Overview
This lecture explains the endospore staining method, focusing on its use for identifying bacteria that form highly resistant endospores and the step-by-step staining procedure.
Endospore Formation and Resistance
- Some bacteria, especially Bacillus genus, form endospores under harsh environmental conditions.
- Endospores are highly resistant to heat and environmental stress due to a tough keratin protein coat.
- The resistance of endospores makes them difficult to stain with normal methods like Gram staining.
Endospore Staining Procedure
- The primary stain used is malachite green, a highly penetrant stain able to enter endospores.
- A bacterial smear is prepared on a slide and heat-fixed to immobilize the bacteria.
- Malachite green is applied to the smear, sometimes with steaming to help the stain penetrate the endospore wall.
- Decolorization is performed to remove the stain from non-endospore cells, as malachite green is water-soluble.
- Endospores retain malachite green, while non-spore forming cells lose the stain.
- A counterstain, safranin, is applied to stain the vegetative cells pink or red.
Staining Results and Microscopic Appearance
- Endospores appear green under the microscope due to retained malachite green.
- Vegetative (non-spore) cells appear pink or red from the safranin counterstain.
- This staining method distinguishes spore-forming bacteria by color differences.
Key Terms & Definitions
- Endospore — a dormant, tough, non-reproductive structure produced by certain bacteria for survival in harsh conditions.
- Keratin — a protein forming the protective coat of endospores, making them resistant to staining.
- Malachite Green — a primary stain that penetrates and stains endospores.
- Safranin — a red counterstain that colors vegetative cells in the staining process.
Action Items / Next Steps
- Review lecture notes and provided resources for further study.
- Practice the endospore staining procedure in the laboratory if assigned.