Environmental Engineering Reference
In-Depth Information
Gram Stain
Microbial cells are nearly transparent when observed by light microscopy and
hence are difficult to see. The most common method for observing cells is by
the use of stained preparations. Dyes are used to stain cells, which increases
their contrast so they can be more easily observed in a light microscope.
Simple cell-staining techniques depend upon the fact that bacterial cells dif-
fer chemically from their surroundings and thus can be stained to contrast
with their environment. Microbes also differ from one another chemically
and physically and therefore may react differently to a given staining proce-
dure. This is the basic principle of
differential staining
—so named because this
type of procedure does not stain all kinds of cells equally.
The Gram staining procedure was developed in the 1880s by Hans
Christian Gram, a Danish bacteriologist. Gram discovered that microbes
could be distinguished from surrounding tissue. Gram observed that some
bacterial cells exhibit an unusual resistance to decolorization. He used this
observation as the basis for a differential staining technique.
Gram differentiation is based on the application of a series of four chemical
reagents: primary stain, mordant, decolorizer, and counterstain. The pur-
pose of the primary stain, crystal violet, is to impart a blue or purple color
to all organisms regardless of their Gram reaction. This is followed by the
application of Gram's iodine, which acts as a mordant (fixer) that enhances
the union between the crystal violet stain and its substrate by forming a
complex. The decolorizing solution of 95% ethanol extracts the complex from
certain cells more readily than others. In the final step, a counterstain (safra-
nin) is applied to reveal organisms previously decolorized by removal of the
complex. Those organisms retaining the complex are Gram positive (blue or
purple), whereas those losing the complex are Gram negative (red or pink).
Gram-Positive Cell Walls
Normally, the thick, homogeneous cell walls of
Gram-positive bacteria are composed primarily of a complex polymer,
which often contains linear heteropolysaccharide chains bridged by pep-
tides to form a three-dimensional netlike structure and envelop the pro-
toplast. Gram-positive cells usually also contain large amounts of
teichoic
acids—typically, substituted polymers or ribitol phosphate and glycerol
phosphate. Amino acids or sugars such as glucose are attached to the ribi-
tol and glycerol groups. Teichoic acids are negatively charged and help give
the Gram-positive cell wall its negative charge. Growth conditions can affect
the composition of the cell wall; for example, the availability of phosphates
affects the amount of teichoic acid in the cell wall of
Bacillus
. Teichoic acids
are not present in Gram-negative bacteria.
Gram-Negative Cell Walls
Gram-negative cell walls are much more complex
than Gram-positive walls. The Gram-negative wall is about 20 to 30 µm thick
and has a distinctly layered appearance under the electron microscope. The
thin inner layer consists of peptidoglycan and constitutes no more than 10%
