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Dr.T.V.Rao MD
Gram staining is one of the oldest procedure in Microbiology, which continues to be
practised by wise and young alike, yet many of us have not perfected with a definitive
opinion, and difference of opinion by different observers leading to many conflicts in
the work place, We teach many of our Undergraduate students in Medicine as the
basis of diagnostic work for understanding the matters when they become interns or
practising physicians.

WHAT IS A GRAM STAINING - 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. Many clinicians ask us whether gram stain of the specimen sent us is positive or negative,
then a question arises , then -is-better-for-my-gram-stain-to-be-positive-or-negative, but not
correct to express without giving further details and expressing the limitations of Gram staining in
clinical decision making. Many years of working on the table clarifies many matters including
conflicts in observers opinion, However the Clinical Microbiologists should be familiar with many
scientific facts before giving valued opinion to make clinical decision making. Many test observations
and the experiment conducted was based upon the known attributes of two different groups of
bacteria, those that are gram positive, and those that are gram negative. Using a specific staining
procedure, it is possible to differentiate the two types under a microscope The gram stain method of
differentiation is possible because of differences in the cell membrane between the two categories
of bacteria The Gram stain differentiates bacteria into two fundamental varieties of cells. Bacteria
that retain the initial crystal violet stain (purple) are said to be ''Gram-positive,'' whereas those that
are decolorized and stain red with carbol fuchsin (or safranin) are said to be ''Gram-negative.'' This
staining response is based on the chemical and structural makeup of the cell walls of both varieties
of bacteria. Gram-positives have a thick, relatively impermeable wall that resists decolourization and
is composed of peptidoglycan and secondary polymers. Gram-negatives have a thin peptidoglycan
layer plus an overlying lipid-protein bilayer known as the outer membrane, which can be disrupted
by decolourization. Some bacteria have walls of intermediate structure and, although they are
officially classified as Gram-positives because of their linage, they stain in a variable manner. A gram
stain continues to guide us on basic bacteriology and no day goes in laboratory without doing this
basic procedure, as A Gram stain and culture of the material from an infected site are the most
commonly performed microbiology tests used to identify the cause of a bacterial infection. Often,
detecting the presence of bacteria and determining whether an infection is caused by an organism
that is Gram-positive or Gram-negative will be sufficient to allow a health practitioner to prescribe
treatment with an appropriate antibiotic while waiting for more specific tests, such as a culture, to
be completed. Absence or presence of white blood cells in the Gram stain can help establish that an
adequate sample was obtained as white blood cells are frequently present with an infection and
presence of neutrophils and lymphocytes express the inflammatory reaction. Many times we forget
to read the association of Neutrophils, pus cells and epithelial cell as the criteria of the specimen is
well flourished with infected material in situ, while it is important or biological elements as mould
and yeast associated with the specimens as colonizers or infectious elements.

WE GET MANY REQEUSTS Gram stain is typically ordered along with a culture when a bacterial or
sometimes fungal infection is suspected. It is also usually performed when the result of a culture is
positive, on a sample of the bacteria grown in the culture. Test results are reported promptly to help
guide treatment , The Gram stain continues to be more requested opinion when we get the CSF,

Body fluids otherwise sterile, it makes a great sense if we observe many fields even finding very few
bacteria makes a greater sense in decision making than awaited culture reports after 24 to 48hours

NEGATIVE FOR MICROBES MEANS - A negative Gram stain is often reported as "no organism
seen." This may mean that there is no bacterial infection present or that there were not enough
bacteria present in the sample to be seen with the stain under a microscope, and subject the
specimens further with culturing on selective, simple or enriched media

POSITIVE FOR MICROBES MEANS- Positive Gram stain results usually include a description of
what was seen on the slide. This typically includes:
Whether the bacteria are Gram-positive (purple) or Gram-negative (pink)
Shape round (cocci) or rods (bacilli)
Size, relative quantity, and/or arrangement of the bacteria, if relevant
Whether there are bacteria present within other cells (intracellular)
Presence of red blood cells or white blood cells
Fungi (in the form of yeasts or molds) may be seen on a Gram stain and are reported. Yeast may
appear as single cells that may have buds, while molds may appear as a wide variety of plant-like
branches called hyphae. Further testing may be performed to identify the specific type

Cautious - when reporting many other specimens Gram stains are not helpful when the diseasecausing bacteria (pathogen) cannot be differentiated from normal bacterial flora. For example, a
Gram stain of a sample from the throat cannot tell the health practitioner what bacteria are causing
the infection because both the "commensal bacteria" and the "pathogenic bacteria" look the same
under the microscope. The idea stands good in reporting of specimen of stools, as we many times
cannot differentiate many pathogens and commensal present in plenty
use objective criteria by Gram stain screening for purulent / septic material before inoculation in to
culture media. Hence the routine sputum Gram stain is essential to provide meaningful culture
report. All the specimens we receive for culture should have a basic evaluation of Gram staining on
1st day of receiving the specimen in the laboratory to avoid much confusion on the 2nd day of culture
reporting, most important specimen for Gram staining continues to be observation of Sputum for
Microscopy. Clinicians are interested in rapid, simple, inexpensive and readily available tests that will
assist them in prescribing proper medications for lower respiratory tract infections. Sputum Gram
stain served this function in the management of lower respiratory tract infection if the
Microbiologists observe the sputum with power to discriminate many uncommon microbes in the
sputum, can be visualised, and more scientific decisions on selecting culture Media as the matters
warrant .
Ref Use of the Gram stain in microbiology- Research Article Biotechnic & Histochemistry Volume 76,
Issue 3, 2001
Dr.T.V.Rao MD Professor of Microbiology Freelance writer