Gram Staining

Question 1

What are the four components of a gram stain?

Answer 1

Crystal Violet
Iodine
Alcohol Acetone
Safranin
Methanol (OSF does not methanol fix, they now heat fix)

Question 2

What is the Crystal Violet used for in a gram stain?

Answer 2

This is the primary stain. This basic dye is positively charged and, therefore, adheres to the cell membranes of both gram negative and positive cells.

Question 3

What is the Iodine used for in a gram stain?

Answer 3

Gram’s iodine is used in Gram staining procedure to differentiate gram positive and gram-negative organisms. Gram’s iodine acts as a mordant that causes the crystal violet to penetrate and adhere to the gram –positive organisms.

Question 4

What is the Alcohol Acetone used for in a gram stain?

Answer 4

A decolorizer such as ethyl alcohol or acetone is added to the sample, which dehydrates the peptidoglycan layer, shrinking and tightening it. The large crystal violet-iodine complex is not able to penetrate this tightened peptidoglycan layer and is thus trapped in the cell in Gram positive bacteria.

Question 5

What is the Safranin used for in a gram stain?

Answer 5

The safranin is also used as a counterstain in Gram’s staining. In Gram’s staining, the safranin directly stains the bacteria that has been decolorized. With safranin staining, gram-negative bacteria can be easily distinguished from gram-positive bacteria.

Question 6

What is the Methanol used for in a gram stain?

Answer 6

Methanol fixation prevents liquid specimens from washing off the slide better than heat fixing, preserves blood cell morphology and results in a clearer background. Flood the entire slide when crystal violet, iodine and safranin are applied. This will ensure that all areas are stained evenly.

Question 7

What is the staining procedure for a gram stain?

Answer 7

Flood the slide with crystal violet for 30 seconds.
Rinse with tap water.
Flood with Gram Iodine for 30 seconds.
Rinse with tap water (Do not use hot water)
Decolorize briefly with acetone.
Immediately rinse with tap water.
Flood with safranin for 30 seconds.
Rinse with tap water
Blot dry with a paper towel and wipe off the back to remove any precipitate. Let dry before adding oil.