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Ex. 4: Gram Stain. Chromophores Basic vs. acidic dyes. Primary stain (stains all cells on slide) Decolorizing step (removes stain from certain types of cells) Counterstain (stains the decolorized cells). Differential Stain. Strongly advised:.

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Ex 4 gram stain
Ex. 4: Gram Stain

  • Chromophores

  • Basic vs. acidic dyes


Differential stain

Primary stain (stains all cells on slide)

Decolorizing step (removes stain from certain types of cells)

Counterstain (stains the decolorized cells)

Differential Stain


Strongly advised
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!



How to label a slide
How to label a slide

Circles on back

Other info on front


Smear preparation
Smear Preparation

From Liquid Medium

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

  • 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


Gram stain is
Gram Stain is ….

…the most important bacterial stain!

Therefore:

Memorize steps as soon as possible


Ex 4 gram stain

Gram Staining Reagents

Decolorizing agent

Primary stain

Counterstain

Mordant


Gram stain mechanism slide from lecture
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
Gram Stain Animations

ASM Microbe Library

YouTube presentation

MacGraw Hill Animation


Artifacts
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.