Sputum Stain for Mycobacteria

What Is Sputum Stain For Mycobacteria?

Sputum stain for mycobacteria is a laboratory test performed on a sample of the patient’s sputum (phlegm). It is also known as an acid-fast bacillus stain (AFB) or a tuberculosis (TB) smear. The test is commonly ordered by a doctor to find out if a patient has tuberculosis (TB) or another type of mycobacterial infection.

Checking your sputum is the best way to find out if you have TB. If you are already taking medication for TB or another mycobacterial infection, the test is also the best way to find out if your medication is working.

Sputum will first be collected by either you or your physician.

Why the Test Is Ordered

Your doctor will order this test if a mycobacterial infection is suspected.

Mycobacteria are a type of microorganism with nearly 100 known species. The most common type, Mycobacterium tuberculosis, causes TB. The general symptoms of TB include:

  • coughing up blood or mucus
  • lack of appetite, weight loss
  • weakness, fatigue
  • fever, chills, night sweats

Another relatively common type, Mycobacterium leprae, causes leprosy. Symptoms of leprosy include:

  • skin discoloration
  • skin lesions, nodules, plaques
  • thickened skin
  • nasal congestion
  • nosebleeds

Except for the two microorganisms that cause TB and leprosy, most mycobacteria live in the water and soil everywhere in the world. These are called nontuberculous mycobacteria (NTM).

NTM are found in city water, bayous, rivers, hot tubs, swimming pools, yard soil, and even in our food. Their tough, waxy cell wall makes them resistant to antibacterial agents.

Although NTM are everywhere, most people are not affected. However, people with immunity problems, such as AIDS, are vulnerable to infection. Some people can have an infection with no symptoms at all. Other people have infections that cause lung symptoms similar to TB.

Medicines can treat these infections, but it often takes more than one medicine to cure them.

How the Stain Test Is Done

The patient’s sputum specimen is spread on a microscope slide. A staining dye is added to the cells of the specimen, and then washed in an acid solution. The cells are then examined under a microscope.

If the cells retain the stain, this confirms the presence of mycobacterium. Most mycobacteria are known to be acid-fast, which means they hold onto the dye when washed in an acid solution.

Another test, called a culture, may be done. The sputum specimen is placed into a culture (nutrient) medium and allowed to grow at room temperature for several days. This makes it possible to see a greater number of bacteria cells in order to confirm results.

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, however, 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 the 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 sample cup, which is sterile. Don’t open the cup until you are ready to collect the sample.

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 one teaspoon).
  • Screw on the cup lid and wash and dry the outside of it.
  • 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 your 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, and 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.
  • For safety, 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, and usually fixes itself), bronchial spasms (a sudden clenching of the muscles in the bronchioles), and irregular heart rhythms.

Results of the Test

If your test results are normal (negative), this means no mycobacterial organisms were found.

If the test is abnormal, it means the stain is positive for one of the following organisms:

  • Mycobacterium tuberculosis
  • Mycobacterium leprae
  • nontuberculous bacteria
  • other acid-fast bacteria

Your doctor will advise you about your test outcome, and the best course of treatment, if needed.

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