Exercise 3-10 Micro Lab: Endospore Stain

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1

endospore

a dominant form of the bacterium that allows it to survive poor environmental conditions

2

keratin

a tough protein that covers the endospore and makes it resistant to heat and chemicals

3

why are extreme measures used to stain endospores?

because keratin covering resists staining

4

In the _________ method, a primary stain of ________ color is forced into the spore by _______ the bacterial emulsion

Schaeffer-Fulton
malachite green
steaming

5

alternatively, how long does the stain need to stay on a spore to stain it?

15 minutes or more

6

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

7

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

8

2 ways spores may be differentiated based on shape:

1. spherical
2. elliptical

9

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)

10

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

11

spore stain

a differential stain used to detect the presence and location of spores in bacterial cells

12

Which 2 genre of bacteria produce spores

1. Bacillus
2. Clostridium

13

4 members of Bacillus spores

1. soil sporophytes
2. freshwater sporophytes
3. marine sporophytes
4. pathogens (B. anthracis)

14

4 members of Clostridium spores

1. soil sporophytes
2. aquatic sporophytes
3. inhabitants of human intestines
4. pathogens (4)

15

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

16

What type of agar is used in spore cultures

48 hour and 5 day sporulating agar

17

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

18

where do you perform the spore staining?

in the fume hood with protective gear