Streptococcus faecalis.
- To understand the impact of cell fixation and cryoprotection on mesosome visualization.
- To optimize methods for observing bacterial mesosomes using freeze-fracture techniques.
Main Methods:
- Freeze-fracture electron microscopy of Streptococcus faecalis.
- Comparison of unfixed, chemically fixed (osmium tetroxide, glutaraldehyde), and glycerol-treated cells.
- Analysis of macromolecular synthesis during fixation.
- Thin section electron microscopy.
Main Results:
- Mesosomes were observed 92% less frequently in unfixed cells compared to fixed cells.
- Chemical fixation (osmium tetroxide, glutaraldehyde) halted all measured synthetic processes.
- Glycerol addition to unfixed, chilled cells significantly increased mesosome observation frequency.
- Cellular autolysis during osmium tetroxide fixation appeared to reduce mesosome numbers.
Conclusions:
- The low frequency of mesosome observation in unfixed cells is likely due to preferential cross-fracturing of mesosomal membranes.
- Glycerol acts as a cryoprotectant, increasing the likelihood of surface-fracturing mesosomal membranes and thus enhancing visibility.
- Chemical fixation and glycerol treatment improve mesosome visualization by altering fracture patterns.