What is being tested?

A Gram stain is a laboratory procedure used to detect the presence of microrganisms, especially bacteria, in a sample taken from the site of a suspected infection. It gives relatively quick results as to the general type of bacteria that may be present. 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 series of special stains and examined under a microscope by trained laboratory staff. It is based on the principle that most bacteria are visualised under the microscope more easily after staining, which gives them a colour, depending on the type of cell wall. Any bacteria that may be present are categorised by colour and shape during the microscopic evaluation:

• Colour - typically bacteria may be either "Gram positive" (purple) or "Gram negative" (pink)
• Shape - the most common shapes include round (cocci) or rod-shaped (bacilli)

Additional information may be obtained by observing the groupings of the bacteria on the slide, such as cocci that are present singly, in pairs, in groups of four, in clusters or in chains, or bacilli that are thick, thin, short, long or have enlarged spores on one end. Any bacteria that are present within the patient's white blood cell (intracellular) are also noted. The Gram stain colour and the bacterial shape give clues as to what microorganism might be causing the infection. Examples of Gram positive cocci include Staphylococcus aureus, the bacterium commonly known as "Golden Staph". An example of a Gram negative bacterium is Escherichia coli (or E. coli), a common cause of urinary tract infections. Fungi (in the form of yeasts or moulds) may also be initially recognised with a Gram stain, but viruses cannot be seen with a Gram stain.

Since its discovery in 1884 by Danish scientist Hans Christian Gram, the Gram stain remains the most simple, fast and cost-effective test in microbiology, providing an incredible amount of information to health practitioners. Particularly in patients who are suspected of septicaemia, pneumonia and meningitis.

How is it used?

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 an infection. Often, detecting the presence of microorganisms and determining whether an infection is caused by an organism that is Gram positive or Gram negative will be sufficient to allow a doctor to prescribe treatment with an appropriate empiric antibiotic while waiting for more specific tests, such as culture and antibiotic sensitivities, 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.

With the exception of vaginal specimens, absence or presence of skin cells (epithelial cells) may represent a contaminated sample or a poor-quality specimen. Careful interpretation of the cells, and or bacteria shown in a Gram stain allow the health practitioners to differentiate between normal flora and true infection.

A Gram stain is often routinely performed as part of the evaluation of a culture. When bacteria grow on 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 needed to identify the causative organism.

When is it requested?

A Gram stain may be performed as part of the bacterial culture when a bacterial infection is suspected. It is performed on the same sample as the culture, and the test results are reported out promptly to help guide treatment.

What does the result mean?

A negative Gram stain is often reported as "no bacteria seen". This may mean that there is no bacterial infection present or that there were not enough microorganisms present in the sample to be seen with the stain under a microscope. Due to the sensitivity of the test being approximately 1000 bacteria in the smear,  a negative Gram stain does not necessarily rule out the presence of infection.

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) as well as their shape — round (cocci) or rods (bacilli). Sometimes size, relative quantity and grouping of the bacteria, if relevant, are also reported. If there are bacteria present within other cells (intracellular), this will also be noted as well as; for example, the presence of red blood cells or white blood cells. This information, along with signs and symptoms and other clinical findings, will help the doctor decide which treatment may be most effective, often before culture results are available.

However, Gram stain results are usually considered preliminary, and results of a culture and/or other tests such as biochemical, mass spectrometry and antigen testing for particular types of bacteria are necessary to confirm a diagnosis. Usually, antibiotic susceptibility testing is necessary to determine which antimicrobial drug will be most effective in treating the infection.

Some body sites such as the blood and cerebrospinal fluid are generally sterile which means bacteria should not be present in these sites when there is not a bacterial infection. They may initially be present in low numbers with an infection, and a 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 laboratory scientist to determine specific features that indicate infection.

Is there anything else I should know?

If left untreated, bacterial infections can spread and may eventually cause tissue and organ damage. Prompt treatment can limit their spread and severity.

If fungi (in the form of yeasts or moulds) are present on the Gram stain, they are reported and further testing is performed as necessary to identify the microorganism. Yeast may appear as single cells that may have buds, while moulds may appear as a wide variety of plant-like branches called hyphae.

Parasites and viruses are not distinguished by Gram staining. Other microscopic and /or molecular and/or serological tests may be used to identify these organisms.

Some bacteria are not able to be identified properly using the Gram stain. For example, if a doctor suspects that a person has a mycobacterial infection, such as Tuberculosis, an AFB (acid fast bacilli) culture may be ordered on a sputum sample instead of, or in addition to, a Gram stain and culture.

Common questions

• What are the treatment options for bacterial infections?

Antibiotics are the main treatment for most bacterial infections. If you have an abscess, incision and drainage of the abscess will be necessary to eliminate the infection because antibiotics may not achieve effective concentrations in the wound without draining the pus first. It is important to know that not all antibiotics treat the same type of infections, several factors are taken into account when prescribing antibiotics. It is important that a trained health practitioner monitors the administration of these substances.

• Why isn't a Gram stain performed on all samples sent for culture?

Gram stains are not helpful when the pathogen cannot be differentiated from your normal bacterial flora. For example, a Gram stain of your stool or your throat cannot tell the physician what bacteria are causing the infection because many "good” or normal bacteria and "bad” or pathogenic bacteria look the same under the microscope.

• What happens if my doctor needs more information than the Gram stain provides?

The Gram stain is usually performed together with a culture. This allows for more accurate identification of the bacteria causing the infection and determination of the most appropriate antibiotic. Furthermore, new technology such as mass spectrometry can provide health practitioners with accurate identification in a fast and cost-effective manner, once the culture has grown.