Negative Staining Flashcards
TRUE or FALSE: All bacteria have capsules
False, bacteria can exist as capsulated bacteria vs non-capsulated bacteria
What are the major function of capsules? (hint: theres two)
- capsule makes the bacteria too large to be engulfed by a phagocyte
- contain carbohydrates which make them sticky and easily bind to host tissue/cells (which promotes infection)
Capsules help differentiate between different types of…
bacteria -> identify bacteria -> make proper diagnosis -> proper treatment -> future prevention
The negative stain technique aka as…?
Why is this?
indirect staining; because we are indirectly looking for the bacterial capsule by staining everything including the background and bacteria. the bacterial capsule will not get stained and will appear colorless/halo.
Be able to label: Background, capsule, bacterium if given a picture of bacteria containing capsules. What color do these appear as?
Background = dark (black; bc india ink)
Capsule = NOT STAINED; clear/halo
Bacterium = purple color (bc CV)
You have begun staining your slide containing bacterial capsules but have only put india ink on it so far. If you were to observe the slide under the microscope, what “colors” would you see?
white (bc of the capsule) and black spots (because of the background)
YOU WOULD NOT BE ABLE TO SEE THE BACTERIA INSIDE THE CAPSULE BECAUSE YOU HAVE NOT STAINED IT WITH CRYSTAL VIOLET
Why arent capsules able to be colored by india ink?
Why aren’t capsules able to be colored by crystal violet?
India ink: India ink is uncharged/non-ionic like the capsule. Dyes bind when it is held by an attraction between opposite charges, so because it is uncharged it cannot bind to capsule (or anything else, including bacterial cw).
Cyrstal Violet: Dyes (like CV) contain chromophores that are positively charged. Capsules are not charged/non-ionic becayse polysaccharides are uncharged. Therefore, they cannot pick up charged dyes and WILL NOT stain = remain colorless
What are the 2 reagents used in the negative stain technique? What is it staining?
India Ink (uncharged)= background (sits in background; dark)
Crystal violet (+ charged) = bacteria
Can india ink stain the bacteria (cell wall)? Why or why not?
No, because india ink is a non-ionic dye, meaning it is uncharged. If it is uncharged, it is not able to bind to anything.
For dyes to stick, it must be of the opposite charge ( + - )
Why is crystal violet able to stain the bacteria?
bacteria cell walls (peptidoglycan) are negatively charged and dyes contains chromophores that are positively charged.
opposite charges attract = cell wall stains
Why do we need to be able to stain the bacteria (using CV)?
to prove that a capsule is really present
without CV, you could not stain the bacteria and would not be able to determine if what you saw was truly a capsulated bacteria or just white space.
Describe the Negative Stain Technique
India Ink
- put one drop of india ink at one side and put 2 loopdools of specimen next to india ink (flame bw loopfools)
- mix india ink and speciment and the end of the slide
- use a second slide and push at an angle to evenly smear the first slide
- air dry = DO NOT HEAT FIX
Crystal violet
- cover entire slide with CV for 60 seconds and rinse with water after
- gently blot and air dry slide
