Lab 1

Question 1

Gram stain method

Answer 1

• cover smear with crystal violet (sit for 1 minute)
• wash with water to remove excess
• cover smear with Gram’s iodine (sit for 1 mintue)
• rinse
• decolorize with 95% alcohol for 10 sec (most critical step)
• wash with water to stop decolorization
• counterstain with safranin for 30 seconds
• dry blot

Question 2

New methylene blue stain

Answer 2

Used to quickly check for presence of bacteria, arrangement, and morphology

• does NOT differentiate the cell wall
• apply NMB stain for 1 min, rinse, blot dry

Question 3

Klnyoun acid-fast stain method

Answer 3

• flood smear with carbol fuchsin (3-5 min)
• wash with water
• decolorize with acid alcohol
• rinse with water
• counterstain with methylene blue or fast green (60 sec)
• blot dry
• employs phenol as a dye carrier, so steaming the dye into the cell is not necessary*

Question 4

Acid-fast organisms

Answer 4

Stain red

• non acid fast organisms and background stain blue or green
• nocardia asteroides, mycobacterium

Question 5

Gram step 1 (unstained)

Answer 5

Both gram pos and gram neg are clear

- fixation

Question 6

Gram step 2 (crystal violet)

Answer 6

Gram pos and gram neg are violet

Question 7

Gram step 3 (iodine)

Answer 7

Both gram pos and gram neg are violet

Question 8

Gram step 4 (decolarization)

Answer 8

Gram pos are violet, gram neg are clear

Question 9

Gram step 5 (safranin)

Answer 9

Gram pos are purple, gram neg are red

Question 10

What is gram stain used to determine?

Answer 10

• gram’s reaction
• arrangement, spatial orientation
• morphology

Question 11

How to determine gram reaction without staining?

Answer 11

Add a colony to 10% KOH on a slide and stir

• if solution is stringy, then it is gram positive
• looking for saponification of phospholipids in outer membrane

Question 12

What is the purpose of Gram’s iodine?

Answer 12

Acts as a mordant to decrease solubility of crystal violet