Wk 1 Lab Gram Stains

Question 1

What are the two main groups of bacteria? How doe we tell them apart?

Answer 1

1. Gram + organisms - stain purple
2. Gram - organisms - stain pink/red

Question 2

Why do the 2 main types of bacteria stain differently?

Answer 2

Outer cell wall of Gram (+) is thick layer of peptidoglycan (no lipids)

Outer layer of cell wall of Gram (-) is lipid w/ thin inner peptidoglycan layer

Question 3

What are the staining steps?

Answer 3

1. Crystal violet (net + charge) - attracted to Gram - and +
2. Iodine added, binds violet, forms larger molecule
3. Alcohol added ->
   -dehdyrates Gram (+) peptidoglycan layer shrinks, tightening matrix, color remains
   -solvent to Gram (-), removes lipid layer -> colorless
4. Safranin used as counterstain so Gram (-) can be seen as pink/red

Question 4

Smear prep steps

Answer 4

1. label frosted end of slide w/ pencil
2. drop of water on surface
3. sterilize wire, touch edge of bacteria colony on agar plate, emulsify bacteria in the water
4. slide on warmer to dry and fix organisms

Question 5

Gram Stain Procedural Steps

Answer 5

1. Crystal violet. 30 s
2. rinse w/ tap water
3. iodine (1% iodine, 2% potassium iodide in water). 1 min
4. Rinse w/ tap water
5. Alcohol-acetone solution or 95% ethanol until colorless fluid. 5 s. Do over dishes, not sink
6. Rinse to stop decolorization. Over sink
7. Safranin. 30 s
8. Rinse w/ tap water. Tap off excess
9. Blot w/ paper towel. Air dry
10. View under microscope w/ 10x then 100x for interpretation

*never allow immersion oil to touch the 40x objective

Question 6

What are two main bacteria morphologies?

Answer 6

1. cocci
2. bacilli

Question 7

How are Gram stain results recorded?

Answer 7

Gram [+/-] [shape][arrangement if notable]

ex. Gram-positive cocci in chains
Gram-negative bacilli
Gram-negative diplococci

Question 8

GPC

Answer 8

Gram-positive cocci

Question 9

GPB or GPR

Answer 9

Gram-positive bacilli or Gram-positive rods

Question 10

GNC

Answer 10

Gram-negative cocci

Question 11

GNDC

Answer 11

Gram-negative diplococci

Question 12

GNB or GNR

Answer 12

Gram-negative bacilli or Gram-negative rods

Question 13

What are the ony acceptable Gram stain abbreviations?

Answer 13

GPC
GPB or GPR
GNC
GNDC
GNB or GNR

Question 14

What are 6 common media used in bacteriology?

Answer 14

1. Sheep Blood Agar (SBR): alpha, beta, and gamma
2. Chocolate Agar (Choc)
3. Colistin-Nalidixic Acid Agar (CNA)
4. MacConkey Agar (MAC)
5. Trypticase Soy Broth (TSB)
6. Modified Thayer-Martin (MTM)

Question 15

What organisms grow on common media?

Answer 15

Question 16

What is the difference b/w alpha, beta, and gamma SBA?

Answer 16

alpha - green to brown discoloration w/ partially lysed RBCs around the colony
beta- clear, colorless zone around colony b/c RBCs completely lysed
gamma - no change in media surrounding colony (“non-hemolytic”)