Microscope Calibration and Staining Techniques

Calibration of Microscopes

• Use a compound microscope with an ocular micrometer for calibration processes.
• Begin with the scanning objective positioned correctly.
• Place a stage micrometer on the stage, aligning its image with the ocular micrometer.
• Record several alignment points between the two micrometers for calibration.
• Switch to the oil-immersion lens; ensure adequate distance to avoid contact with the stage micrometer.
• Average the calibration values for each objective lens and document them for future use without recalibration.

Staining Techniques

• Basic stains contain positively charged chromogens, forming ionic bonds with negatively charged bacterial cells.
• Common basic stains: methylene blue, safranin, and crystal violet.
• For heat-fixing BSL-2 organisms, use a microincinerator to minimize aerosol production.
• Apply stains carefully and avoid excess; let them sit for 30-60 seconds before rinsing.
• Staining observations should document cell morphology, arrangement, and size.

Negative Staining Methodology

• Negative stains utilize negatively charged chromogens that color the background but not the cells, highlighting clear cell outlines.
• This method is crucial for delicate bacteria, such as Treponema, avoiding distortion from heat-fixing.
• Prepare negative stains using nigrosin and eosin, ensuring sterilization of tools after use.
• No heat-fixing is necessary, allowing cellular size and morphology to be assessed accurately.

Laboratory Materials and Safety

• Standard materials for staining procedures include clean glass microscope slides, disposable gloves, chemical eye protection, and specific stains.
• Maintain proper disposal methods for used slides and stains according to laboratory guidelines to ensure safety and cleanliness.

Observations and Record Keeping

• Essential to note down observations during staining procedures, documenting morphology, arrangement, and cellular dimensions for comparison.
• Analyze and compare measurements from negative and simple stains to understand the impact of staining methods on cell appearance.

Questions for Reflection

• Understanding why a negative stain does not colorize the cells is key for grasping staining principles.
• Predicting the outcome of a mixed staining technique (eosin and methylene blue) aids in the understanding of how different chromogens interact with bacterial cells.
• Comparing sizes of different bacteria (e.g., Bacillus cereus, Micrococcus luteus, Rhodospirillum rubrum) assists in learning about species-specific characteristics.