Micro Lab Quiz 1

Gram positive vs. Gram negative

Gram positive

• cell wall = 90% peptidoglycan + 10% periplasm

• no outer membrane or LPS

• contains teichoic acids + lipoteichoic acids

• makes exotoxins

• less resistant to antibiotics

• gram stain color of purple/blue

Gram negative

• cell wall = 45% outer membrane (LPS + phospholipids) + 5% peptidoglycan + 45% periplasm

• outer membrane + LPS present

• no teichoic acids + lipoteichoic acids

• makes endotoxins

• more resistant to antibiotics

  • outer membrane acts like a shield

• gram stain color of pink/red

What is a gram stain

a laboratory technique that differentiates bacteria by their cell wall characteristics

List the steps of the gram stain and their significance

1. Label the slide and heat-fix it

2. Add 2 loopfuls of culture in the center

3. Let it air dry and heat-fix 3 times

4. Add a small drop of water and mix it with the culture

5. Let it air dry

6. Flood with crystal violet for 30 seconds

   1. stains all cells purple

7. Wash excess with water

8. Flood with Gram’s iodine for 1 minute

   1. helps trap stain inside the cells

9. Wash with ethyl alcohol and then water

   1. + stays purple bc of thick wall, - turns colorless

10. Flood with safranin for 1 minute

    1. + stays purple, - turns pink

11. Wash with water

12. Blot with bibulous paper

Mordant

a chemical that binds the dye to the microbes

Primary Stain

the initial stain (crystal violet)

Decolorized

the ethyl alcohol dissolves the lipids found in the cell wall of the gram negative cell, allowing the crystal violet to escape & turn colorless

Counterstain

safranin increases the contrast by coloring the gram negative cells pink

What is a simple stain

one stain is used to dye bacterial cells

• Prepare a smear: Spread bacteria onto a microscope slide

• Heat-fix: Gently heat the slide to attach the bacteria to it

• Apply one dye: Add a stain, such as methylene blue, crystal violet, or safranin

• Rinse and dry: Wash off excess dye and let the slide dry

• Observe: View to see the shape, arrangement, size

What is a capsule stain

identifies capsules around a bacterial cell

• Prepare a smear: Place bacteria on a microscope slide

• Apply a negative stain: Use a dye, such as India ink or nigrosin, to darken the background

• Apply a counterstain: Use a dye to color the bacterial cells

• Observe under a microscope: The capsule appears as a clear halo surrounding the stained bacterial cell against a dark background

What is an acid-fast stain

identify bacteria with waxy cell walls containing mycolic acid (whether they can hold red stain)

What is an endospore stain

identifies endospores (tough & dormant structures that increase survival in harsh conditions)

• add malachite green

• rinse with water

• add safranin

List how the acid-fast stain is done and why

• add red stain w/ heat (heat helps penetrate the waxy cell wall)

• add acid-alcohol (removes dye from non-acid)

• add blue dye (acid-fast bacteria = red/pink + non-acid-fast bacteria = blue)

List how the acid-fast stain is done and why

List how the acid-fast stain is done and why

List the steps of the endospore stain and their significance