LAB: 3
PREPARATION OF A SMEAR
GRAM STAIN
ACID FAST STAIN
BACTERIAL SMEARS
•
•
Before bacteria can be stained a smear must be
made and fixed.
A bacterial smear is made by evenly spreading
bacteria on a clean slide in a drop of liquid
(sterile water or broth).
BACTERIAL SMEARS
Allow the slide to air dry and then:
•
Heat or Chemically fix the sample to the slide:
1. Heat fix:
– Pass it over the incinerator to gently heat fix
2. Chemically fix:
– by flooding the slide with methanol and allow to
evaporate (Denatures bacterial enzymes and
enhances adherence of bacteria to the slide)
THE SLIDE IS NOW READY TO STAIN
GRAM STAIN
• Developed by Hans Christian Gram in
1884 when he was studying bacteria
from different respiratory diseases.
• The Gram Stain is a differential stain
used to classify bacteria as Gram
positive or Gram negative.
• The single most important technique in
the Microbiology Laboratory.
The Gram Stain
The uptake and retaining of the basic stains
depends on the cell wall:
*Gram positive bacteria have many layers of peptidoglycan in their cell wall making it
easier to hold onto the CV-I complex.
*Gram negative bacteria have one layer of peptidoglycan. Thus the CV-I complex is not
retained in the cell.
GRAM STAIN REAGENTS
• 1. Crystal violet (CV): primary basic stain
• 2. Grams Iodine (I): not a stain; it forms a crystal
violet—iodine complex (CV-I) inside the cell wall.
• 3. Decolorizer: ethyl alcohol, used to remove the
primary stain. It removes the CV-I complex from cells
without teichoic acid.
• 4. Safranin: secondary (counter) basic stain
SET UP YOUR SLIDE FOR
STAINING
GRAM STAIN SUCCESS
• DO NOT USE TOO MUCH WATER OR
BROTH. A SMALL LOOPFUL IS ENOUGH.
• DO NOT USE A HEAVY INOCULUM
(AMOUNT OF BACTERIA).
• MAKE AN EVEN, THIN SUSPENSION ON
THE SLIDE. THIS WILL ALLOW FOR
PROPER STAINING AND ESPECIALLY
PROPER DECOLORIZATION.
GRAM STAIN PROCEDURE
1. Flood with
crystal violet for 1
minute. Rinse
2.Flood with
iodine for 1
minute. Rinse
3.Decolorize with
alcohol until no
more purple
comes off the
slide. Rinse
4. Flood with
safranin. Rinse
and dry.
RESULTS
Gram positive
Organisms will
stain purple.
Gram negative
Organisms will
stain pink.
RESULTS
WHAT WOULD HAPPEN IF
WE:
1.) OVER – DECOLORIZE
2.) UNDER – DECOLORIZE
WHY DO WE NEED TO STAIN
MICROORGANISMS???
ACID FAST STAIN
• Developed by Paul Ehrlich in 1882
• Kinyoun published ‘cold-staining’ method in 1915,
which is commonly used today
• Acid fast positive bacteria have fatty acid in their
cell wall (mycolic acid) that makes them ‘acid fast’,
i.e. do not decolorize with acid alcohol
• Acid Fast Stain is a differential stain used to classify
Acid fast positive or Acid Fast negative
microorganisms
ACID FAST STAIN
• The group of microorganisms that are
differentiated by the acid fast stain are the
mycobacteria.
• There are many species of mycobacteria,
but the most important organism in this
group is Mycobacterium tuberculosis, the
causative agent of tuberculosis.
ACID FAST STAIN REAGENTS
• Carbol fuchsin: fuchsin is the red basic
stain and carbol is the acid (carbolic acid)
• Acid alcohol: decolorizer (Hydrochloric acid
and 95% ethanol)
• Methylene blue: a blue counter stain
For Acid-Fast Stain Lab your slide should
look like this.
PROCEDURE – ACID FAST STAIN
COLD KINYOUN METHOD
ACID FAST
STAIN STEPS
1. Primary stain:
Carbol fuchsin
2.Decolorizing agent:
Acid alcohol
3.Counterstain:
Methylene Blue
Color of
Acidfast +
cells
RED
Color of
Acidfast –
cells
RED
RED
COLORLESS
RED
BLUE
Acid-Fast Staining:
Kinyoun Method
ACID FAST POSITIVE - RED
ACID FAST NEGATIVE - BLUE
Acid-Fast Staining:
Kinyoun Method
ACID FAST POSITIVE – RED
ACID FAST NEGATIVE – BLUE
RESULTS
??? OVER – DECOLORIZE
???UNDER – DECOLORIZE
WHY DO WE USE THE
ACID FAST STAIN????