Lab 2B- Gram Stain

Question 1

What is the most widely used staining procedure in bacteriology?

Answer 1

Gram Stain

Question 2

Who devised this staining procedure when trying to differentiate bacteria from a tissue section he was observing?

What time frame?

Answer 2

Christian Gram

1800’s

Question 3

Grams bacterial stains can be characterized by the amount of ___ in the cell wall.

Answer 3

Peptidoglycan

Question 4

Which bacteria have cell walls that contain thick layers of peptidoglycan?

A. Gram Positive
B. Gram Negative

Answer 4

Gram Positive

Question 5

Gram positive bacteria have cell walls that contain peptidoglycan, which accounts for ___% of the cell wall.

Answer 5

90%

Question 6

What color do gram positive bacteria stain?

Answer 6

Purple

Question 7

Which bacteria have walls with thin layers of peptidoglycan and high lipid content?

A. Gram Positive
B. Gram Negative

Answer 7

Gram Negative

Question 8

Gram negative bacteria have walls with thin layers of peptidoglycan which make up ___% of the wall.

Answer 8

10%

Question 9

What color do gram negative bacteria stain?

Answer 9

Pink

Question 10

The gram stain is NOT used for ____ or ____ as both lack peptidoglycans.

Answer 10

Archaea or Eukaryotes

Question 11

The Gram stain has how many basic steps?

Answer 11

4

Question 12

What are the basic steps when performing the Gram Stain?

Answer 12

1. Apply primary stain (crystal violet) to a heat fixed smear
2. Add a mordant (Gram’s Iodine)
3. Rapid decolorization with alcohol or acetone/ OR mix of both
4. Counterstain with safranin

Question 13

Describe the theory behind Gram Positive staining…. (what each step does)

Answer 13

1. The crystal violet enters the thick layer of peptidoglycan of the
   gram positive cell wall.
2. The iodine acts as a mordant and binds to the crystal violet
3. This large complex is caught w/in the complex of the
   peptidoglycan
4. And isn’t washed away by the alcohol, which is used as a
   decolorizer

Question 14

Describe the theory behind Gram Negative staining…. (what each step does)

Answer 14

1. The crystal violet - iodine complex is mainly taken up by the lipids within the outer membrane/ very thin layer of peptidoglycan
2. These lipids are soluble in alcohol so the stain is washed away with the alcohol decolorizer
3. The thin layer of peptidoglycan (found below the lipid layer) can be stained with the secondary stain, Safranin.

Question 15

_____ _____ can also be characterized using Gram’s stain.

Answer 15

Bacterial morphology

Question 16

What are the 3 basic morphological shapes of bacteria?

Answer 16

1. Cocci- Round shaped cells
2. Bacilli- Rod shaped cells
3. Spirilla- Spiral shaped cells

Question 17

E. coli is gram ____ (+ or - )

What color will it stain?

Answer 17

Gram Negative / Pink

Question 18

Bacillus megaterium is gram ___ (+ or - )

What color will it stain?

Answer 18

Gram positive / Purple

Question 19

Why is Gram’s stain a differential stain?

Answer 19

It is used to DIFFERENTIATE between gram (+) and gram (-) cell walls.

Question 20

Gram (+) and Gram (-) organisms are distinguished from each other by differences in their _____.

Answer 20

Cell Walls

Question 21

Gram Positive bacteria have a ______ layer of peptidoglycan

Answer 21

Thick

Question 22

Gram Negative bacteria have a ____ layer of peptidoglycan

Answer 22

thin

Question 23

Why is Gram’s Iodine called a MORDANT?

Answer 23

Because it fixes the primary stain to the cell wall and adds color.

Gram’s iodine binds to crystal violet making a large complex that adheres to the cell membrane.

Question 24

What is a mordant?

Answer 24

“Dye Fixative” - a substance used to set (bind) stains or dyes
It is a trapping agent that complexes with crystal violet, making the crystal violet iodine complex clump & stay contained in thick layers of peptidoglycan in cell walls.