ch.3: staining def

Staining

coloring microorganisms with a dye that emphasizes certain structures

Smear

a thin film of a material containing microorganisms spread over a slide

Fixing

a smear must precede staining

Basic dye

the chromophore is a cation

Acidic dye

chromophore is an anion eosin, acid fuchsin,

Nigrosin

Negative staining

Staining the background instead of the cell

Simple stain

use of a single basic dye

Mordant

may be used to hold the stain or coat the specimen to enlarge it

positive gram stain

bacteria have thick peptidoglycan cell walls

Negative gram stain

bacteria have thin peptidoglycan cell walls and an outer membrane of lipopolysaccharides and phospholipids

Capsules

are a gelatinous covering that do not accept most dyes

what color is a positive gram stain

purple

what color is a negative

pink

fixing

• Attaches microorganisms to the slide

• Kills the microorganisms

• Preserves parts of microbes with minimal distortion

What are the two methods of smear fixation

Heat fixation and chemical fixation

How is heat fixation performed

Slide is passed through a flame several times

What organisms is heat fixation suitable for?

Bacteria, archaea, and sometimes fungi.

How is chemical fixation performed?

Smear is covered with methanol for 1 minute.

What organisms require chemical fixation

Eukaryotes.

What is a stain composed of?

A positive and negative ion—one is the colored chromophore.

What is a basic dye?

A dye where the chromophore is a cation.

A dye where the chromophore is a cation.

Bacterial cells have a negative charge.

Examples of basic dyes?

Examples of basic dyes?

What is an acidic dye?

A dye where the chromophore is an anion.

Examples of acidic dyes?

Eosin, acid fuchsin, nigrosin.

What is negative staining?

Staining the background instead of the cell.

What type of dye is used in simple staining?

Acidic dyes

What does simple staining highlight?

The entire microorganism to visualize cell structure and shape.

Examples of simple stains?

Methylene blue, carbolfuchsin, crystal violet, safranin.

What is a mordant used for?

To hold the stain or coat the specimen to enlarge it.

What are differential stains used for?

To distinguish between bacteria or cells using two or more stains.

What are two main types of differential stains

Gram stain and acid-fast stain.

What are special stains used to detect?

Specific structures like endospores, flagella, and capsules.

What does the Gram stain classify?

Bacteria as gram-positive or gram-negative.

What characterizes gram-positive bacteria?

Thick peptidoglycan cell walls; stain purple.

What characterizes gram-negative bacteria?

Thin peptidoglycan walls and outer membrane of lipopolysaccharides and phospholipids; stain pink/red.

What does the acid-fast stain bind to?

Bacteria with waxy material in their cell walls.

What prevents acid-fast bacteria from being decolorized?

Acid-alcohol.

What is used to stain the waxy cell wall in acid-fast staining?

Acid-alcohol.

What is used to stain the waxy cell wall in acid-fast staining?

Heat.

What organisms are identified using acid-fast stain?

Mycobacterium and Nocardia.

What do special stains detect?

Specific structures like capsules, endospores, and flagella

Examples of special stains?

Capsule stain, endospore stain, flagella stain

What are bacterial capsules?

Gelatinous coverings that do not accept most dyes.

What technique is used to visualize capsules?

Negative staining.

What dyes are used for negative capsule staining?

India ink or nigrosin.

What does the negative stain do?

Contrasts the background with the capsule.

Contrasts the background with the capsule.

A simple stain.

What does a capsule look like under the microscope?

A clear halo around the stained bacterial cell.

What are endospores?

Resistant, dormant structures inside some cells.

Why can't endospores be stained by ordinary methods?

Their tough structure resists dye penetration.

What staining method is used for endospores?

Schaeffer-Fulton endospore stain

What is the primary stain in endospore staining?

Malachite green (usually with heat).

What is used to decolorize the cells?

water

What is the counterstain in endospore staining?

Safranin.

How do stained endospores appear under the microscope?

Green spores inside red or pink cells.