Gram Staining Procedure

GRAM STAINING PROCEDURE
A Gram stain is a laboratory procedure used to detect the presence of bacteria in a

sample taken from the site of a suspected infection. It gives relatively quick results as to
whether bacteria or fungi are present and, if so, the general type(s).
The Gram stain involves applying a sample from the infected area onto a glass slide
and allowing it to dry. The slide is then treated with a special stain...
How is it used?
A Gram stain is used, along with a culture of the material from an infected site, to
identify the cause of a bacterial infection. The Gram stain provides preliminary results
on whether bacteria are present and the general type, such as the shape and whether
they are Gram-positive or Gram-negative.
Often, detecting the presence of bacteria and determining whether an infection is
caused by a microbe that is Gram-positive or Gram-negative will be sufficient to allow a
healthcare 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.
A Gram stain may also be performed as part of the evaluation of a culture. When
bacteria grow on/in a nutrient media in the laboratory, a Gram stain is performed to help
determine the type of bacteria present and to help determine what other tests may need
to be performed to definitively identify the cause of infection.
What does the test result mean?
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
A few examples of some medically significant bacteria and what they look like on a
Gram stain include:
 Gram-positive cocci—Staphylcoccus aureus (Staph aureus) can cause skin
infections and toxic shock syndrome; Streptococcus pneumoniae can
cause pneumonia.
 Gram-negative cocci—Neisseria meningitidis causes meningitis while Neisseria
gonorrhoeae causes the sexually transmitted disease gonorrhea.
 Gram-positive bacilli—Bacillus anthracis (anthrax) can cause skin infections or
pneumonia (also a bioterrorism agent); Listeria monocytogenes can
cause foodborne illnesses.
 Gram-negative bacilli—Escherichia coli is a common cause of urinary tract
infections.
Samples such as blood and cerebrospinal fluid are generally sterile. Bacteria are not
usually present in these samples when there is no bacterial infection. Bacteria may
initially be present in low numbers with an infection, and this type of sample may require
extra processing in order to concentrate the bacteria so that they can be detected by a
Gram stain.
Other body fluids and sites, such as sputum or skin, typically have cells and normal
flora present in addition to any bacteria that are causing an infection. Gram stains on
these types of samples require careful examination by a trained laboratorian to
determine which cells may be normal and which may be due to an infection.
Principle of Gram's Stain
The crystal violet stain is the primary stain, which stains everything in the smear purple- blue. The
Gram's iodine acts as a mordant that causes the crystal violet to penetrate and adhere to the gram-
positive cell. The acetone-alcohol mixture acts as the decolorizer that washes the stain away from
everything in the smear except the gram-positive organisms. The safranine is the counter-stain that
stains everything in the smear that has been decolorized in gram-negative organisms.
DIRECTIONS
There are six basic steps:
1. Apply a smear of bacteria on to a slide. Air dry and then heat fix by passing it through
a flame a few times. Make sure you air dry the bacteria before heat fixing.
2. Add about 5 drops of Crystal Violet to the culture. Let stand for one minute. Bacteria
will stain purple. Wash briefly with water and shake off excess.
3. Add about 5 drops of iodine solution to the culture. Let stand for 30 seconds, wash
briefly with water and shake off excess.
4. Tilt slide and decolorize with solvent (acetone-alcohol solution) until purple color
stops running. Be careful not to over-decolorize. Wash immediately (within 5
seconds) with water and shake off excess.
5. Add about 5 drops of Safraninin Red. Let stand for one minute, wash briefly with
water and shake off excess.
6. Examine under microscope at both 400x and 1,000x oil immersion.

Examples:
Gram-Positive Bacteria: Staphylococcus (Mostly harmless sphere-shaped, skin bacteria);

Streptococcus (rod shaped bacteria: Rheumatic fever, Scarlet fever).
Gram-Negative Bacteria: Escherichia, Salmonella, Shigella (Meningitis, Gonorrhea, Flu and various
Respiratory Diseases)

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GRAM STAINING PROCEDURE

A Gram stain is a laboratory procedure used to detect the presence of bacteria in a

What does the test result mean?

6. Examine under microscope at both 400x and 1,000x oil immersion.

Gram-Positive Bacteria: Staphylococcus (Mostly harmless sphere-shaped, skin bacteria);

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