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Sputum Gram Stain
The sputum Gram stain is a laboratory test ordered when a doctor suspects that a patient's respiratory symptoms may be caused by a bacterial in...

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What Is a Sputum Gram Stain?

The sputum Gram stain is a laboratory test ordered when a doctor suspects that a patient’s respiratory symptoms may be caused by a bacterial infection.

It is the most common preliminary test for pneumonia and other respiratory infections because it provides rapid test results. This allows the physician to begin antibiotic treatment immediately.

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

How the Test Works

The sputum Gram stain detects the thickness of bacteria cell walls using a special staining agent that is sensitive to peptidoglycan. Peptidoglycan is a polymer made of amino acids and sugars, found in the cell walls of bacteria.

The patient’s sputum sample is placed on a slide in a thin layer and allowed to dry. The slide is then treated with stain and examined under a microscope. The Gram stain cannot identify all specific types of bacteria, but it can determine whether they are Gram-positive (thick cell walls), or Gram-negative (thin cell walls). This helps the laboratory identify the specific type of bacteria.

The test can detect bacteria and fungi (yeasts, molds), but viruses cannot be seen with a Gram stain.

The laboratory technician also looks for white blood cells in the sputum. White blood cells are always present in quantity wherever there is an infection.

Why the Test Is Ordered

Sputum Gram stain is often used to begin isolating the cause of pneumonia. The effects of pneumonia can range from moderate to life-threatening illness.

Pneumonia is an infection of the lower respiratory tract, often caused by microorganism invasion. There are many kinds of microorganisms (bacteria, fungi, and viruses) that can cause pneumonia.

According to the Centers for Disease Control and Prevention (CDC), more than one million children and adults are hospitalized with pneumonia each year in the United States. Worldwide, it is the leading cause of death from infectious disease, killing more than 4 million people each year.

Pneumonia symptoms include:

  • shortness of breath
  • wheezing
  • rapid breathing
  • fever and chills
  • cough
  • sputum that is colored or has an odor
  • fatigue
  • headache
  • muscle aches
  • chest pain
  • nausea and vomiting

People most at risk for pneumonia are children below age 5, the elderly over age 65, and those who have weak immune systems. This includes people with AIDS, HIV, cancer, or those who take immunosuppressant medications.

A Gram stain of your sputum is a fast way for your doctor to find out if your respiratory symptoms are being caused by a bacterial infection. This helps your doctor provide the proper treatment.

How to Collect a Sample of Your Sputum

If you are very sick, you may already be in the hospital. If so, the bedside nurse will help you cough up sputum to send to the laboratory for the test. If you have trouble coughing up sputum on your own, the nurse may have you breathe steam.

If you are sick at home, you will need to collect the sputum sample yourself.

Keep in mind that sputum from deep inside your lungs isn’t the same as saliva. Sputum is mucus, and is usually colored and thick in consistency, especially when there is an infection in the lungs. Saliva comes from your mouth and is clear-colored and thin.

Plan to collect sputum first thing in the morning. This makes the test more accurate. Do not eat or drink anything in the morning before collecting your sample. Your doctor will provide a sterile sample cup. Wait until you are ready to collect your sample before opening the lid.

To collect a sputum sample:

  • Brush your teeth and rinse your mouth (don’t use antiseptic mouthwash).
  • Take a couple of long, deep breaths.
  • Breathe deeply again and cough hard until sputum comes up.
  • Spit out the sputum into the sample cup.
  • Keep coughing up sputum until the cup is filled to the marker (approximately 1 teaspoon).
  • Screw on the cup lid and wash and dry the outside of the cup.
  • Write your name and the date on the cup label.

Take the sample to the clinic or laboratory, as instructed. The sample can be refrigerated for up to 24 hours if needed. Do not freeze it or store it at room temperature.

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

If you still cannot cough up sputum, your doctor will do a bronchoscopy to collect sputum directly from the lungs.

How to Prepare for Self-Collection

The night before the test, drink lots of fluids, such as water or tea. This will help your body make more sputum overnight. Collecting your sputum in the morning makes the test more accurate. More bacteria are present first thing in the morning.

How the Doctor Collects Sputum

Bronchoscopy is a simple procedure that takes about 30 to 60 minutes. It is often done in the doctor’s office. The patient usually remains awake.

Do not to take medicines that thin your blood the day before your bronchoscopy. These medicines include aspirin, Motrin, Advil, and Aleve. You may take Tylenol if needed for pain. You will also be asked not to eat or drink anything the night before the procedure.

Bronchoscopy is performed as follows:

  • A local anesthetic will be sprayed into your nose and throat to numb them.
  • You might be given a sedative to help you relax, or medication to put you to sleep.
  • General anesthesia is not usually needed for bronchoscopy.
  • The bronchoscope is a soft, small-circumference tube with a light and magnifying glass on the end.
  • The doctor feeds the scope through your nose or mouth, and into your lungs.
  • Using the magnifying glass, the doctor can see into the lungs and use the scope to remove a sample of your sputum.
  • A nurse will attend you during and after the procedure until you are fully awake.
  • To be safe, you should have someone else drive you home.


There are no risks in collecting a sputum sample yourself. You might feel light-headed when coughing deeply. Rare risks of bronchoscopy include:

  • allergic reactions to sedatives
  • infection
  • bleeding
  • pneumothorax (tearing in the lung which results in a little air being released between the lung and chest wall; usually fixes itself)
  • bronchial spasms (a sudden clenching of the muscles in the bronchioles)
  • irregular heart rhythms.

Test Results

Abnormal results mean that bacteria and white blood cells were seen in the sputum sample. The bacteria found will be either Gram-positive, or Gram-negative.

Common Gram-positive bacteria found by the test include:

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

Common Gram-negative bacteria found by the test include:

  • cyanobacteria
  • spirochaetes
  • green sulfur bacteria
  • certain types of Proteobacteria

A normal test result means that there were very few white blood cells and no bacteria seen in the sputum sample.

Long-Term Outlook

Most bacterial lung infections, including pneumonia, clear up with antibiotic treatment in people who have a healthy immune system.

Bacterial pneumonia can be severe and life-threatening, especially for people who are 65 or older, or those with weakened immune systems. Severe cases can lead to:

  • fluid collecting in the lungs
  • respiratory failure
  • sepsis (bacterial blood infection)
  • scarring (making you susceptible to future infections)
  • abscesses in the lungs
Written by: Sandy Calhoun Rice
Edited by:
Medically Reviewed by: George Krucik, MD
Published By: Healthline Networks, Inc.
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