Gram staining lab and MCQ Flashcards
What is the method of gram staining?
-Add crystal violet cover whole slide
-Incubate on slide for 1 min then wash off until solution goes from purple to clear
- Add gram iodine
- Wash off with water after one minute till clear
-Ethanol/Acetone wash off the slide till it has gone from purple to clear then rinse of with water
- Add safranin incubate for 45 seconds then wash with water
What are the 4 solutions used during grain staining and what order are they
-Crystal violet(Purple) - 1st dye used in staining (1st)
- Gram iodine(Orange)- Trapping dye within the cell (2nd)
- Ethanol/Acetone(Clear)- depolarising agent (3rd)
- Safranin(Pink)- counterstain increase the constant between the gram negative and positive (4th)
What does the crystal violet do in the staining?
Crystal Violet dye dissociates into Crystal Violet ions (CV+) and (Cl-) in aqueous solution, where the CV+ ions bind and stain the negatively charged components of the bacterial cell wall.
What colour does the crystal violet leave
Purple for both + & -
What is the order and structure of the gram negative and postive?
Gram negative= Lipopolysaccharides, Outer membrane and peptidoglycan
Gram positive= Peptidoglycan & Outer membrane
What rare the differences between two bacterial cell types ?
- The amount of peptidoglycan in the cell wall
- 10% for Gram negative (LESS)
- 90% for Gram positive (MORE)
What does grain stain rely on?
- The difference in the thickness of he peptidoglycan layer
What does the gram iodine do?
-Gram’s Iodine is added to fix the dye within the cells.
-They form complexes with crystal violet ions, large complexes which are insoluble in water and become trapped in the cell.
What are the cells after gram iodine staining?
Both purple
What does the Ethanol/Acetone do and what happens during this stage ?
-Ethanol/Acetone is the step that differentiate the gram negative from the gram positive (The crystal in the gram negative is removed but not the gram positive)
-Ethanol/Acetone dissolves the lipid of the outer membrane (Gone away now)
-Ethanol/Acetone dehydrates the peptidoglycan in both gram + & -= now left with thin peptidoglycan outer layer so the crystal violet iodine in the gram negative is removed cause it can’t hold the staining.
-Thick peptidoglycan outer layer in the gram positive so the crystal violet iodine staining stays
What colours are the cells after ethanol/acetone?
-=Colourless
+=Purple
What does the Safranin do ?
- is taken up by both cell types where it binds to the lipid cell membrane.
How is the gram staining technique differentiated?
Techniques differentiates the bacteria based on biochem properties of their cell wall as gram positive or negative
What is gram staining?
Grain staining is the first test performed when identifying the bacteria present in a sample
What colour is gram negative & positive?
Gram negative= Pink/Red
Gram positive= Purple/Blue
The acton of the alcohol during Gram-staining is …?
A) Decolorises the cells
B) Allows the colour
C) It adds colour
D) None of these
A) Decolorises the cells
Bacterial cells here are:
A) Gram positive bacilli
B) Gram negative cocci
C) Gram positive cocci
D) Gram negative bacilli
B) Gram negative cocci
The order of stains in Gram-staining procedure is.
A) Alcohol, Crystal Violet, Iodine solution, Saffranine
B) Alcohol, Crystal Violet, Iodine solution, Alcohol
C) Iodine solution, Crystal Violet, Saffranine, Alcohol
D) Crystal violet, Iodine solution, Alcohol, Saffranine
D) Crystal violet, Iodine solution, Alcohol, Saffranine
Gram positive bacteria appear as
A) Blue
B) Pink
C) Violet
D) Black
C) Violet
Cell-wall is
A) Equal in both
B) In Gram negative cell-wall is absent
C) Thick in Gram positive than Gram negative
D) Thick in Gram negative than Gram positive
C) Thick in Gram positive than Gram negative
Candida albicans when stained by Gram staining, they appear as:
A) Gram positive
B) Gram negative
C) They can be stained either Gram positive or Gram negative
D) They are not bacteria and can not be stained by Gram staining
A) Gram positive
Rickettsiae stained by this technique responds as
A) Gram negative
B) Gram positive
C) both
D) Between positive and negative
A) Gram negative
The percentage of alcohol used in Gram-staining is
A) 25%
B) 60%
C) 90%
D) 75%
C) 90%
The Lipid content present in Gram positive bacterial cell-wall is
A) 2-8 %
B) 1-10 %
C) 1-5 %
D) None of these
C) 1-5 %
Gram negative bacteria appear as
A) Violet
B) Green
C) Both A and B
D) Pink
D)Pink
Lipid contents is more in
A) Gram positive bacteria
B) Gram negative bacteria
C) Same in both A and B
D) None of these
B) Gram negative bacteria
