Ex. 4: Gram Stain

1. Ex. 4: Gram Stain • Chromophores • Basic vs. acidic dyes

2. Primary stain (stains all cells on slide) Decolorizing step (removes stain from certain types of cells) Counterstain (stains the decolorized cells) Differential Stain

3. 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!

7. “Little Finger Technique”

8. How to label a slide Circles on back Other info on front

9. 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

10. Gram Stain is …. …the most important bacterial stain! Therefore: Memorize steps as soon as possible

11. Gram Staining Reagents Decolorizing agent Primary stain Counterstain Mordant

12. 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

13. Gram Stain Animations ASM Microbe Library YouTube presentation MacGraw Hill Animation

14. 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.

15. Examples of Gram Stains

18. Gram Stain of Urine

19. Gram Stain of Urine

20. The End