What is a sputum Gram’s stain?

A sputum Gram’s stain is a laboratory test that allows your doctor to diagnose a bacterial infection in your respiratory tract. They may order it if you have symptoms of a respiratory infection that might be caused by bacteria. It’s the most common preliminary test beyond a chest X-ray for pneumonia and other respiratory infections, and can help your doctor promptly prescribe a treatment plan.

The test is sometimes called a Gram’s stain of sputum. It’s named after its inventor, Hans Christian Gram.

Your doctor may order a sputum Gram’s stain to help identify the cause of pneumonia. This is an infection that can affect your lower respiratory tract. It’s often caused by microorganisms, like bacteria, viruses, or fungi.

The symptoms of pneumonia can range from moderate to life-threatening, and include:

  • wheezing
  • shortness of breath
  • rapid breathing
  • chest pain
  • cough
  • fever
  • chills
  • fatigue
  • headache
  • muscle aches
  • nausea and vomiting
  • sputum that’s colored or has an odor
  • confusion
  • respiratory failure
  • shock (severely low blood pressure)

Children under the age of 5, older adults over the age of 65, and people with a weak immune system are at heightened risk of developing pneumonia.

To complete a sputum Gram’s stain, your doctor will need to a collect a sample of your sputum and send it to a laboratory for testing.

Sputum is a mixture of saliva and mucus that you cough up from your respiratory tract. It’s usually colored and thick in consistency, especially when you have an infection in your lungs.

Plain saliva comes from your mouth and is usually clear.

Your doctor may ask you to collect a sample of your own sputum, but if you can’t they may collect one using a bronchoscopy.


If you’re very sick, you may be in the hospital when your doctor orders a sputum Gram’s stain. A nurse will help you cough up sputum. If you have trouble, they may give you a breathing treatment to help loosen the sputum in your airways.

If you’re at home, your doctor may ask you to collect the sputum sample yourself. They will give you a sterile sample cup to use. Wait until you’re ready to collect your sample before opening the lid.

The night before you provide a sample, try to drink lots of fluids like water or tea. This will help your body make more sputum. Collect your sample first thing in the morning, before eating or drinking anything. There tends to be more bacteria present at this time and can help ensure accurate test results.

To provide a sample of your sputum:

  • Brush your teeth and rinse your mouth. Don’t use antiseptic mouthwash.
  • Take a couple of long, deep breaths. Then breathe deeply and cough hard until sputum comes up.
  • Spit out the sputum into your sample cup. Keep coughing up sputum until the cup is filled to the marker, which should equal approximately 1 teaspoon.
  • Screw the lid onto the cup and wash and dry the outside of it. Write your name and the date on the label.
  • Take the sample to the clinic or laboratory, following your doctor’s instruction. You can refrigerate it for up to 24 hours if needed, but you shouldn’t freeze it or store it at room temperature.

If you can’t cough up enough sputum, try breathing steam in from boiling water, or take a hot steamy shower. The sputum sample must come from deep inside your lungs for the test to be accurate.

If you still can’t cough up enough sputum, your doctor will likely do a bronchoscopy to collect sputum directly from your lungs.


Bronchoscopy is a simple procedure that usually takes about 30 to 60 minutes. You will probably stay awake for it.

Ask your doctor if you should do anything to prepare for your bronchoscopy. They may ask you to avoid taking medicines that raise your risk of bleeding, such as aspirin and warfarin, the day before your procedure. Your doctor may also ask you to avoid eating and drinking anything the night before your procedure.

To perform a bronchoscopy, your doctor or nurse will spray a local anesthetic into your nose and throat. They might also give you a sedative to help you relax, or medication to put you to sleep, but general anesthesia isn’t required.

Your doctor will feed a bronchoscope through your nose or mouth into your lungs. This is a soft, narrow tube with a light and magnifying glass on the end.

Your doctor will use the scope to look into your lungs and collect a sample of your sputum to send to the lab for testing.

You’ll be monitored until you’re fully awake. To be safe, you should have someone drive you home afterward.

At the laboratory, a technician will analyze your sputum sample using a sputum Gram’s stain.

They will place a thin layer of your sputum on a slide and allow it to dry. Then they will treat the slide with a special staining agent that’s sensitive to peptidoglycan.

Peptidoglycan is a polymer made from amino acids and sugars. It’s found in the cell walls of bacteria, and helps laboratory staff learn if bacteria are present in your sputum.

After adding the staining agent, the laboratory technician will examine the slide under a microscope. The Gram’s stain doesn’t allow them to identify every specific type of bacteria, but it can help them tell if there are bacteria with thick cell walls or thin cell walls.

Bacteria with thick cell walls will produce a Gram-positive result. Bacteria with thin cell walls will produce a Gram-negative result.

The test can also help the laboratory technician detect the presence of fungi in your sputum.

And the technician will also look for white blood cells in your sputum, which is a sign of infection.

If the test results from your sputum Gram’s stain are abnormal, it means that bacteria and white blood cells have been detected. The bacteria found will be Gram-positive or Gram-negative.

Common Gram-positive bacteria detected by the test include:

  • Staphylococcus
  • Streptococcus
  • Bacillus
  • Listeria
  • Enterococcus
  • Clostridium

Common Gram-negative bacteria detected by the test include:

  • E. coli
  • Klebsiella species
  • Proteus species
  • Pseudomonas aeruginosa

A normal test result means that few white blood cells and no bacteria have been found in your sputum sample, and your symptoms may be due to other causes.

Depending on your test results, your doctor may prescribe a treatment plan or order more tests. If you have a bacterial lung infection, they will likely prescribe antibiotics.

This will probably be enough to treat your infection if you have a healthy immune system.

In some cases, bacterial pneumonia can be more severe and even life-threatening. It can be particularly serious for older adults and others with weakened immune systems. Sometimes, severe cases of pneumonia can lead to:

  • collection of fluid in your lungs
  • scarring in your lungs, which may make you susceptible to future infections
  • abscesses in your lungs
  • respiratory failure
  • sepsis, which is a bacterial blood infection

Early diagnosis can help your doctor prescribe a treatment plan, which may help improve your outlook.

There are very few risks associated with collecting a sample of your sputum. You might feel slightly light-headed when you cough deeply, or feel discomfort in your lungs or throat.

Side effects from bronchoscopy are also rare, but can include:

  • bleeding
  • infection
  • pneumothorax, which happens when air is released into the space between your lung and chest wall
  • bronchial spasms, which can occur when muscles in your bronchioles suddenly clench
  • irregular heart rhythms
  • allergic reactions to sedatives

Ask your doctor for more information about the potential benefits and risks of providing a sputum sample.

A sputum Gram’s stain is a quick, low-risk laboratory test. Your doctor can use the results to help diagnose the cause of respiratory symptoms. In particular, the presence of disease-causing bacteria.

Determining the cause of your symptoms can help your doctor prescribe an appropriate treatment plan. Your treatment plan will vary, depending on your diagnosis.