Related Experiment Videos
Electron microscopy of malachite green--glutaraldehyde fixed bacteria
Abstract:
Malachite green combined with glutaraldehyde has been used recently as a fixative for preserving and revaling lipid complexes in thin sections of eukaryotic cells examined by electron microscopy. When bacteria were prefixed with the above mixture granular electron dense inclusions were revealed in all cultures tested. These inclusions were replaced by electron transparent areas in cells fixed with glutaraldehyde alone. The structures were frequently located near to or within the nucleoid and adjacent to the cell membrane in Gram-negative bacteria and were associated with the nucleoid and mesosomes in Gram-positive species. Polyhydroxybutyrate granules, generally poorly preserved in thin sections of Aquaspirillum serpens, were well preserved by the malachite green-glutaraldehyde fixative. Malachite green complexes were observed outside of the cells in all preparations. Capsules were neither preserved nor stained.
Insights
Malachite green and glutaraldehyde fixative reveals granular inclusions in bacteria, unlike glutaraldehyde alone. This method effectively preserves polyhydroxybutyrate granules in thin sections for electron microscopy.
Area of Science:
- Microscopy
- Cell Biology
- Bacteriology
Background:
- Lipid complexes in eukaryotic cells are preserved using malachite green and glutaraldehyde fixatives.
- Electron microscopy requires specific fixation techniques for optimal visualization of cellular structures.
Purpose of the Study:
- To investigate the efficacy of malachite green-glutaraldehyde as a fixative for bacterial cell structures.
- To identify and characterize granular inclusions revealed by this new fixation method.
Main Methods:
- Bacteria were prefixed with a mixture of malachite green and glutaraldehyde.
- Comparison of fixation with glutaraldehyde alone versus the combined fixative.
- Electron microscopy was used to examine thin sections of preserved bacterial cells.
Main Results:
- The malachite green-glutaraldehyde mixture revealed granular electron-dense inclusions in all tested bacterial cultures.
- These inclusions were absent or electron-transparent when glutaraldehyde alone was used.
- Polyhydroxybutyrate granules in Aquaspirillum serpens were well preserved.
- Inclusions were located near the nucleoid and cell membrane (Gram-negative) or associated with nucleoid and mesosomes (Gram-positive).
- Malachite green complexes were observed extracellularly; capsules were not preserved or stained.
Conclusions:
- Malachite green-glutaraldehyde is a superior fixative for revealing and preserving specific granular inclusions in bacteria.
- This method enhances the visualization of polyhydroxybutyrate granules, improving their preservation in electron microscopy studies.
- The fixative offers new insights into bacterial ultrastructure and the nature of these inclusions.