🔬 CHAPTER 3 STUDY NOTES: MICROSCOPY & STAINING

Micrometer (μm)

A unit of measurement used to measure bacteria and protozoa.

Nanometer (nm)

A unit of measurement used to measure viruses and molecules.

Simple microscope

A microscope with one lens.

Compound microscope

A microscope with multiple lenses, most common in microbiology.

Total magnification

The product of the ocular lens and the objective lens.

Resolution

The ability to distinguish two close points; for compound light microscopes, it is approximately 0.2 μm.

Oil immersion lens

A lens used at 100× objective to increase resolution by reducing light refraction.

Brightfield microscopy

A type of light microscopy that uses stained specimens to show dark on a light background.

Darkfield microscopy

A microscopy technique that highlights bright organisms on a dark background, suitable for very small or unstained organisms.

Phase-contrast microscopy

A method of microscopy that enhances internal details of living cells without staining.

DIC (Differential Interference Contrast) microscopy

Produces a 3D-like colored image of live cells.

Fluorescence microscopy

Uses fluorescent dyes to make organisms glow under UV light.

Confocal microscopy

Uses laser scanning and fluorescent dyes to produce sharp 3D images.

Two-photon microscopy

Uses long-wavelength light to reduce damage to live cells.

Super-resolution microscopy

A technique that breaks normal light resolution limits to produce highly detailed images.

Scanning acoustic microscopy (SAM)

Uses sound waves to study biofilms and surface-attached cells.

TEM (Transmission Electron Microscope)

A type of electron microscope that uses thin slices of specimens to view internal structures.

SEM (Scanning Electron Microscope)

An electron microscope that provides 3D surface images.

Smears

A thin layer of microbes that is placed on a slide for microscopic examination.

Fixing

The process of using heat or methanol to kill and attach cells to a slide.

Staining purpose

To increase contrast and make cellular structures visible.

Cell charge rule

Bacteria are negatively charged, basic dyes stain cells, and acidic dyes stain the background.

Simple stain

A staining technique that uses one basic dye to show shape, size, and arrangement of cells.

Negative stain

A staining method where an acidic dye stains the background while leaving the cells clear.

Gram stain steps

Steps include crystal violet, iodine, alcohol, and safranin.

Gram-positive results

Cells appear purple after a Gram stain.

Gram-negative results

Cells appear pink/red after a Gram stain.

Acid-fast stain

Used for Mycobacterium and Nocardia; acid-fast bacteria retain carbolfuchsin and appear red.

Endospore stain

A technique that uses malachite green and heat to visualize endospores.

Mordant

A substance used in staining to help the dye bind to the cell.

Counterstain purpose

To color cells that lost the primary stain, especially in acid-fast staining.

Decolorizer purpose

Removes stain from cells that do not retain it, such as in Gram staining.