Exercise 9: Gram Stain

Question 1

Gram stain

Answer 1

• important differential stains
• Gram positive or Gram negative based on cell wall
• 3% KOH is an alternative test

Question 2

Primary Stain

Answer 2

-crystal violet

Question 3

Counterstain

Answer 3

-safranin

Question 4

Iodine

Answer 4

-added as a mordant after primary stain to increase the affinity between the primary stain and the reactive substances in the cell wall

Question 5

Decolorizing Agent

Answer 5

• 95% ethanol
• good lipid solvent and dehydrating agent
• removes crystal violet

Question 6

Gram-Variable

Answer 6

• display both Gram + and Gram - cells of the same size, shape, and arrangement
• Mycobacterium tuberculosis- causes tuberculosis
• Mycobacterium leprae- causes leprosy

Question 7

Gram Positive

Answer 7

• thick peptidoglycan layer (20-80 nm)
• dark blue/purple color
• holds crystal violet solidly
• Staphylococcus epidermidis, B. subtilis

Question 8

Gram Negative

Answer 8

• very thin layer (2-7 nm)
• surrounded by outer membrane consisting of lipopolysaccharides (LPS)
• light red/pink in color
• crystal violet leaks out of thin wall and is thus counterstained
• E. coli

Question 9

Procedure

Answer 9

1. Aseptic Transfer
2. Air dry
3. Heat fix; hold with clothes pin and wave in front of microincinerator
4. Cover smears with crystal violet for 1 minute
5. Rinse w/ water at an angle
6. Cover smears with Iodine for 1 minute
7. Rinse w/ water at an angle
8. Decolorize w/ 95% ethanol for 12-15 seconds
9. Immediately add water
10. Counterstain with safranin for 1 minute
11. Rinse w/ water
12. Observe under microscope