Study Gram Stain Flash Cards

 
Pile Management Card
Gram Stain

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4 steps
1. primary stain 60 sec, cover slide
rinse briefly w/ water
2. mordant 60 sec, droppers full
3. decolorizer dropper full to rinse quickly
rinse immediately w/ water
4. counterstain 30 sec, cover slide
blot slide dry
Problem with old cultures
As cultures age, some cells begin to die and their cell walls break down, and are no longer able to prevent the alcohol from leaching out the crystal violet....creates false reading of gram pos bacteria to look like gram neg
Gram neg cell wall
only a single layer of peptidoglycan surrounded by layers of various types of lipids.

when ethyl alcohol tries to decolorize, lipids are dissolved and crystal violet is leached out of the cell
Gram pos cell wall
thick and composed of many layers of peptidoglycan

when ethyl alcohol tries to decolorize, peptidoglycan becomes dehydrated, shrinks, and becomes impermeable, thus locking in purple stain
permeability theory
explanation for the mechanism of gram stain;

bacteria stain differently because of chemical and structural differences in their cell walls
differentiating step
decolorizer; removes primary stain from some bacteria (gram neg), which now become colorless, but remains in others (gram pos)
counterstain
safranin
decolorizer
ethyl alcohol
mordant
iodine
primary stain
crystal violet
function of mordants
causes a stain to become more tightly bound to the cell; iodine does this by intensifying the ionic chemical bond btw crystal violet and the bacteria
Separates bacteria in 2 groups:
Gram positive
Gram negative
Invented by _____
Year____
Was trying to differentiate ______ from ______
Hans Christian Gram
1884
bacteria; human tissue
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