Lab 4 Lab 3 Goals and Objectives
• Learn methods of differential staining:
– Use two or more stains and categorize cells into groups
– Gram Staining
– Separates bacteria in two different groups – Gram positive and
Gram negative
– Important first test for bacterial identification
– Acid – Fast Staining
– Detects the high mycolic acid content of certain bacterial cell
walls, like those of Mycobacteria
– Mycolic acids are long fatty acids found in the cell walls of a
group of bacteria that includes Mycobacterium tuberculosis.
- Important diagnostic tool
Gram stain – determine bacterial cell wall structure
Figure 15.2
Figure 15.1
GRAM STAIN
1. Crystal Violet = Primary Stain: stains all cell walls purple
2. Iodine = Mordant: combines with crystal violet to create large
insoluble dark purple complex
3. Alcohol = Decolorizer: solubilizes Gram negative outer membrane
flushing iodine/crystal violet complex out of thin wall, thick Gram
positive cells resist decolorization
4. Safranin = Counterstain: stains cell walls pink (obscured by purple
walls of Gram positive, but Gram negative, who had become colorless,
now are pink)
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
Fig 15.3 pg 110
Gram Staining
Add step:
(3.5) Wash
2 seconds
(until water runs clear)
immediately!!!
(until water
runs clear)
Gram stain
Staphylococcus aureus
Bacillus subtilis
Pseudomonas aeruginosa
Kelbsiella pneumonia
Exercise 17: Acid Fast Staining
1. Carbolfuchsin – primary stain
Staphylococcus aureus
2. Aid-alcohol – decolorizer
3. methylene blue – counter-stain
Mycobacterium smegmatis
Wax
Pencil
Each pair make minimum one acid fast slide 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)
2 loops
each organism
mixed together
Fig 17.1 pg 118
Acid Fast Stain
30sec
• ***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!
•
Each pair needs for lab 4:
– 1 broth culture Staphylococcus aureus
– 1 broth culture Pseudomonas aeruginosa
– 1 broth culture Bacillus subtilis (replaces B. megaterium)
– 1 broth culture Escherichia coli
– 1 wet slant 5 day culture Mycobacterium smegmatis
–
–
–
–
–
–
–
Crystal violet
Gram’s iodine
95% Alcohol (Ethanol)
Safranin
Methylene blue
Acid-alcohol
Carbolfuchsin
***95% Ethanol for Gram Stain, Acid-alcohol for Acid Fast Stain!!!