Biology 55 - Quiz #2

gram stain procedure

- prepare smear, air dry, fix with heat
- cover with gram’s crystal violet for 1 min then rinse
- cover with gram’s iodine for 1 min then rinse
- apply acetone-alcohol drop-by-drop briefly for 3-4 secs until color ceases to run then rinse immediately
- cover with gram’s safranin for 1 min then rinse
- blot dry
- gram positive: purple, gram negative: pink

endospore stain procedure (Schaefer-Fulton method)

- prepare smear, air dry, fix with heat
- flood with malachite green, then steam intermittently for 3-4 mins
- rinse thoroughly for 30 secs
- cover with 0.5% safranin for 1 min then rinse
- endospores appear green and pink while vegetative cells stain pink only

acid-fast stain procedure (Ziehl-Neelsen method)

- prepare smear, air dry, fix with heat
- flood with carbolfuchsin, then steam intermittently for 5 mins
- rinse thoroughly
- apply acid-alcohol drop-by-drop briefly for 3-4 secs until color ceases to run then rinse immediately
- cover with methylene blue for 1 min then rinse
- acid-fast: hot pink, non-acid-fast: blue

capsule stain procedure

- prepare smear with loopful of Congo red and a small sample from agar slant
- spread sample evenly over surface of slide using a second slide then air dry
- flood with maneval’s stain for 2 mins then rinse
- blot dry
- capsule is a large uniform clear halo surrounding stained cell amid the red background (negative stain)

You forgot Gram’s iodide for E. Coli. What color do you expect?

pink

You end up with rods from E.Coli that are purple. Why?

decolorizer wasn’t used

What’s the primary stain for the gram stain procedure?

crystal violet

What’s the mordant for the gram stain procedure?

gram’s iodine

What’s the decolorizer for the gram stain procedure?

acetone alcohol

What’s the counter stain for the gram stain procedure?

gram’s safranin

What’s the purpose of the gram stain procedure?

gram-positive is thick and gram-negative is thin

What’s the purpose of the acid-fast stain procedure?

presence of mycolic acid

What’s the primary stain for the acid-fast procedure?

carbolfuchsin

What’s the mordant for the acid-fast stain procedure?

N/A

What’s the deodorizer for the acid-fast stain procedure?

acid alcohol

What’s the counterstain for the acid-fast stain?

methylene blue

What’s the purpose of the endospore stain procedure?

detecting endospore production

What’s the primary stain for the endospore stain procedure?

malachite green

What’s the mordant for the endospore stain procedure?

N/A

What’s the decolorizer for the endospore stain procedure?

N/A

What’s the counterstain for the endospore stain procedure?

0.5% safranin