What is the Acid Fast Stain test? 

Acid-fast staining is a rapid and inexpensive staining method that is mainly used to detect Mycobacteria - a bacteria that causes several diseases in humans, including tuberculosis, leprosy and infections of the respiratory tract. The bacteria can be seen in a sputum (coughed-up mucus) or tissue sample. 

However, because they have a strong cell wall (outer covering of the bacterial cell), mycobacteria do not take up the usual stains that are used to see other bacteria. An acid-fast stain, on the other hand, can penetrate the mycobacterial wall and sticks to the bacterial cells, making it easier to see them under a microscope. 

The primary stain used in the acid-fast staining procedure stains all types of cells - acid-fast and non-acid fast. To differentiate acid-fast bacteria from other bacteria, the lab technician will use a decolourising acid-alcohol solution, which will only remove the original stain from the non-acid fast bacteria. Mycobacteria retain the stain even after decolourising treatment, thus the name acid-fast bacilli. 

Some other acid-fast bacteria include Rhodococcus, Nocardia, Legionella, Cyclospora and Isospora. Once the presence of the bacteria is confirmed in a sample, doctors order further tests to identify the type of Mycobacteria. The result will then help healthcare practitioners to prescribe the appropriate treatment.

The three stains commonly used to detect acid-fast bacteria include:

  • Ziehl-Neelsen (ZN) stain: In this technique, the primary stain is carbol fuschin. It is a red coloured dye that enters the bacteria on being heated. After decolourising, the sample is stained with methylene blue. Therefore, Mycobacteria appear as red-coloured rods amongst the blue non-acid fast bacteria. Ziehl-Neelsen stain can be used to stain sputum or lung tissue. It also aids in staining pleura (the outer membrane covering the lungs) and lymph nodes.
  • Kinyoun stain: It is a modified form of ZN stain. Also referred to as cold stain, this procedure uses a higher concentration of stains than ZN stain and does not include heating the sample.
  • Auramine rhodamine stain: In this technique, the primary stain is rhodamine auramine. Auramine and rhodamine are both fluorescent dyes that specifically bind with the cell wall of acid-fast bacteria and make them appear as bright yellow or orange fluorescent rods under ultraviolet light. The non-acid fast bacilli lose the stain after decolourising and are stained with non-fluorescent potassium permanganate. They don’t show up under the microscope in the dark. Unlike the ZN stain, this procedure does not involve heating. 

Mycobacteria stained using the ZN stain are best be viewed under high power lens (x800-1000), whereas the mycobacteria stained with auramine rhodamine stain can be viewed under a microscope at low power (x450-500) as they appear fluorescent. In addition, auramine rhodamine stain has better sensitivity than ZN stains and can be done faster due to the lower magnification needed for the study. 

The duration of viewing is 20 minutes for ZN stained smears, whereas it is half the time in the case of auramine rhodamine stained smears. Fluorescent stains are less specific than ZN stains; therefore, if an auramine rhodamine stain shows scanty acid-fast bacilli, the results should be reconfirmed using ZN staining method.

  1. Why is Acid Fast Stain test performed?
  2. How do you prepare for Acid Fast Stain test?
  3. How is Acid Fast Stain test performed?
  4. What do Acid Fast Stain test results mean?

Your doctor may order this test if you have any of the following symptoms or conditions:

  • Cough lasting for about two to three weeks or more
  • Prolonged cough along with fever 
  • Weight loss associated with prolonged cough
  • Blood in sputum along with prolonged cough
  • Chest X-ray pointing towards a TB-like condition
  • Positive result on TB skin test
  • Signs of extrapulmonary TB (TB at a site other than lungs)
  • To check the progress of TB treatment

This test is ordered for people who are at a high risk of acquiring a TB infection such as those who have a weak immune system or who have been tested positive for HIV/AIDS (human immunodeficiency virus/acquired immunodeficiency syndrome).

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Your doctor or nurse may suggest you drink fluids before the test to make it easier for you to provide a sputum sample. You will be asked to brush your teeth and rinse your mouth to avoid any food particles, oral bacteria or other contaminants from mixing with the sputum sample. If the sputum is very thick, your doctor may even recommend inhaling warm saline to make the sample slightly less viscous. If you have any questions about the test preparation, please speak to your doctor.

You will be asked to take a few deep breaths and then cough deeply to give a sample of sputum from deep within your chest. About a teaspoon of sputum may be required. 

Usually, the test is performed in the morning before breakfast; this is when the sample is most easily obtained. 

The testing facility may require a repeat test on three consecutive days in order to obtain correct results. Also, collection of three samples increases the chance of isolating the Mycobacteria and early diagnosis of the condition. 

This procedure does not pose any risks.

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Normal results:

The test results are said to be normal (and negative) when no bacteria are detected in the sample. A negative result is an indication that you may not have a mycobacterial infection. 

Abnormal results:

The test result is said to be abnormal (and positive) when mycobacteria are detected in the test sample. A positive result may also be indicated by numbers in some laboratories. The following list shows the numbers and their meaning:

  • 1+ it means that bacteria were present but rare
  • 2+ means that a few bacteria were present in the sample
  • 3+ means that a moderate number of bacteria were present in the sample
  • 4+ means that many bacteria were observed in the sample

A positive result indicate the presence of active mycobacterial infection as is seen in TB or leprosy. It may also be due to HIV/AIDS.

An abnormal result may also be possible if a saliva sample has been taken instead of a sputum sample. 

If the first sample is contaminated by food particles, another sample may be required. Even though this test is rapid and affordable, it has certain drawbacks such as requiring a high bacterial load for detection and not being able to identify the particular species. Therefore, the doctor may have to confirm the results with other tests.

Disclaimer: All results must be clinically correlated with the patient’s complaints to make a complete and accurate diagnosis. The above information is provided from a purely educational point of view and is in no way a substitute for medical advice by a qualified doctor.


  1. Wilson DD. Manual of Laboratory & Diagnostic Tests. Laboratory and diagnostic tests. McGraw Hill. 2008, Pp 8-9.
  2. Wanger A. Microbiology and Molecular Diagnosis in Pathology. 1st edition. Elsevier; 2017. Pages 61-73.
  3. Karimi S. Histopathological findings in immunohistological staining of the granulomatous tissue reaction associated with tuberculosis. Tuberc Res Treat. 2014;2014:858396. PMID: 24511393.
  4. University of Rochester Medical Center [Internet]. Rochester (NY): University of Rochester Medical Center; Acid-Fast Bacteria Culture
  5. Missouri Department of Health and Senior Services [internet]. US; Acid-Fast Bacilli (AFB) Smear and Culture
  6. Bayot ML, Sharma S. Acid Fast Bacteria. [Updated 2018 Dec 28]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020 Jan

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