Gram Stain

What is a Gram stain?

A Gram stain is a laboratory test that checks for bacteria at the site of a suspected infection or in certain bodily fluids. A medical laboratory scientist processes the Gram stain, which gives relatively quick results, so healthcare providers can know if bacteria are present, and, if so, the general type(s). This can help guide further identification tests and treatment options.

Bacteria are a large group of one-celled organisms. They can live in different places in your body and on your skin. While some types of bacteria are harmless or even beneficial, others can cause infections and disease. A Gram stain helps diagnose harmful bacteria.

Under a Gram stain, different kinds of bacteria change one of two sets of colors (pink to red or purple to blue) under a special series of stains and are categorized as “gram-negative” or “gram-positive,” accordingly. Gram staining works by differentiating bacteria by the chemical and physical properties of their cell walls.

However, not all forms of bacteria can be tested using the Gram stain method, and Gram stains don’t usually provide a diagnosis alone. Instead, they help to broadly determine the type of bacteria.

Gram staining is an essential staining technique in microbiology that scientists have used for hundreds of years. It’s named after Danish bacteriologist Hans Christian Gram, who first introduced it in 1882, mainly to identify organisms causing pneumonia.

What are gram-positive and gram-negative bacteria?

If bacteria are present on a Gram stain slide, a medical laboratory scientist classifies them as gram-negative or gram-positive based on which color they turn under a series of stains. There are also gram-variable bacteria, which means they stain irregularly, and there’s a mix of pink- and purple-colored bacteria.

It’s important to note that gram-negative doesn’t mean there are no bacteria, and “positive” and “negative” don’t mean “good” and “bad” in this case.

Gram-positive organisms

Gram-positive bacteria have cell walls that contain thick layers of peptidoglycan, a substance that forms the cell walls of many bacteria. The peptidoglycan forms about 90% of the cell wall in gram-positive bacteria. This causes them to appear blue to purple under a Gram stain.

Gram-positive organisms include:

• Staphylococcus species.
• Streptococcus species.
• Corynebacterium species.
• Clostridium species.
• Listeria species.

Gram-negative organisms

Gram-negative bacteria have cell walls with thin layers of peptidoglycan (10% of the cell wall) and high lipid (fatty acid) content. This causes them to appear red to pink under a Gram stain.

Gram-negative organisms include:

• Neisseria gonorrheae and Neisseria meningitides.
• Moraxella species.
• Escherichia coli (E. coli).
• Pseudomonas species.
• Proteus species.
• Klebsiella species.

What’s the difference between a Gram stain and a bacteria culture?

Healthcare providers use both Gram stains and bacteria cultures if they suspect you have a bacterial infection. However, these tests provide different information.

Providers usually request bacteria culture tests alongside Gram stain tests. In a bacteria culture test, a medical laboratory scientist takes cells from the sample that’s also for the Gram stain and puts it in a special environment to encourage cell growth. Results are often available within a few days, but some types of bacteria grow slowly, which may take several days or longer. Once the cells have multiplied and grown enough, a medical laboratory scientist examines the cultured bacteria to determine their exact identity.

A Gram stain involves applying a stain to a sample in glass microscope slides and looking at it under a microscope to determine if bacteria are present at all. If bacteria are present, the medical laboratory scientists can see if they’re gram-negative or gram-positive based on if the bacteria appear pink to red or purple to blue under the stain. Different types of bacteria are either gram-negative or gram-positive.

While Gram stains provide quicker results than bacteria cultures, in most cases, a Gram stain can’t diagnose the type of bacterium, unlike a bacteria culture. However, Gram stains can help point healthcare providers in the right direction for prescribing treatment.

What conditions do Gram stains help diagnose?

Healthcare providers order Gram stains to help diagnose and treat certain bacterial infections, such as urinary tract infections (UTIs) and bacterial pneumonia. Providers may not always request a Gram stain to help diagnose conditions caused by bacteria. For example, they usually don’t Gram stain for food poisoning, but it’s possible to do so through a stool (poop) sample.

Common gram-negative bacteria and their associated conditions

Examples of gram-negative bacteria (in italics) and the conditions they can cause include:

• Escherichia coli: Urinary tract infections (UTIs) and food poisoning.
• Legionella pneumoniae: Legionnaires’ disease.
• Salmonella: Food poisoning and Typhoid fever.
• Klebsiella: Pneumonia, bloodstream infections, wound or surgical site infections and bacterial meningitis.
• Neisseria gonorrheae: Gonorrhea.
• Proteus: UTIs.
• Morganella morganii: UTIs, sepsis, pneumonia, wound infections, musculoskeletal infections, central nervous system infections and pericarditis.
• Aeromonas: Eye infections, osteomyelitis, bacterial meningitis, respiratory infections following "near drowning" and joint infections.

Common gram-positive bacteria and their associated conditions

Examples of gram-positive bacteria (in italics) and the conditions they can cause include:

• Streptococcus pneumoniae: Bacterial pneumonia.
• Staphylococcus aureus: Bacterial pneumonia, Methicillin-resistant Staphylococcus aureus (MRSA) and toxic shock syndrome.
• Group A Streptococcus species: Strep throat infections.
• Listeria: Food poisoning.
• Clostridium: Food poisoning
• Clostridium botulinum and clostridium butyricum: Botulism.

When is a Gram test performed?

Healthcare providers typically request a Gram stain along with a bacteria culture when they suspect you have a bacterial, or sometimes fungal, infection. It’s one of the most common ways to quickly diagnose bacterial infection in your body.

Who performs a Gram stain test?

Depending on what kind of infection you may have, many types of healthcare providers could collect a sample for a Gram stain test. For example, a gynecologist may collect a swab for a gonorrhea Gram stain test and a pulmonologist may collect a sample of your spit or mucus from your cough to use for a bacterial pneumonia Gram stain test.

Your provider then puts the sample in a sterile container and sends it to a laboratory for testing. A medical laboratory scientist applies the sample to microscope slides, applies a series of stains and looks at the sample under a microscope. They put together a report and share it with your healthcare provider.

How do I prepare for a Gram stain test?

You don’t need to do anything special to prepare for a Gram stain test.

How is a Gram stain test performed?

There are three general steps involved in a Gram stain test, including:

• Collecting the sample.
• Processing the sample.
• Examining the sample.

Collecting the Gram stain sample

A Gram stain test involves your healthcare provider collecting a sample from a site of suspected infection. Some of the ways a provider can collect Gram stain test samples include:

• Brushing or scraping tissue from the surface of a part of your body.
• Collecting fluid or discharge samples from your body.
• Using fine-needle aspiration to draw a fluid sample from an area in your body.

Sites collected by a swab with brushing and scraping may include your:

• Throat.
• Nostrils.
• Genitals.
• Skin wounds.

Samples that you can collect directly into a sterile container include:

• Spit (sputum).
• Urine (pee).
• Stool (poop).

Areas in your body that may require the use of fine-needle aspiration include:

• Synovial fluid in your joints.
• Fluid around your heart (pericardial fluid).
• Fluid around your lungs (pleural fluid).
• Cerebrospinal fluid (CSF) around your spinal cord.

Your provider then puts the sample in a sterile container and sends it to a laboratory for testing.

Gram staining procedure

At the lab, a medical laboratory scientist smears or spreads the sample on glass microscope slides. These slides are known as smears. They then apply a series of stains to the smear to perform a Gram stain.

The Gram staining process includes four basic steps, including:

1. Applying a primary stain (crystal violet).
2. Adding a mordant (Gram’s iodine).
3. Rapid decolorization with ethanol, acetone or a mixture of both.
4. Counterstaining with safranin.

Examining the Gram stain

The medical laboratory scientist then categorizes any bacteria that may be present by color and shape during the microscopic evaluation:

• Color: Typically, bacteria that are gram-positive appear purple to blue, and bacteria that are Gram-negative appear pink to red.
• Shape: The most common shapes include round (cocci) or rod-shaped (bacilli).

The medical laboratory scientist also looks for additional characteristics of the sample by observing the groupings of the bacteria on the slide. Examples include:

• Cocci that are present singly, in pairs, in groups of four, in clusters or in chains.
• Bacilli that are thick, thin, short, long or have enlarged spores on one end.
• If bacteria are present within white blood cells.

The medical laboratory scientist then puts together a report and sends it to your healthcare provider.

What are the risks of a Gram stain test?

There’s no risk to having a Gram stain that involves a swab or fluid collection of sputum (spit), urine or stool.

There’s very little risk to having blood tests. You may have slight tenderness or a bruise at the site of the blood draw, but this usually resolves quickly.

Possible fine-needle aspiration risks include:

• Discomfort or soreness.
• Bleeding.
• Infection.
• Damage to nearby structures.

What do the results of a Gram stain mean?

Gram stain test results reveal one of two categories: a negative Gram stain or a positive Gram stain. This is not to be confused with gram-negative bacteria or gram-positive bacteria.

Negative Gram stain

If your test result reveals a negative Gram stain or “no organism seen,” it usually means that there are too few bacteria present to be able to be seen using the Gram stain method. Bacteria might still be detected by culture if a culture is performed on the specimen.

Positive Gram stain

If your test result reveals a positive Gram stain, it means that bacteria were present in your sample. If your result is positive, it usually includes information about what kind of organism was present on the sample slide, including:

• Type of bacteria: Gram-positive or gram-negative.
• Shape of bacteria: Round (cocci) or rods (bacilli).
• Other bacteria characteristics: Size, relative quantity (number) and/or arrangement of the bacteria, if applicable.
• Other cells: Whether there are bacteria present within other cells (intracellular) and if there’s a presence of red blood cells or white blood cells.
• Fungi: Gram stains can check for the presence of fungi in the form of yeasts or molds. You may need further testing to identify the specific type.

This information, along with signs and symptoms and other clinical findings, will help your healthcare provider determine which treatment may be most effective, sometimes before bacteria culture results are available.

When should I know the results of the test?

Gram stains generally provide quick results. However, Gram stain results are usually considered preliminary, meaning they can’t always provide a diagnosis alone. Results of a bacteria culture and/or other tests, such as antigen and antibody testing for particular types of bacteria, might be necessary to confirm a diagnosis.

When should I call my doctor?

If you’ve been diagnosed with an infection and are having issues with your treatment or your infection isn’t improving, contact your healthcare provider.

A note from Cleveland Clinic

Gram stains are a very common and often painless way to determine the presence of a bacterial or fungal infection. While it can be stressful to wait for the results of a diagnostic test, know that your healthcare team is there to support you no matter what the results are. Don’t be afraid to ask your healthcare provider questions about your test or results.