The Whys and Whats of Acid-Fast Stain Tests

What Is an Acid-Fast Stain Test?

An acid-fast stain is a laboratory test performed on a sample of blood, sputum (phlegm), urine, stool, bone marrow, or skin tissue.

Your doctor would typically order this test to find out you have tuberculosis (TB) or another type of bacterial infection.

TB was very common at one time, however it is now rare in the United States. According to the Centers for Disease Control and Prevention (CDC), only 3.6 cases of TB per 100,000 people were reported in the United States in 2010. This is the lowest rate since national reporting began in 1953. (CDC)

While bacterial infections of this type are no longer common, people who have weak immune systems are still vulnerable to them.

To perform the test, a staining dye is added to a bacterial culture and then washed in an acid solution. After the acid wash, the cells of certain types of bacteria retain the dye either completely or partially. This test is able to isolate specific types of bacteria by their “acid fastness,” or their ability to remain dyed.

What Does the Acid-Fast Stain Test?

Based on the type of bacteria found in the body substance culture, there are two types of results from this test—either an acid-fast stain, or a partial or modified acid-fast stain. The type of results depends on the bacteria being tested.

Sputum, or phlegm, is often used to test for Mycobacterium tuberculosis, to find out if a patient has TB. This bacterium is completely acid-fast (meaning that the entire cell holds onto the dye). A positive test result from the acid-fast stain confirms the patient has TB.

With other types of Mycobacterium, such as Nocardia, only certain parts of each cell retain the dye (such as the wall of the cell only). A positive test result from partial or modified acid-fast stain identifies these types of infections.

Although Nocardia is not common, it is dangerous. Nocardia infection starts in the lungs, and can spread to the brain, bone, or skin of people with weak immune systems.

How Are the Body Substance Samples Collected?

If a mycobacterial infection is suspected, your doctor will need a sample of one or more body substances. Your doctor will collect samples using some of the following methods:

Blood Sample

A healthcare provider will draw blood from your vein, usually from one at the inside of the elbow. The area is first cleaned with a germ-killing antiseptic. Then an elastic band is wrapped around your arm, causing the vein to swell with blood.

The healthcare provider gently inserts a needle syringe into the vein. Blood collects in the syringe tube. When the tube is full, the needle is removed. The elastic band is then removed and the needle puncture site is covered with sterile gauze to stop any bleeding.

This is a low-risk test. In rare cases, blood sampling can have risks such as:

  • excessive bleeding
  • fainting (or feeling light-headed)
  • hematoma (blood pooling under the skin)
  • infection (a slight risk any time the skin is broken)

However, these side effects are uncommon.

Sputum Sample

Your healthcare provider will give you a special plastic cup for collecting your sputum. Brush your teeth and rinse your mouth as soon as you wake up in the morning (before eating or drinking anything). Do not use mouthwash.

Take a deep breath and hold it for five seconds. Slowly breathe out. Take another breath and cough hard until some sputum comes up into your mouth. Spit the sputum into a cup. Screw the cup’s lid on tightly. Rinse and dry the outside of the cup. Write the date you collected the sputum on the outside of the cup.

If necessary, the sample can be refrigerated for 24 hours. Do not freeze it or store at room temperature. Take the sample to where your doctor instructed you as soon as you can.

There are no risks involved with taking a sputum sample.


If you are unable to produce sputum, the doctor may collect it using a procedure called a bronchoscopy. This simple procedure takes about 30 to 60 minutes.

Patients usually remain awake for the procedure. First, your nose and throat will be sprayed with a local anesthetic to make it numb. In some cases, you may also be given a sedative to help you relax or to put you to sleep.

A bronchoscope, a long, soft tube with a magnifying glass and light on the end, will be gently passed through your nose or mouth, and down into your lungs. The tube is about as wide as a pencil. The doctor will then be able to see and take samples of sputum or tissue for biopsy through the scope tube.

You will be observed closely by a nurse during the test and afterward, until you are fully awake and able to leave. For safety reasons, you should have someone else drive you back home.

Rare risks of bronchoscopy include:

  • allergic reactions to sedatives
  • infection
  • bleeding
  • tearing in the lung
  • bronchial spasms
  • irregular heart rhythms

Urine Sample

Your doctor will give you a special cup to collect “clean-catch midstream” urine. It is best to collect the sample the first time you urinate in the morning. At that time, the bacterial levels will be higher.

First, wash your hands. Remove the cup’s lid and set it down, with the inside surface up. Use sterile towelettes to clean the area inside and around the penis and foreskin, or the folds of the vagina. Begin urinating into the toilet or urinal. For women, hold apart the folds of skin around the vagina while you are urinating.

After the urine has flowed for several seconds, place the collection container in the stream and collect about 2 fluid oz. of this "midstream" urine, without stopping the flow. Then, carefully replace the lid on the container.

Wash the cup and your hands. If you are collecting the urine at home and cannot get it to the lab within one hour, refrigerate the sample. It can be refrigerated for up to 24 hours.

There are no risks associated with taking a urine sample.

Stool Sample

Make sure to urinate before providing a stool sample so that no urine will get into the sample.

Put on gloves before handling your stool. It contains bacteria that can spread infection. Pass the stool (but no urine) into the dry container that your healthcare provider gave you. You may be given a plastic basin that can be placed under the toilet seat to catch the stool.

Either solid or liquid stool can be collected. If you have diarrhea, a clean plastic bag can be taped to the toilet seat to catch the stool. If you are constipated, you may be given a small enema to help you pass stool.

It is important that you do not collect the sample from the water in the toilet bowl. Do not mix toilet paper, water or soap with the sample.

After collecting the sample, you should remove your gloves and throw them away. Wash your hands. Place the lid on the container. Label it with your name, your doctor’s name, and the date the sample was collected. Place the container in a plastic bag and wash your hands again. Take the sample to the location your doctor instructed as soon as you can.

There are no risks associated with taking a stool sample.

Bone Marrow Biopsy

Bone marrow is the soft fatty tissue inside the body’s larger bones. Bone marrow is usually collected from the pelvis (hip bone) or the sternum (breastbone) in adults, or the tibia (shin bone) in infants and children. The site is first cleaned with an antiseptic, such as iodine, and then injected with a local anesthetic.

Once the site is numb, the doctor inserts a needle through the skin and into the bone. The doctor uses a special needle that draws out a core sample (or cylindrical section). After the needle is removed, a sterile bandage is placed over the site and pressure is applied.

Following the biopsy, you should lie down quietly until your blood pressure, heart rate, and temperature return to normal. You should keep the site dry and covered for about 48 hours.

Some rare and uncommon risks of bone marrow biopsies include:

  • persistent bleeding
  • infection
  • pain
  • reaction to local anesthetic or sedative

Skin Biopsy

There are several methods of skin biopsy, including shave, punch, and excisional. The procedure is usually done at an outpatient clinic or in a doctor’s office.

The shave biopsy is the least invasive technique. In this case, your doctor simply removes the outermost layers of skin.

With a punch biopsy, the doctor removes a small round “plug” of skin (about the size of a pencil eraser) using a sharp, hollow instrument. The area may need to be closed with stitches afterward.

An excisional biopsy removes a larger area of skin. First, a numbing medicine is injected into the area. Then, the skin section is removed and the area is closed with stitches. Pressure is applied to stop the bleeding. If a large area is biopsied, a skin graft (or flap of normal skin) may be used to replace the skin that was removed.

Risks from skin biopsies include:

  • infection
  • excessive bleeding
  • scarring

Preparing for Your Test

No preparations are needed when taking blood, urine, or stool samples. For bone marrow or skin biopsies:

  • Your doctor may instruct you not to eat or drink liquids before the procedure.
  • Be sure to tell your doctor about any medications you are taking (including vitamins and supplements, herbal remedies, and over-the-counter prescriptions).
  • Notify your doctor about all allergies, any previous reactions to medications or bleeding problems in the past, or if you are pregnant.

What Happens in the Lab?

After collection, your sample is sent to a laboratory where it is allowed to grow in culture at room temperature for up to two days. During this time, any present bacteria grow and multiply. The culture is then stained with dye, heated, and washed in an acid solution.

Results of the Test

If your test results are normal and no acid-fast bacteria are found, this means you may not be infected with acid-fast bacteria, or with partial or modified acid-fast bacteria.

If the test is abnormal, it means you may be infected. Your doctor will advise you about your test outcome and the best course of treatment, if needed.

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