GRAM STAINING: Mechanism , Limitations and Modified techniques
Gram stain or Gram staining, also
called Gram’s method, is a method of staining used to classify
bacterial species into two large groups: gram-positive
bacteria and gram-negative bacteria. The name comes from the Danish
bacteriologist Hans Christian Gram, who developed the technique.
Gram staining differentiates bacteria
by the chemical and physical properties of their cell walls. Gram-positive
cells have a thick layer of peptidoglycan in the cell wall that retains the
primary stain, crystal violet. Gram-negative cells have a thinner peptidoglycan
layer that allows the crystal violet to wash out with the addition of ethanol. They
are stained pink or red by the counterstain, commonly safranin or fuchsine.
Lugol’s iodine solution is always added after the addition of crystal violet to
strengthen the bonds of the stain with the cell membrane. Gram staining is
almost always the first step in the preliminary identification of a bacterial
organism. While Gram staining is a valuable diagnostic tool in both clinical
and research settings, not all bacteria can be definitively classified by this
technique. This gives rise to gram-variable and gram-indeterminate groups.
Staining mechanism
Gram-positive bacteria have a thick
mesh-like cell wall made of peptidoglycan (50–90% of cell envelope), and as a
result are stained purple by crystal violet, whereas gram-negative bacteria
have a thinner layer (10% of cell envelope), so do not retain the purple stain
and are counter-stained pink by safranin. There are four basic steps of the
Gram stain:
1. Applying a primary stain (crystal
violet) to a heat-fixed smear of a bacterial culture. Heat fixation kills some
bacteria but is mostly used to affix the bacteria to the slide so that they
don’t rinse out during the staining procedure.
2. The addition of iodine, which binds
to crystal violet and traps it in the cell
3. Rapid decolorization with ethanol
or acetone
4. Counterstaining with safranin.
Carbol fuchsin is sometimes substituted for safranin since it more intensely
stains anaerobic bacteria, but it is less commonly used as a counterstain.
Crystal violet (CV)
dissociates in aqueous solutions into CV+ and chloride (Cl−) ions. These ions penetrate the cell walls of
both gram-positive and gram-negative cells. The CV+ Ion interacts with
negatively charged components of bacterial cells and stains the cells purple.
Iodide (I− Or I−3)
interacts with CV+ and forms large complexes of crystal violet and iodine (CV–I)
within the inner and outer layers of the cell. Iodine is often referred to as a
mordant, but is a trapping agent that prevents the removal of the CV–I complex
and, therefore, colors the cell.
When a decolorizer such as alcohol
or acetone is added, it interacts with the lipids of the cell membrane. A
gram-negative cell loses its outer lipopolysaccharide membrane, and the inner
peptidoglycan layer is left exposed. The CV–I complexes are washed from the
gram-negative cell along with the outer membrane. In contrast, a gram-positive
cell becomes dehydrated from an ethanol treatment. The large CV–I complexes
become trapped within the gram-positive cell due to the multilayered nature of
its peptidoglycan. The decolorization step is critical and must be timed correctly;
the crystal violet stain is removed from both gram-positive and negative cells
if the decolorizing agent is left on too long (a matter of seconds).
After decolorization, the
gram-positive cell remains purple and the gram-negative cell loses
its purple color. Counterstain, which is usually positively charged
safranin or basic fuchsine, is applied last to give decolorized gram-negative
bacteria a pink or red color. Both gram-positive bacteria and
gram-negative bacteria pick up the counterstain. The counterstain,
however, is unseen on gram-positive bacteria because of the
darker crystal violet stain.
Gram-positive bacteria
Gram-positive bacteria generally have
a single membrane (monoderm) surrounded by a thick peptidoglycan. This rule is
followed by two phyla: Firmicutes (except for the classes Mollicutes and
Negativicutes) and Actinobacteria.In contrast, members of the Chloroflexi
(green non-sulfur bacteria) are monoderms but possess a thin or absent (class
Dehalococcoidetes) peptidoglycan and can stain negative, positive or
indeterminate; members of the Deinococcus–Thermus group stain positive but are
diderms with a thick peptidoglycan.
Historically, the gram-positive forms
made up the phylum Firmicutes, a name now used for the largest group. It
includes many well-known genera such as Lactobacillus, Bacillus, Listeria,
Staphylococcus, Streptococcus, Enterococcus, and Clostridium. It has also been
expanded to include the Mollicutes, bacteria such as Mycoplasma and
Thermoplasma that lack cell walls and so cannot be Gram-stained, but are
derived from such forms.
Some bacteria have cell walls that
are particularly adept at retaining stains. These will appear positive by Gram
stain even though they are not closely related to other Gram-positive bacteria.
These are called acid-fast bacteria, and can only be differentiated from other
gram-positive bacteria by special staining procedures.
Gram-negative bacteria
Gram-negative bacteria generally
possess a thin layer of peptidoglycan between two membranes.
Lipopolysaccharide (LPS) is the most abundant antigen on the cell surface of
most Gram-negative bacteria, contributing up to 80% of the outer membrane of E.
coli and Salmonella. Most bacterial phyla are gram-negative, including the
cyanobacteria, green sulfur bacteria, and most Proteobacteria (exceptions being
some members of the Rickettsiales and the insect-endosymbionts of the
Enterobacteriales).
Gram-variable and Gram-indeterminate
bacteria
Some bacteria, after staining with
the Gram stain, yield a gram-variable pattern: a mix of pink and purple cells
are seen. In cultures of Bacillus, Butyrivibrio, and Clostridium, a decrease in
peptidoglycan thickness during growth coincides with an increase in the number
of cells that stain gram-negative. In addition, in all bacteria stained using
the Gram stain, the age of the culture may influence the results of the stain.
Gram-indeterminate bacteria do not
respond predictably to Gram staining and, therefore, cannot be determined as
either gram-positive or gram-negative. Examples include many species of
Mycobacterium, including Mycobacterium bovis, Mycobacterium
leprae, and Mycobacterium tuberculosis, the latter two of
which are the causative agents of leprosy and tuberculosis, respectively.
Bacteria of the genus Mycoplasma lack a cell wall around their cell membranes,
which means they do not stain by Gram’s method and are resistant to the
antibiotics that target cell wall synthesis.
Limitations of Gram
staining:
Some Gram-positive bacteria may lose
the stain easily and therefore appear as a mixture of Gram-positive and
Gram-negative bacteria (Gram-variable). When over-decolorized,
even Gram-positive bacteria may appear pink and when under-decolorized
gram-negative bacteria may appear Gram-positive.
The Gram reaction also depends on the
age of the cell. Old cultures of Gram-positive bacteria (where
cell walls may be weakened) may readily get decolorized. Gram-positive cells
affected by cell wall active agents such as lysozyme or antibiotics may become
Gram-negative. Gram-positive bacteria such as Actinomyces, Arthobacter,
Corynebacterium, Mycobacterium, and Propionibacterium have
cell walls particularly sensitive to breakage during cell division,
resulting in Gram-negative staining of these cells. In cultures of Bacillus,
and Clostridium a decrease in peptidoglycan thickness during cell growth may
cause some of them to appear Gram-negative.
Certain groups of bacteria can display
variable responses to the stain, which can be due to growth stress
(e.g., unsuitable nutrients, temperatures, pHs, or electrolytes) that
results in a number of nonviable, gram-negative cells in a gram-positive
culture, but certain bacterial species are known for their gram variability
even under optimal growth conditions. Some bacteria tend to appear Gram-negative when grown in an acidic medium.
Loss of cell walls in Gram-positive
bacteria may render them Gram-negative (L-forms). Bacteria totally devoid of
cell walls (Mycoplasma) are always Gram-negative. Bacteria such as Mycobacterium
that have extra waxy content in their cell wall are difficult to stain. Small
and slender bacteria such as Treponema, Chlamydia, and Rickettsia are often
difficult to stain by Gram’s method. Gram-positive bacteria that have been phagocytosed
by polymorphs may also appear Gram-negative.
MODIFIED GRAM STAINING TECHNIQUES
:
Following are the modified gram
staining techniques :
1.
Kopeloff and Beerman’s modification
2. Jensen’s
modification
3. Preston’s
and Morrell’s modification
4.
Weigert modification
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