2 loops each organism Wax Pencil mixed together

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2 loops each organism Wax Pencil mixed together 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 on slide then mix and spread 2. Bacillus subtilis & Escherichia coli: two loops of each broth mixed ***Make all smears on slides first so they can dry before heat fixing!!! You can split the work to save time but make sure everyone gets to see all slides! 2 loops each organism mixed together Wax Pencil

Figure 15.1 Figure 15.2

Crystal Violet = Primary Stain: stains all cell walls purple Iodine = Mordant: combines with crystal violet to create large insoluble dark purple complex in peptidoglycan 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)

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

2 loops each organism Wax Pencil mixed together 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 on slide then mix and spread 2. Bacillus subtilis & Escherichia coli: two loops of each broth mixed ***Make all smears on slides first so they can dry before heat fixing!!! You can split the work to save time but make sure everyone gets to see all slides! 2 loops each organism mixed together Wax Pencil