Gram stain

Question 1

What is a gram stain?

Answer 1

A gram stain is a sequential procedure involving crystal violet and iodine solutions followed by alcohol that allows the rapid classification of organisms into groups such as: Gram-Positive or Gram-negative baccilli (rods) or cocci, The culture procedures identify specific organisms, but they require 24-28 hours for completion .
Often AB selection is empirical and is based on the presciber’s clinical impression. Once culture and sensitivity results are available, the antibiotic therapy might be changed. This is referred to as directed therapy. (Page 914, Pharmacology)

Question 2

What are the noticeable differences on a cellular level of gram negative and gram positive bacteria?

Answer 2

Gram Negative:

• Inner most plasma membrane
• Thin Peptidoglycan cell wall
• Another Plasma membrane
• outer capsule
• Harder to treat with ABs
• Stains red/pink after Gram stain

Gram Positive:

• Inner most plasma membrane
• thick peptidoglycan cell wall
• Outer capsule
• More easily to treat with AB’s
• Stains purple/violet

Question 3

Which type of bacteria is more easily treatable and why?

Answer 3

Gram positive bacteria is more easily treatable because it lacks the second phospholipid bilayer (Plasma membrane) compared to Gram negative.

Question 4

What is the purpose of gram stain?

Answer 4

It’s a way to identify the type of infectious bacteria

Question 5

Why is gram staining so important?

Answer 5

Because antibiotic treatment will differ based upon outcome of gram stain

Question 6

What are the six steps of gram stain?

Answer 6

1.) Smear bacteria sample across glass slide
2.) Heat fix bacteria to slide (pass over open flame)
3.) Apply crystal violet to the bacteria
4.) Apple iodine to the bacteria (Iodine molecules bind with crystal violet molecules)
5.) Wash Bacteria with alcohol (alcohol is a destaining chemical)
**At this stage Gram positive: Cell wall will shrink, capsule will dissolve/wash away.. because these cells would have retained the crystal violet dye they will appear purple/violet in appearance.
**At this stage Gram Negative: Cell wall will shrink because of the thin cell wall, crystal violet and iodine molecules will also get washed away. Therefore Gram negative will loose their colour and look transparent.
6.) Apply safranin (pink in colour) to the bacteria
RESULT:
Gram positive:Purple in appearance
Gram Negative:Pink in appearance.

Question 7

Why does Gram positive bacteria turn purple/violet after Gram stain? and Why does Gram Negative turn Pink in appearance after the gram stain?

Answer 7

Gram positive turns purple/violet because these cells have retained the crystal violet dye (after adding safranin (pink) to the cells appear purple/violet after gram stain. gram positive remember - no 2nd plasma membrane and more easily treatable with AB’s.

Gram negative turns pink in appearance because the crystal violet and iodine molecules get washed away by the destaining chemical (alcohol) due to the THIN cell wall of gram negative bacteria, therefore loose their colour and appear transparent until Safranin (pink) is added to the slide. Therefore Gram negative only retains the pink colour. **gram negative remember - harder to treat with AB’s due to 2nd plasma membrane.

Question 8

Why does the iodine and crystal violet get washed away for the gram negative but not the gram positive?

Answer 8

Gram negative has a thin cell wall, Gram positive has a thick cell wall

Question 9

What are the chemical used in the gram stain procedure?

Answer 9

Crystal violet (synthetic Violet dye), Iodine(brownish non metallic element), Alcohol (colourless flammable liquid) and Safranin (Pink synthetic dye)