Ex. 4: Gram Stain • Chromophores • Basic vs. acidic dyes
Feb 24, 2016
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