Negative Stain Quiz (4pts)

What are the 2 reagents in the negative capsule stain, in order?

1. India Ink → 2. Crystal Violet

Function of India Ink?

Makes the dark background. It's non-ionic, so it does NOT bind the capsule.

Function of Crystal Violet?

Stains the bacterium inside the capsule. Covers the entire slide for 60 seconds.

Why does crystal violet stain the bacterium but not the capsule?

The cell wall (peptidoglycan) is negatively charged and the dye's chromophore is positively charged, so they attract.

• The capsule is uncharged, so the dye doesn't stick.

Why doesn't the capsule stain?

It's made of polysaccharides, which are non-ionic (uncharged), so it can't pick up charged dyes.

What do the background, capsule, and bacterium look like?

Background is dark, the capsule is a clear "halo," and the bacterium is purple.

Why is this called "negative" or "indirect" staining?

The capsule itself isn't stained. You see it because of what surrounds it, the dark background.

How do you confirm a true capsule?

You must be able to see the bacterium inside the halo!

What do you do after the India Ink smear, and why?

Air dry. Do NOT heat fix, because heat shrinks or destroys the capsule.

What are 2 functions of capsules?

1. Anti-phagocytic: limits phagocytes' ability to engulf the bacteria.
   
   

2. Attachment to host tissue, which promotes infection.

Why does detecting capsules matter clinically?

It helps identify bacteria, which leads to diagnosis, then treatment, then prevention.