Exercise 3 Studyguide

What does staining provide?

The contrast necessary to visualize samples using bright field microscopy.

What are the three different types of stains?

Simple

Differential

Structural stain

What is a simple stain?

one dye used to stain all cells present

What is a differential stain?

detects differences between organisms through the use of many dyes and reagents

What is the differential stain we did in class?

Gram Stain

What is a structural stain?

confirm structural characteristics of cells

Who developed the gram stain and when?

Hans Christian Gram in mids 1880s

What was Hans Christian Gram trying to do and what was his problem?

Trying to differentiate bacteria causing pneumonia from the lung tissue of deceased patients.

-He only used crystal violet only realized some cells didn’t remain purple after ethanol

The gram stain is now the most ____ etc…

widely used and important stain in bacteriology and others built on this.

Differentiates between gram positive and gram negative bacteria based on

amount of cell wall peptidoglycan 

Gram positive cell wall has what and what color?

Thick Peptidoglycan, appears purple

Gram negative cell wall has what and what color? (also a piece that positive doesn’t have?)

Thin peptidoglycan, appears pink. Has outer membrane.

Gram staining materials:

Primary Dye

-Crystal Violet

-stains all

Gram staining materials:

Mordant

-Gram’s Iodine

-Binding agent

Gram staining materials:

Decolorizer

-95% Ethanol

-makes differential

-if we did not decolorize all cells would be purple

Gram staining materials:

secondary dye

-Safranin

-Counterstain

Gram stain procedure:

Crystal violet- 1 minute

RINSE

gram’s iodine- 1 minute

RINSE

95% ethanol- 10-12 drops

(gram pos retain purple, thick peptigoglycan)

RINSE RIGHT AWAY

(gram neg becomes clear, needs counterstain)

Safranin- 1 minute

RINSE

BLOT DRY

What percent ethanol do we use to wipe benches?

70% ethanol