is important in order to identify bacteria and other microbes because cells,
due to their high water content, are naturally translucent. Light will pass
through them. Contrast is needed in order to visualize and identify cells.
Staining is the simplest way to increase contrast in order to visualize cells.
simple stain, using a single staining agent, provides contrast to a biological
specimen, allowing a normally transparent biological sample to be visualized
with a light microscope. While a single stain allows cells to be differentiated
from the background, it does not give very much information about the cellular
structures or properties; cells of different species can have different
physical and chemical properties. These differences can be exploited using
staining techniques so that cellular differences can be easily visualized under
a bright field microscope. Differential stains use two or more different types
of stains to show the different parts of the microorganism.
Heat-fixing bacteria to the slide prior to staining is
important because in order to stain microbiological specimens, the organisms
must be adhered to the microscope slide; otherwise, the cells will be
rinsed/washed off the slide. What follows is a brief description of the
heat-fixing process. Before heat-fixing, organisms must be applied to the glass
slide using the wet mount technique. The specimen is then be allowed to air dry.
Next, the slide must be heated to adhere the cells to the slide. This is done
by quickly passing the center of the slide, where the specimen is located, over
the flame of a Bunsen burner two to three times. The slide should be warm, not
hot to the touch; overheating will cause the cells to burst, ruining the
The Gram stain includes the following components, whose
basic functions are described. The primary
stain used in the Gram stain is crystal violet, a basic dye. Crystal violet
is soluble in water, and when applied to a specimen smear, will quickly make
its way through the cell walls of Gram-positive and Gram-negative bacteria, and
associates with the negatively charged cell wall. Crystal violet will be
trapped in the thick outer peptidoglycan layer of Gram-positive bacteria;
Gram-negative bacteria, lacking this thick outer layer, do not retain crystal
violet. After the dye has been allowed to absorb and is rinsed off, a mordant (Gram’s iodine) is applied to
the specimen. The mordant helps the crystal violet to “set” into the bacterial
cell wall; it aids penetration and binding of the dye. After the mordant has
been given time to work, with the excess being rinsed off, a decolorizer (ethanol or acetone) is
used to remove the primary stain. If it has set, primarily due to the action of
the mordant, then the cell will remain colored with crystal violet; if not, the
crystal violet will be washed away by the decolorizer. At this stage, the
Gram-positive cell retains crystal violet in the peptidoglycan outer layer; the
Gram-negative cell is stripped of crystal violet with its outer lipid membrane.
Once the decolorizer has been removed, a counter
stain, safranin, is applied to the specimen. After decolorization, Gram-negative
cells that are unable to retain crystal violet become colorless, therefore the
counter stain will colorize the cell. Safranin does not colorize the
Gram-positive cells already stained with crystal violet, because in order to
bind, the cell wall must be free of other cations. The result is a differential
stain, showing the difference in bacterial cell wall structure.
The step with the decolorizer is the most likely to lead to
poor results if done incorrectly. The decolorizer can eventually remove even a
set-in stain, so it is critical that it is only in contact with the specimen briefly.
The acid-fast stain includes the following components, whose
basic functions are described. The primary
stain used in acid-fast staining, carbolfuchsin, is lipid-soluble and
contains phenol, which helps the stain penetrate the waxy cell wall. The smear
is then rinsed with a very strong decolorizer,
an acid alcohol, which strips the stain from all non-acid-fast cells but does
not permeate the cell wall of acid-fast organisms. A strong decolorizer is
needed because the primary stain used binds strongly to the peptidoglycan in
the cell walls of non-acid-fast organisms. The decolorized non-acid-fast cells
then take up the counter stain,
methylene blue. This creates a differential stain useful for identifying structures
or organisms that are difficult to characterize using other staining
is a medically genus of bacteria identifiable by the
acid-fast stain. Mycobacteria have
cell walls with a high content of mycolic acids, compounds which are very
hydrophobic and thus resistant to traditional hydrophilic dyes.