Staining Techniques (Lab) Flashcards
Why do we need to stain bacterial cells?
They’re transparent and hard to see
What are the 4 types of stains we used in this lab?
- Simple stains
- negative stains
- Differential stains
- structural stains
What are simple stains?
General stains that stain whole cells and stain every cell
How do simple stains work?
They are often positively charged and will interact with the negatively charged membrane
What type of stain is methylene blue?
Simple stain
What type of stain is crystal violet?
Simple stain
What type of stain is safranin?
Simple stain
What are negative stains?
Stains that stain the background instead of the cell, so the cell sticks out like a silhouette against a dark background
What are the 2 types of negative stains?
Colloid stains and acidic stains
How do colloid stains work?
Dark particles of ink that are neutrally charged but are physically too big to get into the cell
How do acidic stains work?
Negatively charged stains that are repelled by the cell envelope
What type of stain is India Ink?
Colloid negative stain
How do differential stains work?
Use multiple stains that will bacteria with different properties differently
What type of staining is gram staining?
Differential. Gram-positive bacteria look purple because they retain the CV-I complexes, and gram-negative bacteria look pink because they become counterstained by safranin
What type of staining is acid fast staining?
Differential. Acid-fast bacteria look pink and everything else is counterstained by methylene blue
What are the 3 dyes used in gram staining?
Crystal violet, iodine, safranin
What does the crystal violet stain do in gram staining?
Primary stain. Stains everything
What does the iodine do in gram staining?
Mordant. Forms complexes with the crystal violet
What does the ethanol wash do in gram staining?
Dehydrates the cell membrane and causes gram-negative cells to lose the CV-I complexes while gram-positive cells have thicker cell walls and retain them
What does the safranin do in gram staining?
Counterstains the gram negative cells that lost the CV-I complexes
What causes over-decolourization in gram-staining?
Washed with ethanol for too long, and caused the gram-positive cells to lose the CV-I complexes
What happens to a gram stain if there was too much heat fixation?
The cell walls and membranes get destroyed and the CV-I complexes don’t get retained by anything, so everything looks gram negative
What happens to a gram stain if the smear was too thick ?
The stains can’t get inside the layers, so we get a mixture of purple and pink
What happens to a gram stain if there was improper washing between the steps?
The extra CV-I complexes don’t get washed away and everything looks purple
How does culture age affect gram stains?
Older cultures have more dead cells, which don’t have intact membranes and look pink from the counterstain
What happens to a gram stain if there was an impure culture?
Mixture of both colours
Why can’t acid fast bacteria be gram stained?
The mycolic acid cell wall keeps all the stains out
How are acid fast bacteria stained?
The cells are heated with carbolfushin in phenol, which stains them pink. Wash with a mixture of acid and alcohol so only the acid fast cells retain the stain, then counterstain everything else with methylene blue
What do structural stains stain?
Structural features like flagella, capsules, or endospores
How do you get a stain into an endospore?
Malachite green + heat and steam
