1. Staining Lab 3 and 4 Notes and Pictures

2. Smears Air dry first to prevent lysis (boiling) Heat Fixing –Kill –Stops autolysis –Adherence to slide

3. Staining Chromophore –Positive: basic stain Attaches to cell wall and bacterium appears colored Direct Stain –Negative: acidic stain Repelled from cell wall; stains background Negative Stain

4. Simple Stain To observe basic external structures of cell with brightfield scope (cellular morphology) –Reagent: Methylene Blue

6. Gram Stain Purpose –To view cellular morphology –Diagnostic purposes: What type of cell wall a bacterium has Gram Positive –Thicker peptidoglycan layer and no outer membrane Gram Negative –Thinner Peptidoglycan and an out membrane

7. Gram Type Cell Walls

8. Reagents for Gram Stain Crystal Violet (purple) –Primary stain; positive stain –Stains cell wall purple Iodine –Mordant –Combines with CV to form an insoluble complex that gets trapped in thicker peptidoglycan layers –What happens if you skip this step? Ethanol –Decolorizer –CV-I complex washed out of Gram negative organisms because it cannot be trapped by peptidoglycan layer; flows right through outer membrane Safranin (pink) –Counterstain –Simple positive stain that provides contrasting dye for decolorized cells (Gram negative) –Stains all cells, but only the negative ones actually appear pink.

11. Gram Stain Gram + Coccus and Human cells Gram – Coccobacilli and Human cells

12. Acid Fast Stain Purpose –To view cellular morphology –Diagnostic purposes; some diseases are caused by acid fast organisms (which ones?) Mycobacterium –Mycolic acids: Wax like lipid (hydrophobic) –Requires special stain Mycobacterium tuberculosis Mycobacterium leprae Ziehl-Neelsen method with Kinyoun modification

13. Acid Fast Reagents Carbolfuchsin (red) –Primary stain –Lipid soluble; phenol Acid alcohol –Decolorizer –Removes carbol fuchsin that has not bound to a mycolic acid. Methylene blue –Counterstain –Cannot penetrate mycolic acid; provides contrast to non acid fast cells.

14. Acid Fast

15. Endospore Stain Genera that form endospores –Bacillus –Clostridium Survival mechanism; not for reproduction Vegetative cell versus endospore Purpose –Diagnostic purposes –Some diseases caused by spore forming bacterium (which diseases?)