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endospore stain purpose
detects presence of endospores
endospores
highly resistant, dormant structures formed by certain bacteria as a survival strategy under unfavorable conditions
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
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.
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
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
what happens if a pathogen loses its ability to form capsules?
it ceases to be pathogenic
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)
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
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
flagella
slender, whip-like structures responsible for motility
categories of flagellation
peritrichous, atrichous, monotrichous, amphitrichoua, lophotrichoua
monotrichous flagella
single flagellum
peritrichous
flagella all around
amphitrichous
flagella at both ends
lophotrichous
tuft of many flagella at one end or both ends
atrichous
without flagella, non motile
motility
can be identified in a couple of different ways:
- the hanging drop wet mount
- motility agar media (SIM & tetrazolium motility agars used later)
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.
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
spores
highly resistant, metabolically inactive cell type
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
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
free spore
endospore that is released from a degenerating vegetative cell & becomes an independent cell
spore coats
impervious layer around an endospore