Ex 7: Gram Staining Flashcards
What is a differential stain?
one that distinguishes between microroganisms or parts of same organism due to their ability to retain dyes
Name the three main steps in differential staining
primary stain, decolourization step and counterstain
What do gram-positive cell walls have?
a thick peptidoglycan layer with teichoic acids
What do gram-negative cell walls have?
thin peptidoglycan layer surrounded by an outer membrane containing lipopolysaccharide and lipoproteins
What is the first step in gram staining?
primary stain, crystal violet is added, stains cells deep purple
What is the second step in gram staining?
mordant/fixative, iodine is added. increases cells’ affinity for stain
How does iodine increase cells’ affinity?
by binding to the primary stain forming an insoluble complex. results in crystal violet-iodine complex (CV-I)
What is the third step in gram staining?
A decolourizer, isopropanol and acetone is added
What does decolourizer do in gram negative bacteria?
alcohol increase porosity of cell wall by dissolving lipids CV-I complex removed from thinner and less highly linked peptioglycan. cell is unstained
What does decolourizer do in gram positive bacteria?
alcohol dehydrates thicker peptidoglycan making pores smaller, resulting in stringent retention of CV-I complex, cell is violet
What is the final step in gram staining?
counterstain, safranin used. gram -ve underwent decolourization so will absorb stain to turn pink
What are some examples of gram-variables?
Neisseria and Moraxella, gram-ive but resist decolourization
What are gram-nonreactives. Give example
bacteria that stain poorly or do not. Mycobacterium
What happens when we use old cells for gram staining?
old cells lose ability to hold onto stain, appear gram -ive. cultures less than 24 hours are best
What happens when we overheat cells for gram staining?
disrupts cell walls, make gram +ive appear gram -ive
