Overview
This lecture covers the fundamentals of microscopy in microbiology, types of microscopes, and staining techniques used to visualize and differentiate microorganisms.
Introduction to Microscopy
- Microorganisms are too small to be seen with the naked eye, requiring microscopes for visualization.
- The compound light (brightfield) microscope is the most common tool in microbiology.
Compound Light Microscope Structure & Function
- Uses a light source beneath the stage passing through a glass slide to illuminate specimens.
- Has two magnifying lenses: objective (changeable, 4x–100x) and ocular (10x).
- Total magnification = objective lens magnification x ocular lens magnification (max 1,000x).
- The condenser lens focuses light onto the specimen but does not affect magnification.
- Only objective and ocular lenses change magnification; other parts adjust clarity and illumination.
Image Clarity: Resolution and Refraction
- Resolution (resolving power) is the ability to distinguish two separate points; not the same as magnification.
- Refraction (bending of light) can blur images at high magnification.
- Oil immersion at 100x objective lens matches refractive index to glass, reducing light loss and improving clarity.
Types of Microscopes
- Brightfield: Requires staining (kills cells); most common and inexpensive.
- Phase Contrast: Uses differences in refractive index; allows viewing of living, unstained cells.
- Interference (DIC): Uses two light sources to create 3D images of living cells.
- Dark Field: Highlights living, unstained organisms against a dark background.
- Fluorescence: Uses tagged antibodies for specific identification of organisms; cells may be dead.
- Confocal: Uses laser to compile images from layers, creating 3D reconstructions.
- Scanning Electron Microscope (SEM): Metal-coated, dead cells; provides detailed 3D surface images.
- Transmission Electron Microscope (TEM): Thinly sliced, dead cells; reveals internal structures.
- Atomic Force Microscopy: High-resolution surface imaging of living cells; no special preparation needed.
Slide Preparation and Staining Techniques
- Heat fixation attaches cells to the slide (kills them but allows for staining).
- Negative Stain: Acidic, negatively charged dye repelled by cell surface, stains background but not cells.
- Simple Stain: Basic, positively charged dye binds to negatively charged cells, coloring all cells the same.
- Differential Stains: Distinguish different types of cells.
- Gram Stain:
- Gram-positive: purple (thick cell wall retains dye).
- Gram-negative: pink/red (dye removed by decolorizer, counterstained).
- Critical decolorizing step determines result.
- Acid-Fast Stain: Detects cells with waxy lipid coats, e.g., Mycobacterium; standard stains won’t work.
- Special stains detect unique structures (capsules, endospores, flagella) or use fluorescent antibodies for identification.
Key Terms & Definitions
- Resolution — Ability to distinguish two nearby points as separate.
- Refraction — Bending of light as it passes between materials.
- Heat Fixation — Using heat to adhere cells to the slide, killing them.
- Negative Stain — Stains background, leaving cells clear.
- Simple Stain — Stains all cells the same color.
- Differential Stain — Staining that differentiates between cell types (e.g., Gram stain).
- Acid-Fast Stain — Special stain for cells with waxy outer layers.
Action Items / Next Steps
- Review the microscope summary handout and identify which microscopes are for living vs. dead cells.
- Practice the steps and interpretation of Gram and Acid-Fast staining procedures.
- Prepare for lab by understanding slide preparation and stain application methods.