Lab.5
Staining techniques
Bacteria
are semi transparent difficult to visualized in unstained condition.
Identification of bacteria is aided by their staining reaction.
Stain:
are coloring agent used for biological purposes to identify microscopical
agents. There are alkaline and acidic stains. There are three types of staining
methods:
Simple
stain: is composed of one type of stain, e.g. crystal violet (C.V.), safranin.
Differential
stain: it gives different color to different bacteria e.g. Gram’s stain, Albert’s
stain and Ziehle Neelsen (ZN) stain.
Special
stain: e.g.:
Negative
staining (have background colored in which bacteria remain unstained).
India
ink stain for capsule staining.
Special
stains used for staining of some bacterial components like spore stain and
flagella stain.
Gram stain technique:
The
Gram stain procedure was originally developed by the Danish Physician Hans
Christian Gram in 1884 to differentiate pneumococci fro Klebsiella pneumonia.
In brief, the procedure involves the application of a solution of iodine
(potassium iodide) to cells previously stained with crystal violet or gentian
violet. This procedure produces purple colored iodine-dye complexes in the
cytoplasm of bacteria. The cells that are previously stained with crystal violet
and iodine are next treated with a decolorizing agent such as 95% ethanol or a
mixture of acetone and alcohol. The difference between Gram-positive and Gram
negative bacteria is in the permeability of the cell wall to these purple
colored iodine-dye complexes when treated with the decolorizing solvent. While
Gram-positive bacteria retain purple iodine dye complexes after the treatment
with the decolorizing agent. Gram-negative bacteria do not retain complexes
when decolorized. To visualize decolorized Gram-negative bacteria, a red
counter stain such as safranin is used after decolorization treatment.
The
first consideration is the correct preparation of the smear. Make a thin film
of the material on a clean glass slide, using a sterile loop. Air dry, then heat
fix the slide by passing it several times through a flame (the slide should not
become too hot to tough). Failure to follow these directions may disrupt the
normal morphology of bacteria and cells.
To
be visible on a slide, organisms that stain by the Gram method must be present
in concentrations of a minimum of 104 to 105 organisms/ml of unconcentrated
staining fluid. At lower concentrations, the Gram stain of a clinical specimen
seldom reveals organisms even if the culture is positive. Smears that are not
properly fixed tend to be washed away during staining and washing resulting in
the absence of stained bacteria.
Staining procedure:
Flood
slide with crystal (or gentian) violet 10 seconds. (Wash with running tap
water).
Flood
with Gram’s iodine 10 seconds. (Wash with water).
Carefully
decolorize with 95% ethanol until thinnest parts of the smear are colorless.
(Wash with water). This third step is the most critical and also the one most
affected by technical variations in timing and reagents.
Flood
with safranin (pink color) 10 seconds. (Wash with water). Air dry, or blot with
absorbent paper.
Results:
Organisms
that retain the violet-iodine complexes after washing in ethanol stain purple
and are termed Gram-positive, those that lose this complex stain red from the safranin
counter stain are termed Gram-negative.
Notes:
In
the diagnosis of patients with acute infections, the decision to send specimens
of sputum, urine, cerebrospinal fluid or material from wounds or abscesses for
culture should automatically trigger a response to first examine a Gram-stained
smear.
There
are some specimens that are not suitable for routine Gram staining, such
examples are routine throat and stool specimens in which the pathogen usually
cannot be distinguished from the normal flora. Blood specimens are rarely
Gram-stained.
Certain
bacteria stains only feebly or not at all with the Gram staining technique
requiring special staining techniques. Such examples are acid-fast staining for
mycobacteria and nocardia, and immunofluorecent stain for Legionella.
Interpretation of Gram stain reaction:
Most
cell walls contain Peptidoglycan, a molecule made of amino acids and sugar. A
distinguishing factor among Gram-positive bacteria is that 90% of their cell
wall is comprised of Peptidoglycan and a Gram-positive bacteria can have more
than 20 layers of Peptidoglycan stacked together to form the cell wall.
The
blue-violet color reaction in Gram-positive bacteria is caused by
crystal-violet, complexing with the iodine mordant. When the decolorizer is
applied, a slow dehydration of the crystal-violet/iodine complex is observed
due to the closing of pores running through the cell wall. Because the
crystal-violet is still present in the cell, the counter stain is not
incorporated, thus maintaining the cell’s blue-violet color.
Unlike
Gram-positive bacteria, which appear a violet color in Gram staining, Gram
negative bacteria incorporate the counter stain rather that the primary stain.
Because the cell wall of Gram-negative bacteria is high in lipid content and
low in Peptidoglycan content, the primary crystal-violet escapes from the cell
when the decolorizer is added. This is because primary stains like to bind with
Peptidoglycan- sometimes the Gram negative cell has very little of. This
pathogenic capacity of Gram negative bacteria is usually associated with
certain components of their cell walls, particularly the lipopolysaccharide
(endotoxin) layer. Examples of common Gram-positive cells are Staphylococcus
aureus and Stryptococcus. Examples of common Gram-negative cells are E. coli
and Salmonella.
Errors in Gram stain technique:
Over
decolorization.
Old
culture.
Too
much exposure to safranin.
Thick
smear.