Gram staining, also called Gram's method, is a method of differentiating bacterial species
into two large groups.
The name comes from its inventor, Hans Christian Gram.
Gram staining differentiates bacteria by the chemical and physical properties of their
cell walls by detecting peptidoglycan, which is present in a thick layer in gram-positive
bacteria.
In a Gram stain test, gram-positive bacteria retain the crystal violet dye, while a counterstain
added after the crystal violet gives all gram-negative bacteria a red or pink coloring.
The Gram stain is almost always the first step in the 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 as well.
History The method is named after its inventor, the
Danish scientist Hans Christian Gram, who developed the technique while working with
Carl Friedländer in the morgue of the city hospital in Berlin in 1884.
Gram devised his technique not for the purpose of distinguishing one type of bacterium from
another but to make bacteria more visible in stained sections of lung tissue.
He published his method in 1884, and included in his short report the observation that the
typhus bacillus did not retain the stain.
Uses Gram staining is a bacteriological laboratory
technique used to differentiate bacterial species into two large groups based on the
physical properties of their cell walls.
Gram staining is not used to classify archaea, formerly archaeabacteria, since these microorganisms
yield widely varying responses that do not follow their phylogenetic groups.
The Gram stain is not an infallible tool for diagnosis, identification, or phylogeny, and
it is of extremely limited use in environmental microbiology.
It still competes with molecular techniques even in the medical microbiology lab.
Some organisms are gram-variable; some organisms are not susceptible to either stain used by
the Gram technique.
In a modern environmental or molecular microbiology lab, most identification is done using genetic
sequences and other molecular techniques, which are far more specific and informative
than differential staining.
Gram staining has proven as effective a diagnostic tool as PCR, particularly with regards to
gonorrhoea diagnosis in Kuwait.
The similarity of the results of both Gram stain and PCR for diagnosis of gonorrhea was
99.4% in Kuwait.
Medical
Gram stains are performed on body fluid or biopsy when infection is suspected.
Gram stains yield results much more quickly than culturing, and is especially important
when infection would make an important difference in the patient's treatment and prognosis;
examples are cerebrospinal fluid for meningitis and synovial fluid for septic arthritis.
Staining mechanism Gram-positive bacteria have a thick mesh-like
cell wall made of peptidoglycan, and as a result are stained purple by crystal violet,
whereas gram-negative bacteria have a thinner layer, so do not retain the purple stain and
are counter-stained pink by the Safranin.
There are four basic steps of the Gram stain: Applying a primary stain 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.
The addition of iodide, which binds to crystal violet and traps it in the cell,
Rapid decolorization with ethanol or acetone, and
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 dissociates in aqueous solutions into CV+ and chloride ions.
These ions penetrate through the cell wall and cell membrane 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 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, color 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.
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.
Some bacteria, after staining with the Gram stain, yield a gram-variable pattern: a mix
of pink and purple cells are seen.
The genera Actinomyces, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium have
cell walls particularly sensitive to breakage during cell division, resulting in gram-negative
staining of these gram-positive cells.
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.
Examples Gram-positive bacteria
Gram-positive bacteria generally have a single membrane surrounded by a thick peptidoglycan.
This rule is followed by two phyla: Firmicutes and the Actinobacteria.
In contrast, members of the Chloroflexi are monoderms but possess a thin or absent 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 Bacillus, Listeria, Staphylococcus, Streptococcus,
Enterococcus, and Clostridium.
It has also been expanded to include the Mollicutes, bacteria like Mycoplasma that lack cell walls
and so cannot be stained by Gram, but are derived from such forms.
Normally, if Gram stain is done on acid-fast bacteria, they show up as if they are gram-positive,
mostly because of their thick cell wall.
Gram-negative bacteria
Gram-negative bacteria generally possess a thin layer of peptidoglycan between two membranes.
Most bacterial phyla are gram-negative, including the cyanobacteria, spirochaetes, and green
sulfur bacteria, and most Proteobacteria and Escherichia coli..
Gram-indeterminate bacteria Gram-indeterminate bacteria, also known as
gram-variable bacteria, do not respond predictably to Gram staining and, therefore, cannot be
determined as either gram-positive or gram-negative.
They tend to stain unevenly, appearing partially gram positive and partially gram negative,
or even unstained.
Staining older cultures can lead to false gram-variable results, probably due to changes
in the cell wall with aging.
Gram-indeterminate bacteria are best stained using acid-fast staining techniques.
Examples include many species of Mycobacterium, including M. tuberculosis and M. leprae.
See also Bacterial cell structure
Staining Trichrome stain
References
External links Gram staining technique video