1. Lab 4 Goals and Objectives: ***Use wax pencils (China Marker): Sharpie will wash off with alcohol! Be sure to resuspend broth cultures completely! Exercise 15: Gram Stain: Each pair make 2 slides each mix and stain: 1. Staphylococcus aureus & Pseudomonas aeruginosa: two loops of each broth mixed 2. Bacillus subtilis & Escherichia coli: two loops of each broth mixed ***95% Ethanol for Gram Stain, Acid-alcohol for Acid Fast Stain!!! 2 loops each organism mixed together Wax Pencil Exercise 17: Acid Fast Staining Each pair make 2 slides with: Staphylococcus aureus (use 2-4 loops of broth on slide first) Mycobacterium smegmatis (slant, use needle, get tiny amount and mix into broth on slide very well: break up all clumps) ***Make all smears on slides first so they can dry before heat fixing!!! Split the work to save time but make sure everyone gets to see all slides!

2. Figure 15.2 Figure 15.1

3. Crystal Violet = Primary Stain: stains all cell walls purple Iodine = Mordant: combines with crystal violet to create large insoluble dark purple complex Alcohol = Decolorizer: solubilizes Gram negative outer membrane flushing iodine/crystal violet complex out of thin wall, thick Gram positive cells resist decolorization Safranin = Counterstain: stains cell walls pink (obscured by purple walls of Gram positive, but Gram negative, who had become colorless, now are pink)

4. Lab 4 Goals and Objectives: ***Use wax pencils (China Marker): Sharpie will wash off with alcohol! Be sure to resuspend broth cultures completely! Exercise 15: Gram Stain: Each pair make 2 slides each mix and stain: 1. Staphylococcus aureus & Pseudomonas aeruginosa: two loops of each broth mixed 2. Bacillus subtilis & Escherichia coli: two loops of each broth mixed ***95% Ethanol for Gram Stain, Acid-alcohol for Acid Fast Stain!!! 2 loops each organism mixed together Wax Pencil Exercise 17: Acid Fast Staining Each pair make 2 slides with: Staphylococcus aureus (use 2-4 loops of broth on slide first) Mycobacterium smegmatis (slant, use needle, get tiny amount and mix into broth on slide very well: break up all clumps) ***Make all smears on slides first so they can dry before heat fixing!!! Split the work to save time but make sure everyone gets to see all slides!

5. On Website

6. Figure 15.3 Gram Staining (until water runs clear) immediately!!!

8. Acid Fast Stain Figure 17.1 30sec

10. Lab 4 Goals and Objectives: ***Use wax pencils (China Marker): Sharpie will wash off with alcohol! Be sure to resuspend broth cultures completely! Exercise 15: Gram Stain: Each pair make 2 slides each mix and stain: 1. Staphylococcus aureus & Pseudomonas aeruginosa: two loops of each broth mixed 2. Bacillus subtilis & Escherichia coli: two loops of each broth mixed ***95% Ethanol for Gram Stain, Acid-alcohol for Acid Fast Stain!!! 2 loops each organism mixed together Wax Pencil Exercise 17: Acid Fast Staining Each pair make 2 slides with: Staphylococcus aureus (use 2-4 loops of broth on slide first) Mycobacterium smegmatis (slant, use needle, get tiny amount and mix into broth on slide very well: break up all clumps) ***Make all smears on slides first so they can dry before heat fixing!!! Split the work to save time but make sure everyone gets to see all slides!