Microbiology Lab 10

Gram-negative cell wall

A single layer of peptidoglycan surrounded by various types of lipids.

Permeability theory

*Bacteria stain differently because of chemical & structural differences in their cell walls. *When ethyl alcohol attempts to decolorize a GP bacteria , the peptidoglycan becomes dehydrated, physically shrinks, & becomes impermeable. The purple stain is locked in the cell. *When ethyl alcohol comes in contact with GN cell walls, the lipids are dissolved and the crystal violet is leached out of the cell.

Materials

*Crystal violet stain *Gram's Iodine (should be brown, not pale yellow) *Ethyl alcohol (also called ethanol or ETOH) *Safranin stain (red)

Slant cultures

*Staphylococcus epidermidis *Escherichia coli *Bacillus megaterium *Unknowns

The 4 steps of gram staining

1. Primary stain (stains all bacteria purple) 2. Mordant (Gram's iodine causes a stain to become more tightly bound to the cell. A chemical bond between the crystal violet and the bacteria) 3. Decolorizer (removes the stain from gram-negative bacteria becoming colorless) 4. Counterstain (staining with safranin turns gram-negative bacteria red)

Gram stain origin

Invented by Hans Christian Gram in 1884 while he was attempting to devise a method to differentiate bacteria from human tissue in biopsies.

Gram stain purpose

It separates all bacteria into 2 major groups: Gram-positive & Gram-negative

Old cultures vs. The Permeability Theory

Old cultures of GP bacteria sometimes come out red instead of purple. As the culture ages, cells begin to die and their walls break down. The broken down cell wall is no longer able to prevent the alcohol from leaching out the crystal violet.

Gram-positive cell wall

Thick & composed of many layers of peptidoglycan.