Gram staining lab and MCQ

Question 1

What is the method of gram staining?

Answer 1

-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

Question 2

What are the 4 solutions used during grain staining and what order are they

Answer 2

-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)

Question 3

What does the crystal violet do in the staining?

Answer 3

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.

Question 4

What colour does the crystal violet leave

Answer 4

Purple for both + & -

Question 5

What is the order and structure of the gram negative and postive?

Answer 5

Gram negative= Lipopolysaccharides, Outer membrane and peptidoglycan

Gram positive= Peptidoglycan & Outer membrane

Question 6

What rare the differences between two bacterial cell types ?

Answer 6

• The amount of peptidoglycan in the cell wall
• 10% for Gram negative (LESS)
• 90% for Gram positive (MORE)

Question 7

What does grain stain rely on?

Answer 7

• The difference in the thickness of he peptidoglycan layer

Question 8

What does the gram iodine do?

Answer 8

-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.

Question 9

What are the cells after gram iodine staining?

Answer 9

Both purple

Question 10

What does the Ethanol/Acetone do and what happens during this stage ?

Answer 10

-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

Question 11

What colours are the cells after ethanol/acetone?

Answer 11

-=Colourless
+=Purple

Question 12

What does the Safranin do ?

Answer 12

• is taken up by both cell types where it binds to the lipid cell membrane.

Question 13

How is the gram staining technique differentiated?

Answer 13

Techniques differentiates the bacteria based on biochem properties of their cell wall as gram positive or negative

Question 14

What is gram staining?

Answer 14

Grain staining is the first test performed when identifying the bacteria present in a sample

Question 15

What colour is gram negative & positive?

Answer 15

Gram negative= Pink/Red
Gram positive= Purple/Blue

Question 16

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

Answer 16

A) Decolorises the cells

Question 17

Bacterial cells here are:

A) Gram positive bacilli
B) Gram negative cocci
C) Gram positive cocci
D) Gram negative bacilli

Answer 17

B) Gram negative cocci

Question 18

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

Answer 18

D) Crystal violet, Iodine solution, Alcohol, Saffranine

Question 19

Gram positive bacteria appear as

A) Blue
B) Pink
C) Violet
D) Black

Answer 19

C) Violet

Question 20

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

Answer 20

C) Thick in Gram positive than Gram negative

Question 21

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

Answer 21

A) Gram positive

Question 22

Rickettsiae stained by this technique responds as

A) Gram negative
B) Gram positive
C) both
D) Between positive and negative

Answer 22

A) Gram negative

Question 23

The percentage of alcohol used in Gram-staining is
A) 25%
B) 60%
C) 90%
D) 75%

Answer 23

C) 90%

Question 24

The Lipid content present in Gram positive bacterial cell-wall is
A) 2-8 %
B) 1-10 %
C) 1-5 %
D) None of these

Answer 24

C) 1-5 %

Question 25

Gram negative bacteria appear as
A) Violet
B) Green
C) Both A and B
D) Pink

Answer 25

D)Pink

Question 26

Lipid contents is more in
A) Gram positive bacteria
B) Gram negative bacteria
C) Same in both A and B
D) None of these

Answer 26

B) Gram negative bacteria