Lab 4 Goals and Objectives:
Wax
Pencil
***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 loops
each organism
mixed together
2. Bacillus subtilis & Escherichia coli: two loops of each broth mixed
***95% Ethanol for Gram Stain, Acid-alcohol for Acid Fast Stain!!!
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!
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
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)
Lab 4 Goals and Objectives:
Wax
Pencil
***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 loops
each organism
mixed together
2. Bacillus subtilis & Escherichia coli: two loops of each broth mixed
***95% Ethanol for Gram Stain, Acid-alcohol for Acid Fast Stain!!!
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!
On Website
Figure 15.3
Gram Staining
(until water runs clear)
immediately!!!
Figure 17.1
Acid Fast Stain
30sec
Lab 4 Goals and Objectives:
Wax
Pencil
***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 loops
each organism
mixed together
2. Bacillus subtilis & Escherichia coli: two loops of each broth mixed
***95% Ethanol for Gram Stain, Acid-alcohol for Acid Fast Stain!!!
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!