Exercise 3-10 Micro Lab: Endospore Stain
endospore
a dominant form of the bacterium that allows it to survive poor environmental conditions
keratin
a tough protein that covers the endospore and makes it resistant to heat and chemicals
why are extreme measures used to stain endospores?
because keratin covering resists staining
In the _________ method, a primary stain of ________ color is forced into the spore by _______ the bacterial emulsion
Schaeffer-Fulton
malachite green
steaming
alternatively, how long does the stain need to stay on a spore to stain it?
15 minutes or more
what is it about malachite green that makes it such a good stain?
it is water soluble and has a low affinity for cellular material so vegetative cells and spore mother cells can be decolorized with water and counterstained with safrannin
Spores may be located in which three locations:
1. central - middle of cell
2. terminal - end of the cell
3. subterminal - the end and middle of the cell
2 ways spores may be differentiated based on shape:
1. spherical
2. elliptical
Three ways spores may be differentiated:
1. location
2. size
3. shape relative to the cell (do they cayuse the cel to look swollen or not)
4 steps to endospore staining
1. stain with malachite green
2. heat used to force stain into cells
3. decolorization with water removes stain from cells but not spores
4. safrannin is used to counterstain vegetative and spore mother cells red
spore stain
a differential stain used to detect the presence and location of spores in bacterial cells
Which 2 genre of bacteria produce spores
1. Bacillus
2. Clostridium
4 members of Bacillus spores
1. soil sporophytes
2. freshwater sporophytes
3. marine sporophytes
4. pathogens (B. anthracis)
4 members of Clostridium spores
1. soil sporophytes
2. aquatic sporophytes
3. inhabitants of human intestines
4. pathogens (4)
The 4 types of Clostridium pathogens and the diseases they produce
1. C. tetani - tetanus
2. C. botulinum - botulism
3. C. perfringens - gas gangrene
4. C. difficile - colitis
What type of agar is used in spore cultures
48 hour and 5 day sporulating agar
Contents of sporulating agar (8):
1. pancreatic digest of gelatin
2. pancreatic digest of casein
3. yeast extract
4. beef extract
5. dextrose
6. agar
7. manganous sulfate
8. distilled or deionized water
where do you perform the spore staining?
in the fume hood with protective gear