Microbio Lab Quiz 1

Steps in a simple stain

1. make 2 wax circles on the slide

2. add a loop of water to each circle

3. add a loop of bacterial and mix in the first circle

4. mix whatever remains on your loop into the second circle

5. allow it to dry completely

6. heat fix by passing the slide (bacteria side facing away) across the incinerator 4-5 times

7. stain (methylene blue) for 1 minute

8. rinse with distilled water and dry

What does simple stain show you?

morphology, arrangement, lack/presence of cells

what are disadvantages of heat fixing?

cells are dead and you can’t see motility, cell shape may get distorted

how does staining work?

Bacterial cells are typically negatively charged. Dyes, when ionized (gain/lose an electron) they are either attracted to the bacterial cell or repelled away. Basic dyes (like methylene blue) enter and dye the cell so that it is visible. Acidic dyes (negrosin) repel from the cell but dye the background.

Steps to an endospore stain

1. create a smear

2. heat fix

3. add malachite green

4. incubate at 60 degrees celcius

5. rinse with water

6. stain with safranin for 1 minute

7. rinse with water

8. dry

purpose of endospore stain?

to differentiate between an endospore and vegetative cell

steps to a nigrosin stain

1. put a drop of negrosin on one end of a slide

2. place a loop of bacteria onto negrosin drop

3. mix

4. use a second slide to drag negrosin across the surface of the slide

5. dry

Steps to a gram stain

1. prepare a heat fixed smear

2. stain with crystal violet 1 minute

3. rinse with water

4. stain with gram idione 1 minute

5. rinse with ethanol until clear

6. rinse with water

7. stain with safranin 1 minute

8. rinse with water

9. dry

what does mannitol salt agar plate select and differentiate for?

select: salt-tolerant organisms (halophiles)

differentiates: mannitol fermentation (turns yellow)

what does eosin methylene blue plate select and differentiate for?

selects: gram negative bacteria; eosin+methylene blue is inhibitory to gram positive bacteria

differentiates: lactose fermentation (color of colonies)

what does phenol red carbohydrate broth test for?

fermentation and gas production

• yellow broth = lower pH(fermentation)

• durham tube catches any gasa

what does Voges- Proskauer Test for?

production of organic acids into neutral end products acetoin and 2.3 butanediol

• barritts reagents reacts to acetoin and turns red

What does kligers agar deep slant test for?

cysteine desulferase activity

• turns all yellow: glucose and lactose fermentation

• half yellow: lactose fermentation

• turns black: cysteine desulferase Activity

what does indole production test for?

enzyme tryptophanase by detecting indole production

• yellow ring = negative

• red ring = positive

how does indole production test work?

tryptophan is degraded by tryptophanase into indole, pyruvic acid, and ammonia

what does a Phenylalanine deaminase slant test for?

activity of phenylalanine deaminase and presence of phenylpyruvic acid

• yellow liquid: negative

• green liquid: positive

is E. coli gram positive or gram negative?

gram negative

is S. epidermidis gram positive or gram negative?

gram positive

What is the bacteria quantification formula?

(# of CFUs/ volume plated) * (1/Dilution Factor)

what is resolution?

the ability of the microscope to distinguish two adjacent objects as separate

what objective lens should you start with?

10X

While is oil immersion important?

It has the same refraction levels as glass and prevents light rays form getting lost, potentially blurring the image