| - |
primary stain: crystal
violet |
| |
o |
stains both gram-positive & gram-negative
bacteria |
| |
o |
enters bacterial cell & forms iodine-crystal violet
complexes |
| - |
decolorizer: acetone-alcohol |
| |
o |
dehydrates thick cell wall of gram-positive bacteria;
iodine-crystal violet dye complex is trapped in cells: gram-positive cells
remain purple |
| |
o |
disrupts outer membrane & partially deteriorates thin
cell wall of gram-negative bacteria; iodine-crystal violet dye complex
escapes cells:
gram-negative cells become
clear |
| |
o |
stains cell wall of cells |
| |
|
• |
purple of gram-positive cells masks
pink color & remain purple |
| |
|
• |
unstained gram-negative cells become
pink |
| |
o |
factors that could cause gram-positive bacteria to appear
gram-negative: |
| |
|
• |
old cultures; damage to cell wall |
| |
|
• |
overzealous heat fixation |
| |
|
• |
overzealous decolorization |
Gram-positive
bacteria
(Staphylococcus
epidermidis) |
|
Gram-negative
bacteria
(Escherichia
coli) |
| (Ziehl-Neelsen)
Acid-Fast Stain |
| - |
primary stain: carbolfuchsin |
| |
o |
carbolfuchsin (basic fuchsin in phenol), with mild heating, is able to
penetrate the waxy layer of mycolic acids present in the cell walls of acid-fast
bacteria, & enters both acid-fast & non-acid-fast bacteria
(acid-fast bacteria are difficult to stain as most staining reagents are
unable to penetrate the cell wall; phenol & heat
assists dye entry into the cell) |
| - |
decolorizer: acid-alcohol
(3% HCl in ethanol) |
| |
o |
carbolfuchsin remains in acid-fast bacteria as the cell wall is
resistant to the acid wash:
acid-fast cells remain
pink |
| |
o |
without a resistant layer of mycolic acids in the cell wall, the acid
wash removes the carbolfuchsin from the non-acid fast bacteria: non-acid-fast cells become
clear |
| - |
counterstain: methylene
blue |
| |
o |
acid-fast cells
retain the carbolfuchsin & remain pink |
| |
o |
non-acid-fast cells
take up the methylene blue & become blue-green |
| - |
acid-fast bacteria: genus
Mycobacterium |
| |
o |
genus Nocardia is generally weakly acid-fast, & may not
retain the primary stain following the typical acid wash (a weak acid wash
(using 1% sulfuric acid) is sometimes used, which will usually allow these
bacteria to retain the primary stain while still removing the primary
stain from non-acid-fast bacteria) |
| |
o |
common acid-fast bacteria: Mycobacterium tuberculosis,
Mycobacterium leprae, & Mycobacterium
avium-intracellulare complex |
Mix of acid-fast & non-acid-fast bacteria
(Mycobacterium gordonae &
Staphylococcus epidermidis) |
| (Schaeffer-Fulton)
Endospore Stain |
| - |
primary stain: malachite
green |
| |
o |
heating allows malachite green to enter the tough spore coat of
endospores... cooling traps the dye inside the spore coat (spore coats,
like acid-fast cell walls are resistant to most staining reagents);
vegetative cells take up malachite green as well |
| |
o |
spore coats of endospores retain stain: endospores remain
green |
| |
o |
water washes malachite green from vegetative cells: vegetative cells become
clear |
| |
o |
endospores retain
the malachite green & remain green |
| |
o |
vegetative cells
take up the safranin & become pink |
| - |
common endospore-forming bacteria:
genus Bacillus; genus
Clostridium |
Endospore-forming
bacterium Bacillus subtilis |
Capsule Stain
- Place a drop of Enterobacter aerogenes, grown for two days in skim milk broth, onto a slide.
- Spread across the slide, allow to air dry.
- Cover smear with crystal violet for 1 minute, rinse with either water or 20% copper sulfate. Blot dry and observe under oil (100X).
Considerations:
1) Both the stain and capsule are more soluble in water than in copper sulfate so you may like your results better with the copper sulfate rinse.
2) Heat fixing doesn't seem to be helpful, so don't.
3) Blot, don't wipe the stain off the slide.
Water Wash
Heat Fix, Water Wash
Copper Sulfate Wash
Copper Sulfate Wash
