microbiology - w2: differential stains; structural stains

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25 Terms

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endospore stain purpose

detects presence of endospores

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endospores

highly resistant, dormant structures formed by certain bacteria as a survival strategy under unfavorable conditions

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Schaeffur-Fulton technique

Identifies pathogenic spore-forming bacteria (ex: Bacillus, Clostridium species)

1. Apply malachite green to stain endospores

2. Heat the stain (due to impervious coats of free spore, acceptance of stain without heat is not easy)

3. Use water (decolorizer) to remove stain from vegetative cell components

- spore remains green, cannot be decolorized

2. Apply counterstain (safranin) to stain vegetative cells

Spores: green

Vegetative cells: red

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Endospore Stain Procedure

1. Flood smears with malachite green & place on top of a beaker of water sitting on a warm hot plate, allowing the preparation to steam for 2 to 3 minutes (Do NOT allow stain to evaporate; replenish stain as needed); Prevent the stain from boiling by adjusting the hot plate temperature.

2. Remove slides from hot plate, cool, & wash under running tap water

3. Counterstain with safranin (30 seconds)

4. Wash with tap water

5. Blot dry with bibulous paper & examine under oil immersion.

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capsule stain purpose

visualize gelatinous outer layer (capsule) that surrounds some bacterial cells

- associated with virulence, as it helps bacteria evade the host immune system

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capsule

gelatinous outer layer that surrounds some bacterial cells & adheres to cell walls

- uncommon to all organisms

- most composed of polysaccharides (levans, dextrans, celluloses) or polypeptides (thick, detectable, & discrete layer) outside the cell wall

- some have well-defined boundaries, some have fuzzy, trailing edges

- virulent & capable of producing disease

protects bacteria from phagocytic action of immune cells & allow pathogens to invade the body

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what happens if a pathogen loses its ability to form capsules?

it ceases to be pathogenic

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capsule stain technique

Identifies capsule appears as a clear halo around the stained cell against a dark background, providing a distinct contrast; identifies encapsulated pathogens (Streptococcus pneumoniae & Klebsiella pneumoniae)

1. Use a combination of a simple stain (cell) & a negative stain (background)

- Simple stain (Crystal violet/safranin)

- Negative stain (India ink/congo red)

2. Use a primary stain (Crystal violet (1% aqueous)) to a non-heat-fixed smear

2. Use a decolorizer agent (Copper sulfate (20%))

- capsule nonionic, primary stain adheres to it (does not bind)

- decolorizer washes out primary WITHOUT removing stain bound to cell wall

- capsule absorbed copper sulfate (blue)

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Capsule Stain Procedure

1. Obtain one clean glass slide; place several drops of crystal violet stain on the slide.

2. Using aseptic technique, add three loopfuls of a culture to the stain & gently mix with the inoculating loop.

3. With a clean glass slide, spread the mixture over the entire surface of the slide to create a very thin smear. Let stand for 5 to 7 minutes. Allow smears to air-dry (Do not heat fix)

4. Wash smears with 20% copper sulfate solution

5. Gently blot dry & examine under oil immersion

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flagella stain purpose

visualize bacterial flagella, the slender, whip-like structures responsible for motility

- thickened coat around the flagella, making them more easily seen with a light microscope

- are extremely thin & of small diameter, so they are below the resolution of the light microscope if unstained

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flagella

slender, whip-like structures responsible for motility

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categories of flagellation

peritrichous, atrichous, monotrichous, amphitrichoua, lophotrichoua

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monotrichous flagella

single flagellum

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peritrichous

flagella all around

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amphitrichous

flagella at both ends

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lophotrichous

tuft of many flagella at one end or both ends

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atrichous

without flagella, non motile

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motility

can be identified in a couple of different ways:

- the hanging drop wet mount

- motility agar media (SIM & tetrazolium motility agars used later)

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Flagella Stain Procedure

1. These stains are bought & ready to use.

2. Although they have cover slips, you still use oil when on 100X magnification.

3. Be sure to remove the oil with the lens paper.

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flagella stain technique

allows for the observation of the number, arrangement, & length of flagella, which are important characteristics for the classification & identification of motile bacteria

1. Use mordant (tannic acid) to coat the flagella

2. Use staining agent (pararosaniline or crystal violet)

2. Increase diameter to stain & observe under a light microscope

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spores

highly resistant, metabolically inactive cell type

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making of an endospore

when environmental conditions become unfavorable for continuing vegetative cellular activities (exhaustion of a nutritional carbon source)

- these cells have the capacity to undergo sporogenesis & give rise to a new intracellular structure called the endospore, which is surrounded by impervious layers called spore coats

- the endospore is released from the degenerating vegetative cell & becomes an independent cell called a free spore

- because of the chemical composition of spore layers, the spore is resistant to the damaging effects of excessive heat, freezing, radiation, desiccation, & chemical agents + commonly employed microbiological stains

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making of a free spore

an endospore released from the degenerating vegetative cell & becomes an independent cell

- because of the chemical composition of spore layers, the spore is resistant to the damaging effects of excessive heat, freezing, radiation, desiccation, & chemical agents + commonly employed microbiological stains

MAY revert back to metabolically active & less resistant vegetative cell through germination due to favorable environmental conditions

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free spore

endospore that is released from a degenerating vegetative cell & becomes an independent cell

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spore coats

impervious layer around an endospore