1. 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

2. Figure 15.1
Figure 15.2

3. 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)

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

6. 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