Micro Biology Lab Packet
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Week3CapsuleFlagellaSporeStains1-30-18.pptEx 3.9 Capsule Stain
Ex 3.10 Endospore Stain
Ex 3.12 Flagella Stain
Week 3
Today we’re doing:
Differential stains on some reference organisms:
Capsule stain
Endospore stain
Flagella stain
Finish Gram Staining
Differential Stains: Endospore Stain
- Differentiates bacteria based on ability to form endospores or not
- Can often visualize the presence, shape, and location of spores within a vegetative cell
- Spores are resistant forms; heat is needed to drive the stain into the spores
- Spore-forming bacteria include Bacillus species and Clostridium species
The Genus Clostridium
- Gram-positive,
spore-forming rods - Anaerobes
- > 100 species
- Pathogens:
- C. tetani - tetanus
- C. botulinum - botulism
- C. perfringens – gas gangrene
- C. difficile - antibiotic-associated colitis
- Nonpathogens:
- Live in large bowel as commensals
Clostridium tetani
Clostridium difficile
Spore stain (p 217)
- Endospores are resistant; stain does not readily penetrate spores
- Spores will stain with Malachite Green (primary stain) and heat
- Decolorize with water; vegetative cells will lose the malachite green
- Counterstain with safranin
- Spores will appear _________ and vegetative cells will appear ________.
blue-green
red/pink
Spore
Stain
Spores will appear _________ and vegetative cells will appear ________.
blue-green
red/pink
The Genus Bacillus
- Gram-positive,
spore-forming rods - Aerobes
- 40 species
- Only pathogens:
- B. anthracis – anthrax
- B. cereus – food poisoning
- Nonpathogens:
- B. subtilis – source of enzymes
- B. polymyxa – source of antibiotics
Bacillus anthracis
Spore Stain – think about it
- When do spore-forming bacteria undergo sporulation?
- Are spores likely to be observed in fresh cultures or on a rich medium?
- If spores are not observed:
- it might mean that spores weren’t produced under the conditions of growth
- or, it might mean this organism is not a spore-former
Expectations – Work as individuals
Spore stain – p. 217
- Prepare a smear on a slide each for:
Bacillus subtilis and Bacillus cereus
- Observe slides using 100X oil immersion objective
- Observe Flagella stained slides on instructors table at front
- After that, continue with your prepared simple and Gram stained slides
p. 217
Steam w/ Malachite Green stain for 10-15 min
Differential Stains: Capsule Stain
- Differentiates bacteria based on ability to produce a large capsule or very little/no capsule
- Capsule is one kind of glycocalyx
- Usually composed of polysaccharide
- Protects cell from drying and from host defenses (anti-phagocytic)
- Caution: Capsules are somewhat fragile:
- Destroyed by heat-fixing
- Can be washed away
- Staining for a capsule is useful for identification and classification
Capsule stain (p 212)
- Small amount of bacteria is suspended in Congo red – an acidic dye (Ignore the serum)
- Acidic dye will stain/not stain the bacterial cell
- Draw out the drop to create a smear
- Do not heat fix your smear! Let it air dry
- Flood the smear with Maneval’s stain
- Basic dye will stain/not stain the bacterial cell
- Gently rinse with water.
- Observe using oil immersion lens (100X)
- Distinguish between a tiny halo (from cell shrinkage) and a true capsule.
(p 212)
Capsule
Stain
Flagella
- Flagella allow for motility
- Bacterial flagella rotate rather than whip
- Arrangements
- Monotrichous
- Peritrichous
- Lophotrichous
- Amphitrichous
- Presence/absence, number & placement of flagella are useful for identification & classification
Monotrichous
Amphitrichous
Lophotrichous
Peritrichous
A
B
C
D
How would you determine if a bacterium has flagella?
- Look for flagella microscopically
- But hard to see with the light microscope
- Look for motility
- an indirect assessment of the presence of flagella
- So, if a cell is motile, it must have flagella
- We’ll look for motility using a wet mount
Motility Test Medium
- A semisolid medium, containing a reduced amount of agar
- Inoculate vertically, using an inoculating needle
- Motility is apparent if organisms grow and spread away from the line of inoculation
- Nonmotile organisms will only grow at the line of inoculation
Motility Test Medium
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