Gram Stain

Question 1

Step 1 of Gram stain

Answer 1

heat fix organism to slide via Bunsen burner or bacticinerator

Question 2

Step 2 of Gram stain

Answer 2

cover slide with crystal violet for 1 minute; rinse with distilled water thoroughly to remove excess stain

Question 3

Step 3 of Gram stain

Answer 3

cover slide with iodine (mordant) for 1 minute; rinse with distilled water thoroughly

Question 4

Step 4 of Gram stain

Answer 4

decolorize slide with alcohol, only 2-3 passes of alcohol needed; immediately rinse with distilled water

Question 5

Step 5 of Gram stain

Answer 5

counterstain slide with safranin for 2 minutes; rinse with distilled water

Question 6

What color will Gram-positive cells be?

Answer 6

darker purple

Question 7

What color will Gram-negative cells be?

Answer 7

lighter pink

Question 8

Why does alcohol work as a decolorizer?

Answer 8

the alcohol ruptures the cell membrane of Gram-negative cells while the peptidoglycan cell wall of Gram-positive cells remains intact (for longer)

Question 9

What is the purpose of iodine (mordant)?

Answer 9

iodine “fixes” the crystal violet dye to the peptidoglycan cell wall

Question 10

Why might the Gram stain lead to Gram-positive cells having a pink color?

Answer 10

• the culture of the organism is old and the cell wall has degraded over time
• too much decolorizer (alcohol) was used