Microscopy and Staining Techniques in Microbiology ~ Chp 3

Microscope

Parts of the Compound Light Microscope

The compound light microscope is a fundamental tool in microbiology, allowing for the visualization of small specimens using visible light and multiple lenses. Understanding its components is essential for proper operation and accurate observation.

• Illumination: The light source that provides the necessary illumination for viewing specimens.

• Condenser: Focuses light onto the specimen, enhancing image clarity and resolution.

• Objective Lens: The primary lens that magnifies the specimen; typically available in several magnifications (e.g., 4x, 10x, 40x, 100x).

• Ocular Lens (Eyepiece): The lens through which the viewer observes the magnified image, usually 10x magnification.

• Stage (Mechanical Stage): The platform where the specimen slide is placed and can be moved precisely.

• Diaphragm: Regulates the amount of light passing through the specimen.

• Course Adjustment: Allows for rapid, coarse focusing of the image.

• Fine Adjustment: Permits precise, fine focusing for detailed observation.

Properties of Microscopes

Microscopes are defined by their ability to magnify and resolve small objects. Resolution is the minimum distance at which two points can be distinguished as separate entities. It is limited by the physics of light and optics.

• Resolution Formula: $d = \frac{\lambda}{2n \sin \theta}$ where d is the minimum resolvable distance, λ is the wavelength of light, n is the refractive index of the medium, and θ is the angle of light collected by the objective lens.

• Magnification: The number of times larger an image appears compared to the actual object.

• Refraction: The bending of light as it passes from one medium to another, affecting image clarity.

• Refractive Index: A property of each medium that determines the degree to which light bends when moving from one medium to another (e.g., air has a refractive index of 1.0).

Types of Microscopes

Different types of microscopes are used in microbiology, each with unique capabilities for observing specimens.

Light Microscopes

Light microscopes use visible light to illuminate specimens and can resolve objects up to 0.2μm with magnifications up to 1,500X.

Type	Description	Applications
Bright Field	Large cells easily observed; small cells difficult to see without staining.	General observation of stained cells.
Dark Field	Disc blocks direct light; only refracted light enters. Unstained (live) cells easily visualized.	Examination of unstained bacterial cells.
Phase Contrast	Disc partially blocks direct light, allowing for direct and diffracted light to reinforce. Internal structures become more sharply defined.	Observation of unstained eukaryotic cells.

Electron Microscopes

Electron microscopes use beams of electrons instead of light, allowing for much greater resolution and magnification.

Type	Resolution	Magnification	Application
Transmission Electron Microscope (TEM)	10 nm	10,000,000X	Internal structures, greatest magnification and resolution.
Scanning Electron Microscope (SEM)	10 nm	500,000X	3D, exterior morphology revealed.

Key Terms and Concepts

• Magnification: The process of enlarging the appearance of an object.

• Refraction: The change in direction of light as it passes from one medium to another.

• Refractive Index: A measure of how much light bends when entering a medium.

Staining

Principles of Staining

Staining is a technique used to enhance the contrast of microscopic specimens, making cellular structures more visible. It is essential for observing cell morphology and distinguishing between different types of cells.

• Smear: Spreading a thin layer of specimen on a slide.

• Fix: Preserving the specimen's structure, often by heat or chemicals.

• Stain: Applying dyes to color the specimen.

• Mordant: A substance that helps fix the dye to the specimen.

• Decolorizer: Removes excess stain, differentiating cell types.

Types of Stains

• Simple Stain: Uses a single dye to color cells, revealing cell shape and arrangement.

• Gram Stain: Differentiates bacteria into Gram-positive and Gram-negative based on cell wall properties.

• Capsule Stain: Highlights the presence of a capsule surrounding some bacteria.

Staining Mechanism

Stains are classified as basic or acidic depending on the charge of the dye. Basic dyes (e.g., crystal violet, methylene blue) are positively charged and bind to negatively charged cell components. Acidic dyes (e.g., eosin) are negatively charged and stain the background.

• Most cells have a slight negative charge, so basic dyes are commonly used.

• Acidic dyes are used to stain the background or can be used if the cell is made more positive by acidification (adding H+).

Example: Gram Staining

1. Apply crystal violet (basic dye).

2. Add iodine (mordant).

3. Decolorize with alcohol.

4. Counterstain with safranin (basic dye).

Gram-positive cells retain the crystal violet and appear purple; Gram-negative cells lose the violet and take up the safranin, appearing pink.

Additional info: Staining techniques are crucial for identifying and classifying microorganisms in clinical and research settings.