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Nucleoid structure in freeze fractures of Streptococcus faecalis: effects of filtration and chilling
Abstract:
With the techniques used in this study, the nucleoid of Streptococcus faecalis could not be seen in freeze-etch preparations unless glutaraldehyde had been added to cultures of cells before they were frozen. With time, the nucleoid became visible as a network of fibers, apparently as a result of the aggregation of individual chromosomal elements in the presence of glutaraldehyde. When glutaraldehyde was added to undisturbed cultures, the fibers that became visible were observed in small patches that were seemingly scattered throughout the cytoplasm. However, if cells were chilled or placed on filters before glutaraldehyde was added, the fibers which then developed were seen in large central areas. The appearance of centralized nucleoids in freeze fractures of cells that had been chilled or filtered could be correlated with a decrease in the central density of the cytoplasm, as seen by light microscopy, in cells embedded in gelatin or bovine serum albumin. These observations are discussed in relation to a model for the normal structure of the nucleoid which suggests that the treatments routinely used to study the morphology-physiology of cells (chilling, filtration, and fixation) result in a reorganization of the cytoplasm, leading to an increase in the centralization of nuclear material.
Insights
Glutaraldehyde fixation reveals the nucleoid of Streptococcus faecalis as a fiber network. Cell treatments like chilling or filtration before fixation cause nucleoid centralization, impacting observed cell structure.
Area of Science:
- Microbiology
- Cell Biology
- Microscopy Techniques
Background:
- The bacterial nucleoid, comprising chromosomal DNA, is crucial for cell function.
- Studying nucleoid structure requires specific preparation techniques that can influence observations.
- Freeze-etching is a microscopy method used to visualize cellular ultrastructure.
Purpose of the Study:
- To investigate the visibility and structural organization of the Streptococcus faecalis nucleoid using freeze-etch electron microscopy.
- To determine the effect of glutaraldehyde fixation and other sample preparation methods on nucleoid morphology.
Main Methods:
- Utilized freeze-etch electron microscopy to examine Streptococcus faecalis cells.
- Applied glutaraldehyde fixation at various stages of sample preparation.
- Investigated the impact of cell chilling and filtration prior to fixation.
Main Results:
- Nucleoid structures were not visible in freeze-etch preparations without glutaraldehyde.
- Glutaraldehyde induced the aggregation of chromosomal elements into visible fiber networks.
- Chilling or filtering cells before glutaraldehyde fixation resulted in the centralization of nucleoid fibers.
Conclusions:
- Glutaraldehyde fixation is essential for visualizing the Streptococcus faecalis nucleoid via freeze-etching.
- Standard cell preparation techniques (chilling, filtration, fixation) can induce cytoplasmic reorganization and nucleoid centralization.
- These findings suggest that observed nucleoid morphology may be an artifact of preparation methods, influencing interpretations of cell structure and physiology.