Lab 5 Goals and Objectives:
Special Stains
Exercise 13: Negative Staining:
Exercise 14: Capsular Staining:
Exercise 16: Spore Staining
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Make all smears first so they can dry before heat fixing!!!
Split the work to save time: make sure everyone sees all.
Exercise 13: Negative Staining: Used to determine morphology and cellular
arrangement in bacteria which are too delicate to withstand heat-fixing
No FIX
Use a dye ( Nigrosin) solution in which the chromogen is acidic and carries a negative
charge – the cell reminds unstained
Each pair makes slides with:
Staphylococcus aureus (slant)
Bacillus subtilis (slant, 24hr) (Substitute)
Mix both together on same slide. Use Nigrosine NOT India Ink
No smears first: organism in ink
At least one slide-spread slide (Fig 13.2)
Oral organisms optional
Fig 13.1 pg 101
Negative Staining
No smear first: mix organisms in ink, spread, and dry
Do NOT touch
dropper to slide
Exercise 14: Capsular Staining: Use 11th edition instructions in supp.
packet!!!
Capsules are composed of mucoid polysaccharides or polypeptides that
repel most stains – staining around the cell
Heat destroy capsules
Capsule stain
An acidic stain nigrosin (stains the background)
FIX the cells gently
A simple (basic stain - crystal violet)
Each pair makes slides with:
Klebsiella pneumoniae (broth)
Klebsiella pneumoniae (slant) (additional culture) On separate slides.
Use Nigrosine NOT India Ink, No smears first: organism in ink
Minimum one slide each of slant and broth (Fig 14.1 in 11th edition)
Fig 14.1 pg 103 from 11th ed.
Capsular Staining
Do NOT touch
dropper to slide
nigrosin
paper towel
Exercise 16: Spore Staining
Endospore is dormant form of the bacterium
Resistant to heat or chemicals
Malachite green – water soluble and has a low affinity for cellular
material
Procedure
Primary stain – malachite green
Decolorized with water from vegetative and spore mother cells
Counterstaining with safranin
Each pair make at least 2 separate slides with:
Bacillus subtilis (slant, 36hr)
Clostridium sporogenes (slant, 4 day)
Schaeffer-Fulton Method only (Fig 16.2)
Make all smears first so they can dry before heat fixing!!!
Split the work to save time: make sure everyone sees all.
Endospore Stain
• Endospore is dormant form of the bacterium
– Resistant to heat or chemicals
• Malachite green – water soluble and has a low affinity for cellular
material
• Procedure
– Primary stain – malachite green
– Decolorized with water from vegetative and spore mother cells
• Counterstaining with safranin
• Clostridium
• Bacillus
Fig 16.1 pg 114
Endospore Staining
Make heat fixed smear slide first!
Steam tray
No paper towel
paper towel
On Website
Each pair needs for lab 5:
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Slant culture of Staphylococcus aureus
Milk broth culture of Klebsiella pneumonia
Milk slant culture of Klebsiella pneumonia
“New” 24 hr slant culture of Bacillus subtilis
“Old” 36 hr slant culture of Bacillus subtilis
“Old” 96 hr slant culture of Clostridium sporogenes
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Nigrosine
Crystal violet
Malachite green
Safranin