What is being tested?
Acid-fast bacilli (AFB) are rod shaped bacteria that can be seen and counted under the microscope on a specially stained sample on a glass slide, called an AFB smear. The most common acid-fast bacilli are members of the genus Mycobacterium.

Mycobacterium tuberculosis is the most prevalent species of mycobacteria, and the most infectious. Most samples that are submitted for AFB smears and cultures are collected because the doctor suspects that the patient has TB. Only a few of the more than 60 species of mycobacteria that have been identified cause infections in humans. They include:

• M. africanum, causes a disease similar to TB in certain parts of the world
• Mycobacteria avium-intracellulare complex (MAC), can cause a lung infection in immunosuppressed patients, such as the elderly and those with AIDS; this infection is not contagious, but can be difficult to treat as it tends to be highly resistant to antibiotics
• Other mycobacterial species, such as Mycobacterium marinum, grow in water, such as fish tanks, and can cause skin infections, while other rapidly growing mycobacteria can infect wounds and prosthetic devices 
• A few mycobacteria, such as M. bovis, can sometimes be transferred from animal to human

Several AFB smears from different samples should be screened for AFB since the number of bacilli may vary day to day. If acid-fast bacilli are present on any of the smears, a mycobacterial infection is likely. Since M. tuberculosis is the most common cause of respiratory infections with mycobacteria, a presumptive diagnosis of TB can be made but other follow-up testing must be done to positively identify the acid-fast bacilli as either M. tuberculosis, or another mycobacteria species.

Patient samples are processed for AFB cultures at the same time as the smears..  The cultures are used to grow acid-fast bacilli in the laboratory. Body fluid or tissue samples are decontaminated to remove the normal respiratory bacteria, digested to emulsify the mucus, and concentrated to increase the number of bacteria before being introduced into a nutrient environment and incubated. Since mycobacteria grow slowly, positive identification of the species that is/are present may take days to several weeks, while negative results (no mycobacterial growth) can take up to 6 to 8 weeks to confirm.

How is the sample collected for testing?

Since M. tuberculosis and M. avium  most frequently infect the lungs (pulmonary), sputum is the most commonly tested sample. Sputum is phlegm, thick mucous that is coughed up from the lungs. Usually, three to five early morning samples are collected (on consecutive days) in individual sterile cups.

If you are unable to produce sputum, your doctor may collect respiratory samples using a procedure called a bronchoscopy. Bronchoscopy allows your doctor to look at, and collect samples from, your bronchi and bronchioles. These branching tubes connect the trachea in your throat to your lungs, providing a pathway for air to enter your lungs. Once a local anaesthetic has been sprayed onto the trachea, your doctor can insert a tube into your bronchi and smaller bronchioles and aspirate fluid samples for testing. Sometimes, they will introduce a small amount of saline through the tubing and into the bronchi and then aspirate it to collect a bronchial washing.

Since young children can not produce a sputum sample, gastric washings/aspirates may be collected. This involves introducing saline into the stomach through a tube, followed by fluid aspiration.

If your doctor suspects extrapulmonary TB (outside of the lungs, fairly common in AIDS patients), he may test the body fluids and tissues most likely affected. For instance, you may collect one or more urine samples if he suspects TB has infected your kidneys. A needle may used to collect fluid from your joints or from other body cavities, such as the pericardium (lining around the heart) or abdomen. Occasionally, your doctor may need to use a needle to collect a sample of cerebral spinal fluid (CSF) or perform a minor surgical procedure to obtain a tissue biopsy.