Lab 5 Goals and Objectives: Special Stains Exercise 13: Negative Staining: Exercise 14: Capsular Staining: Exercise 16: Spore Staining 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: Each pair makes slides with: 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 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

Fig 13.2 pg 102 Negative Staining Spread well!

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 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

FIG. 2. Encapsulated Streptococcus lactis stained using Anthony's capsule stain

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 Steam tray No paper towel paper towel Make heat fixed smear slide first!

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Each pair needs for lab 5: –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 –Nigrosine –Crystal violet –Malachite green –Safranin