BIOL 2041 (pre-lab quiz2)

Endospore stain alternate name

Schaeffer-Fulton stain

Endospore stain purpose

Differential stain used to detect endospores

Primary stain in endospore stain

Malachite green

What color does malachite green stain endospores?

Green

Counterstain in endospore stain

Safranin

What color does safranin stain vegetative cells?

Red or pink

Decolorizer in endospore stain

Water

Why is heat used in endospore staining?

Heat helps malachite green penetrate the thick endospore coat

Why are endospores hard to stain?

They have a tough spore coat outside the cell

Endospores are

Specialized dormant resting cells

Vegetative cells are

Active cells that can reproduce

Endospores can reproduce?

No, endospores are dormant and cannot reproduce

When do endospores form?

During adverse or unfavorable conditions

Important endospore-forming genera

Bacillus and Clostridium

Endospores may be located

Terminally, sub-terminally, or centrally

Organism used for endospore stain

Bacillus megaterium

B. megaterium cultures compared

2–3 day old culture and 7 day old or 10+ day old culture

Older B. megaterium culture is expected to have

More endospores

Younger B. megaterium culture is expected to have

More vegetative cells

Endospore stain result

Endospores appear green; vegetative cells appear red/pink

Endospore stain first step

Prepare smear, air dry, and heat fix

Endospore stain heating setup

Place slide over boiling water on mesh/beaker

Why use paper towel during endospore staining?

To hold malachite green over the smear while heating

How long is malachite green applied in endospore stain?

About 5 minutes

Important warning during malachite green step

Do not let the paper towel dry

After malachite green in endospore stain

Remove paper towel and rinse with water

How long is safranin applied in endospore stain?

About 1 minute

Final step in endospore stain

Blot dry with bibulous paper

Acid-fast stain alternate name

Kinyoun stain

Acid-fast stain purpose

Differential stain used to identify acid-fast organisms

Acid-fast organism used in lab

Mycobacterium smegmatis

Other medically important acid-fast organisms

Mycobacterium tuberculosis and Mycobacterium leprae

Why are Mycobacterium acid-fast?

They have mycolic acids in their waxy cell wall

Mycolic acids are

Waxy materials in the cell wall of Mycobacterium

Why can’t Mycobacterium be Gram stained well?

Its waxy cell wall layer is not water soluble

Primary stain in acid-fast stain

Carbolfuchsin

What color does carbolfuchsin stain Mycobacterium?

Red

Decolorizer in acid-fast stain

Acid alcohol

Counterstain in acid-fast stain

Methylene blue

What does methylene blue stain?

Non-acid-fast cells blue

Acid-fast stain result for Mycobacterium smegmatis

Red/pink acid-fast rods

Non-acid-fast organism used in lab

Staphylococcus saprophyticus

Acid-fast stain result for Staphylococcus saprophyticus

Blue non-acid-fast cocci

Why can carbolfuchsin enter Mycobacterium?

It is phenolic and lipid soluble, so it can penetrate the waxy layer

Is heat used in Kinyoun acid-fast stain?

No heat is used

How long is carbolfuchsin applied in acid-fast stain?

About 5 minutes

How long is acid alcohol used?

About 15–20 seconds

How long is methylene blue applied?

About 1 minute

Final step in acid-fast stain

Blot dry with bibulous paper

Acid-fast smear preparation

Add M. smegmatis colonies and 1 loopful of S. saprophyticus to the slide and mix

Saliva in acid-fast smear

No saliva is added

Why are acid-fast organisms resistant?

Their waxy mycolic acid cell wall helps resist desiccation, chemicals, and antibiotics

General smear prep from plate

Draw circles under slide, add 1–2 drops water, touch colony, mix gently, air dry, heat fix

Smear prep from broth

Use 1 loop of broth, air dry, then heat fix

Why air dry before heat fixing?

To prevent splattering and preserve the smear

Why heat fix?

To attach cells to the slide and help kill cells

Bibulous paper purpose

Used to blot dry the slide

Which plate should show the highest number of endospores?

The older B. megaterium culture

Which plate should show the highest number of vegetative cells?

The younger B. megaterium culture

What shape and color is Mycobacterium in acid-fast stain?

Red or pink rods

What shape and color is Staphylococcus saprophyticus in acid-fast stain?

Blue cocci