Ex. 4: Gram Stain

• Chromophores• Basic vs. acidic dyes

Differential Stain

1. Primary stain (stains all cells on slide)

2. Decolorizing step (removes stain from certain types of cells)

3. Counterstain (stains the decolorized cells)

Strongly advised:When doing any of the microbiology

labs, make sure to carefully follow the procedure outlined in the Materials and Methods section . . . And any advice given by your instructor!

From now on: read the labs before coming to class!

Have fun!

“Little Finger Technique”

How to label a slide

Circles on backOther info on front

Smear Preparation

From Liquid Medium• Suspend bacterial

sediment in culture tube by gently tapping on it.

• Transfer one loopful of the bacterial suspension to slide and spread out (You may exceed the limits of the circle)

• Air-dry and heat-fix

From Solid Medium• Transfer one loopful of H2O

into circle area of slide. • Using loop, suspend very

small quantity of colony of interest in that drop of water and spread the suspension evenly. It should only appear slightly milky. Do NOT use too many bacteria

• Air-dry and heat-fix

Gram Stain is ….

…the most important bacterial stain!

Therefore:

Memorize steps as soon as possible

Primary stain Mordant

Decolorizing agent

Counterstain

Gram Staining Reagents

Gram Stain Mechanism (slide from lecture)

• Crystal violet-iodine crystals form in cell.• Gram-positive

– Alcohol dehydrates peptidoglycan– CV-I crystals do not leave

• Gram-negative– Alcohol dissolves outer membrane and leaves holes

in peptidoglycan.– CV-I washes out

Gram Stain Animations

ASM Microbe Library YouTube presentation

MacGraw Hill Animation

Artifacts Precipitated crystal violet stain may resemble cocci. Clues: dense clumping, small size of the gram-positive dots. Paucity of organisms elsewhere in the field.

Examples of Gram Stains

Gram Stain of Urine

Gram Stain of Urine