microbiology - w2: differential stains; cell wall

differential stains

use more than one strain

- cells of different bacterial species can have different appearances based on their chemical/structural properties

gram stain history

developed by Christian Gram (1884), shows gram-positive or gram-negative bacteria

gram-positive

thick layers of peptidoglycan (substance of carbohydrates. protein subunits) in their cell walls

- teichoic acids

gram-negative

little peptidoglycan

- no teichoic acids

- outer membrane containing lipolysaccharides (LPS), released as endotoxins when cell dies

when are gram stains best performed?

on fresh culture; older cells may have damaged cell walls

gram-variable

gram-positive & gram-negative reactions are visible on your slide

gram stain procedure

1. Crystal Violet (primary) enters the peptidoglycan of all bacteria, giving a purple color

2. Gram's iodine (mordant) combines with primary to make a bigger complex in peptidoglycan

3. Ethanol (decolorizer) REMOVES primary & mordant from THIN peptidoglycan, but STAYS in THICK peptidoglycan (length of time crucial)

4. Gram-positive & gram-negative will either keep the purple crystal violet color or lose it, respectively

5. In gram-negative, use Safarin (counterstain) to stain the colorless gram-negative cells pink a color.

acid-fast stain

differential stain to identify acid-fast organisms

acid-fast organisms

wax-like, nearly impermeable cell walls

- mycolic acids, large amounts of fatty acids, waxes, & complex lipids

- resistant to most compounds

carbol fuschin

primary stain in acid-fast organisms that penetrates the cell wall

- assisted by heat (steam) that loosens up waxy layer & promotes entry

what happens when carbon fuschin is rinsed with decolorizer?

- acid-fast organism: decolorizer does not permeate the cell wall

- non acid-fast organism: decolorizer strips stain

what happens to non-acid fast cells?

they take up the methylene blue (counterstain)