Microbiology Lab Quiz; Gram Stain

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What does the Crystal Violet stain do within the cell wall?

Forms a purple complex

What is the purpose of the mordant, Iodine?

Keeps the Crystal Violet from being removed from the Gram-positive bacteria

What does the Decolorizing Solution, Ethanol, do to Gram-negative bacteria? Why?

Washes out the purple complex. Cell walls are too thin

What does the Decolorizing Solution, Ethanol, do to Gram-positive bacteria?

Nothing, because of the thick cell walls

What is the purpose of Safranin to Gram-negative bacteria?

Turns it pink/red

What happens to the cell wall if they are old or are stressed by heat or cold?

The cell walls become thin

It is best to use cultures for Gram staining within what time period?

24 hours

What are the two staining techniques?

Simple and differential

Bacteria react different based on what on their cells?

Surface composition

Name the four staining reagents.

Crystal Violet, Iodine, Ethanol, Safranin

Why is gram staining important?

Identifies unknown bacteria

What are the two groups of bacteria and their colors?

Gram-positive and Gram-negative bacteria; purple and pink, respectively

What does Gm-/Gm+ do help us determine?

Appropriate antibiotic treatments

What year was the gram stain invented?

1882

Who invented the gram stain?

Hans Christian Gram

What did we use as the primary stain?

Crystal Violet

What did we use as the mordant?

Iodine

What did we use as the decolorizing agent?

Ethanol

What did we use as the counter-stain?

Safranin

The bacterial cell wall is composed of what? Where is it located?

Peptidoglycan; on top of the plasma membrane

Gram-positive cell walls contain only one...?

Membrane

Gram-negative cell walls contain two...? Which are? What's in the middle?

Membranes; inner and outer; peptidoglycan

What kind of dye is Crystal Violet? Cationic or Anionic?

Cationic

The exterior of the cells have what kind of charge; anionic or cationic?

Anionic

What is the purpose of the mordant?

Iodine complexes the crystal violet to the peptidoglycan layer

Gm- cell walls have a higher _____ content than Gm+ cell walls?

Lipid

What does CFU stand for and what does it mean?

Colony Forming Units; the number of colonies grown during incubation

What does it mean to aliquot something?

Take a smaller volume of a sample

What is a diluent? Give an example.

Fluid used to dilute concentration sample; water

What is a dilution factor?

Fraction by which original is diluted

What does TNTC stand for and what are the numerical values?

Too numerous to count; >300

What does TFTC stand for and what are the numerical values?

Too few to count; <30

How much sterile water do you put in the 7 test tubes?

9.0 mL

How many test tubes do you use for dilution?

7

What do you label the test tubes for dilution?

10^-1 --> 10^-7

How much of the broth sample do we obtain and where do we put it?

We obtain 1.0 mL; put it in the 10^-1 test tube

What do we do each time we transfer the broth from test tube to test tube?

Use a new pipette

What do you do after diluting the test tubes? How much do we obtain? 10^-4 turns into?

Dilute again into petri dish; 0.1 mL; 10^-5

What are the steps to using the spreading rod?

Dip in alcohol, pass through burner; let cool; spread specimen

How do you solve for Final Concentration?

CFU/0.1 mL

How do you solve for O.T. Concentration?

[CFU/0.1 mL]/[1/10,000]

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