Lab 2B- Gram Stain Flashcards
What is the most widely used staining procedure in bacteriology?
Gram Stain
Who devised this staining procedure when trying to differentiate bacteria from a tissue section he was observing?
What time frame?
Christian Gram
1800’s
Grams bacterial stains can be characterized by the amount of ___ in the cell wall.
Peptidoglycan
Which bacteria have cell walls that contain thick layers of peptidoglycan?
A. Gram Positive
B. Gram Negative
Gram Positive
Gram positive bacteria have cell walls that contain peptidoglycan, which accounts for ___% of the cell wall.
90%
What color do gram positive bacteria stain?
Purple
Which bacteria have walls with thin layers of peptidoglycan and high lipid content?
A. Gram Positive
B. Gram Negative
Gram Negative
Gram negative bacteria have walls with thin layers of peptidoglycan which make up ___% of the wall.
10%
What color do gram negative bacteria stain?
Pink
The gram stain is NOT used for ____ or ____ as both lack peptidoglycans.
Archaea or Eukaryotes
The Gram stain has how many basic steps?
4
What are the basic steps when performing the Gram Stain?
- Apply primary stain (crystal violet) to a heat fixed smear
- Add a mordant (Gram’s Iodine)
- Rapid decolorization with alcohol or acetone/ OR mix of both
- Counterstain with safranin
Describe the theory behind Gram Positive staining…. (what each step does)
- The crystal violet enters the thick layer of peptidoglycan of the
gram positive cell wall. - The iodine acts as a mordant and binds to the crystal violet
- This large complex is caught w/in the complex of the
peptidoglycan - And isn’t washed away by the alcohol, which is used as a
decolorizer
Describe the theory behind Gram Negative staining…. (what each step does)
- The crystal violet - iodine complex is mainly taken up by the lipids within the outer membrane/ very thin layer of peptidoglycan
- These lipids are soluble in alcohol so the stain is washed away with the alcohol decolorizer
- The thin layer of peptidoglycan (found below the lipid layer) can be stained with the secondary stain, Safranin.
_____ _____ can also be characterized using Gram’s stain.
Bacterial morphology
What are the 3 basic morphological shapes of bacteria?
- Cocci- Round shaped cells
- Bacilli- Rod shaped cells
- Spirilla- Spiral shaped cells
E. coli is gram ____ (+ or - )
What color will it stain?
Gram Negative / Pink
Bacillus megaterium is gram ___ (+ or - )
What color will it stain?
Gram positive / Purple
Why is Gram’s stain a differential stain?
It is used to DIFFERENTIATE between gram (+) and gram (-) cell walls.
Gram (+) and Gram (-) organisms are distinguished from each other by differences in their _____.
Cell Walls
Gram Positive bacteria have a ______ layer of peptidoglycan
Thick
Gram Negative bacteria have a ____ layer of peptidoglycan
thin
Why is Gram’s Iodine called a MORDANT?
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.
What is a mordant?
“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.
