Download Staining of microorganisms (focus on bacteria)

Staining of microorganisms
(focus on bacteria)
Staining: coloring a microbe with a
dye that binds to and emphasizes
certain structures
Preparation: Fixing
•  Before staining, a sample of bacteria must
be fixed to a microscope slide.
•  A smear of bacteria (thin film containing
cells) is spread over the slide, and the cells
are killed and fixed in place by exposure to
dry heat.
•  Now we are ready to STAIN!
What are dyes?
•  Dyes are salts composed of a positive
and negative ion, one of which is
colorful - this is called the chromophore.
•  Basic dye: chromophore is positive ion
•  Acidic dye: chromophore is negative ion
Bacteria are somewhat negatively
charged, so…
Basic dyes bind to bacterial structures
Examples: crystal violet, methylene blue
Acidic dyes cause negative staining, since they may bind to background and avoid binding to the bacterium
(charge repulsion)
Staining techniques
•  Simple stain - solution of single basic dye
Generally highlights entire microorganism.
Mordant improves binding of dye to
sample
•  Differential staining - divides bacteria into
groups according to their reaction to the
staining procedure - most popular = •  Gram stain!
The Gram Stain
•  Divides bacteria into two large groups, the
gram-positive and the gram-negative.
•  Developed by Hans Christian Gram in
1884 to aid in bacterial identification.
•  So, how do you do it?...
Gram stain procedure
•  1. Apply a basic dye
(usually crystal violet).
This stains all the cells
purple or blue and is
called the primary dye.
•  2. After a short time,
wash off dye and apply
iodine, the mordant: after
it is washed off all cells
appear purple.
Gram staining cont’d
•  3. Wash preparation with
alcohol, a decolorizing agent
which removes the stain from
SOME bacteria.
•  4. Apply another basic stain,
safranin, which colors the
unstained bacteria red or pink.
Safranin is the counterstain.
•  When it is all over, –  Gram-positive cells look purple
–  Gram-negative cells look red Why do bacteria react differently?
•  Structural differences in their cell walls
account for the different reactions to the
Gram stain. More about this soon!
•  Also popular, especially clinically, is the
ACID-FAST STAIN. Why? Because it
preferentially distinguishes bacteria of the
genus Mycobacterium, which cause
tuberculosis.
Acid-fast Stain
•  Procedure is generally similar to the Gram
stain.
•  1. Apply a basic dye (carbolfuchsin). This
stains all the cells red and is called the
primary dye.
•  2. Carbolfuchsin preferentially binds to cell
walls rich in a certain type of wax.
Acid-fast cont'd
•  3. Wash preparation with acid-alcohol, a
decolorizing agent which removes the stain
from SOME bacteria (those without the
waxy substance)
•  4. Apply another basic stain, methylene
blue, which colors the unstained bacteria
blue. Methylene blue is the counterstain.
•  When it is all over, –  Acid-fast cells look red
–  Non acid-fast cells look blue Special stains exist to...
•  Visualize microbial capsules (negative
capsule staining); use an acid stain (colors
background), then safranin (colors entire
bacterium EXCEPT for capsule; capsule
appears as a halo). (Capsules are related to
virulence of pathogens)
•  Highlight endospores
•  Highlight flagella