Flashcards in Lab 4 Deck (10):
What are the reagents used in gram stain?
-Primary stain: crystal violet
-Mordant: grams iodine
-Decolorizing agent: acetone/ alcohol
What are the 5 steps of the gram stain?
1. Cover with crystal violet for 1 minute, rinse with water
2. Apply grams iodine for 1 minute, rinse with water
3. Rinse with acetone/alcohol until colorless, rinse with water
4. Apply safranin for 1 minute, rinse with water
5. Blot dry
What is the function of the primary stain crystal violet?
stains gram positive and negative purple
What is the function of the mordant grams iodine?
Enhances crystal violet and keeps them purple
What is the function of decolorizing agent acetone/alcohol?
removes crystal violet from gram negative cells
What is the function of the counter stain safranin?
counterstains gram-negative cells reddish/pink
What cell component is responsible for the different stain outcomes in a gram negative?
High lipid content, thin peptidoglycan layer.
Acetone/alcohol extracts lipid making the cell wall more porous and not capable of retaining crystal violet or iodine.
What cell component is responsible for the different stain outcomes in a gram-positive?
The decolorizer dehydrates the gram-positive cell wall and the thick peptidoglycan and cross-linking with teichoic acids traps the crystal violet and iodine making the decolorizer not work (keeps it purple).
What are the 5 steps in preparing a smear?
1. small drop of water on slide (if broth culture skip this step)
2. add bacteria to water (multiple smears can be made on one slide)
3. let smear dry
4. heat fix, pass through flame 2-3 times
5. let slide cool