Gram and Acid-Fast Staining

Question 1

How does the Gram stain classify bacteria?

Answer 1

into gram-positive and gram-negative

Question 2

What kind of stain is Gram staining and acid-fast?

Answer 2

a differential stain

Question 3

What is the difference between gram-positive and gram-negative bacteria in regards to their susceptibility to antibiotics?

Answer 3

Gram-positive bacteria tend to be killed by penicillin and detergents while gram-negative are more resistant to antibiotics.

Question 4

Describe the cell wall of a gram-positive bacteria.

Answer 4

1. peptidoglycan cell wall

2. plasma membrane

Question 5

Describe the cell wall of a gram-negative bacteria.

Answer 5

1. outer membrane
2. peptidoglycan cell wall
3. periplasmic space
4. plasma membrane

Question 6

What are the four steps/components of Gram stain? What do they do?

Answer 6

1. Primary stain: Crystal violet - stains both Gram-positive and Gram-negative cells
2. Mordant: Iodine - enhances the crystal violet staining by forming a crystal violet-iodine complex.
3. Decolorizing agent: Alcohol-acetone (Ethyl Alcohol) - most important step that removes the stain from Gram-negative cells only.
4. Counterstain: Safranin - stains the Gram-negative cells reddish-pink (red stain) but because the Gram-positive cells are already purple, they do not stain.

Question 7

What color will the primary stain, crystal violet, turn gram-positive cells? gram-negative cells?

Answer 7

Gram-positive: purple

Gram-negative: purple

Question 8

What color will the mordant, iodine, turn gram-positive cells? gram-negative cells?

Answer 8

Gram-positive: purple

Gram-negative: purple

Question 9

What color will the decolorizing agent, alcohol-acetone, turn gram-positive cells? gram-negative cells?

Answer 9

Gram-positive: purple

Gram-negative: colorless

Question 10

What color will the counterstain, safranin, turn gram-positive cells? gram-negative cells?

Answer 10

Gram-positive: purple

Gram-negative: red

Question 11

What are the three organisms we looked at using Gram stain? What was the cellular morphology and arrangement? Color? Gram reaction (+-)?

Answer 11

1. Escherichia coli (Ec): Single rods, pink, gram-negative
2. Staphylococcus epidermidus (Se): Clustered spheres, purple, gram-positive
3. Bacillus cereus (Bc): rod, purple, gram-positive

Question 12

What qualifies a cell as acid-fast? What is acid-fast staining?

Answer 12

Cells that retain a basic stain in the presence of acid-alcohol.
Acid-fast stain is a differential stain used to detect cells capable of retaining a primary stain when treated with an acid alcohol.

Question 13

What type of bacteria is always acid-fast?

Answer 13

mycobacteria

Question 14

All mycobacteria is what? (2 things)

Answer 14

aerobic and acid-fast

Question 15

Why are mycobacterial infections notoriously difficult to treat?

Answer 15

They are hardy due to their cell wall which is neither truly Gram negative nor positive and unique to the family. This makes them naturally resistant to a number of antibiotics that utilize the destruction of cell walls, such as penicillin.

Question 16

Along with antibiotics, what else are acid fast organisms highly resistant to?

Answer 16

disinfectants and dry conditions

Question 17

What are two distinct pathogens of mycobacteria?

Answer 17

1. M. tuberculosis (organisms that causes tuberculosis)

2. leprae (organism that causes leprosy)

Question 18

What is the primary stain used in acid-fast staining?

Answer 18

carbolfuchsin

Question 19

How does the primary stain in acid-fast staining penetrate the cell wall?

Answer 19

Carbolfuchsin is lipid soluble and contains phenol which helps penetrate the cell wall which is further assisted by the addition of heat.

Question 20

What are the steps to acid-fast staining and what does each step do?

Answer 20

1. Primary stain - Carbolfuchsin (penetrates the cell wall)
2. Apply heat (assists in penetration of the cell wall)
3. Rinse with strong decolorizer - acid alcohol (strips the stain form all non-acid-fast cells but does not permeate the cell wall of acid-fast organisms)
4. Counterstain - methylene blue (taken up by the non-acid-fast cells)

Question 21

What are the two organisms we looked at using acid-fast stain? What was the cellular morphology and arrangement? Color? Acid-fast reaction (+-)?

Answer 21

1. Mycobacterium phlei - rod shaped; red/pink; acid-fast positive
2. Staphylococcus epidermidus (Se) - clustered spheres; blue; acid-fast negative

Question 22

List two genera which stain acid fast positive.

Answer 22

Mycobacterium and Nocardia

Question 23

What types of microbes are susceptible to acid fast staining?

Answer 23

Organisms with mycolic acids in their cell walls

Question 24

What are acid-fast stains useful in identifying?

Answer 24

They are useful in identifying acid-fast bacilli (AFB) and in rapid, preliminary, and provisional diagnosis of tuberculosis.