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AFB (Acid-Fast Bacillus)

The acid-fast bacillus (AFB) is a type of bacteria that causes tuberculosis andv some other infections. Tuberculosis, commonly known as TB, is a serious bacterial infection that mainly affects the lungs. It can also affect other parts of the body, including the brain, spine, and kidneys.

Related ArticlesSputum For AFB
ZN Stain
Sputum Culture
MTB PCR
Bronchial Washing for AFB
CSF For AFB
Pleural Fluid For AFB
SpecimenSputum, Bronchial Washing, CSF, Plueral Fluid
MethodsZN Stain, Fluorescent Microscopy, Conventional Microscopy, Auramine Stain
Acid Fast bacilli
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Why To Get Tested:

When To Get Tested:

  • When you have signs and symptoms of a lung infection, such as a chronic cough, weight loss, fever, chills, and weakness, which may be due to TB or a nontuberculous mycobacterial (NTM) infection.
  • When you have a positive IGRA blood test or tuberculin skin test (TST) and are in a high risk group for progressing to active TB.
  • When you have an infection on the skin or other parts of the body that may be due to mycobacteria.
  • When you are being treated for tuberculosis.

Morphology of MTB

  • Straight or slightly curved thin rod-shaped bacilli.
  • Non-sporing, non-motile, non-capsulated bacteria.
  • Acid-fast bacilli, neither gram +ve nor gram –ve.
  • During acid-fast stain, they appear bright red to intensive purple with green/blue background.
  • They measure 0.5 µm x 3 µm.
  • Appears in single, in pairs, or small chumps.
  • The high content of mycolic acid (50 to 60 %).
  • The cell wall is rich in lipids and waxes.
  • They are wrapped together due to the presence of fatty acids.
  • Capable of intracellular growth.
  • They are resistant to disinfectants, detergents, common antibiotics, dyes, stains, osmotic lysis, lethal oxidation, etc.

Cultural Characteristics of MTB

  • Culture is the gold standard for laboratory confirmation of Tuberculosis.
  • Growing bacteria are required to perform drug-susceptibility testing and genotyping.
Cultural Characteristics of MTB

In the Lowenstein-Jensen (LJ) medium

  • It is an egg-based medium and growth is quite slow.
  • It takes 6-8 weeks to get visual colonies on this type of media.
  • Colonies are non-pigmented, dry, rough, raised, irregular with a wrinkled surface
  • They are creamy-white initially, becoming yellowish or buff-colored on further incubation.
  • Growth is eugonic (grows more luxuriantly in culture).
  • The optimum temperature is 35-37°C and the optimum pH is 6.4 to 7.
  • It is an obligate aerobe.
  • Increased carbon dioxide (5-10%) enhances the growth of Mycobacterium tuberculosis.
  • 5% Glycerol also stimulates the growth.
  • LJ media contain inhibitors to keep contaminants from out-growing Mycobacterium tuberculosis.
  • The green color of the medium is due to the presence of malachite green which is one of the selective agents to prevent the growth of most other contaminants.
  • A faster result can now be obtained using the Middlebrook medium or BACTEC.

Other Culture Media used for MTB

Solid media

  1. Egg-based – Petragnini medium and Dorset medium
  2. Middlebrook 7H10 Agar
  3. Middlebrook 7H11 Agar
  4. Blood-based – Tarshis medium
  5. Serum based – Loeffler medium
  6. Potato based – Pawlowsky medium

Liquid media

  1. Dubos’ medium
  2. Middlebrook 7H9 Broth
  3. Proskauer and Beck’s medium
  4. Sula’s medium
  5. Sauton’s medium

Biochemical Characteristics of MTB

Sr #Biochemical TestResult
15% NaCl ToleranceNegative (-ve)
268°C Catalase TestNegative (-ve)
3Acid PhosphataseNegative (-ve)
4Amidase TestPositive (+ve)
5Arylsulphatase TestNegative (-ve)
6Growth on P-Nitrobenzoic AcidNegative (-ve)
7Growth on TCH (10mg/ml)Positive (+ve)
8Iron UptakeNegative (-ve)
9Neutral Red TestPositive (+ve)
10Nicain TestPositive (+ve)
11Nitrate Reduction TestPositive (+ve)
12Peroxidase TestPositive (+ve)
13Pyrazinamidase TestPositive (+ve)
14Semi-Quantitative Catalase TestNegative (-ve)
15Tellurite Reduction TestVariable
16Tween 80 HydrolysisNegative (-ve)
17Urease TestVariable

Laboratory diagnosis of MTB

Specimen and processing

  • sputum, bronchial washings, brushings or biopsies or early morning gastric aspirates, Cerebrospinal Fluid (CSF), urine
  • Specimens from sputum and other nonsterile sites should be liquefied with N-acetyl-L-cysteine decontaminated with NaOH (kills many other bacteria and fungi), neutralized with buffer, and concentrated by centrifugation.
  • Specimens from sterile sites, such as cerebrospinal fluid, do not need the decontamination procedure but can be directly centrifuged, examined, and cultured.

Direct Detection Methods

1. Microscopy

  • Detection of the acid-fast property of mycobacteria to detect them in sputum and other clinical material by the Ziehl–Neelsen (ZN) staining technique.
  • Red bacilli are seen against the contrasting background color.
  • Slender, straight, or slightly curved rods with a barrel or beaded appearance.
  • Fluorescence microscopy, based on the same principle of acid-fastness, is increasingly used and is much less tiring.

2. Culture

  • As sputum and certain other specimens frequently contain many bacteria and fungi that would rapidly overgrow any mycobacteria on the culture media, decontamination methods make use of the relatively high resistance of mycobacteria to acids and certain disinfectants.
  • The deposit is used to inoculate the Lowenstein Jensen (LJ) medium.
  • Specimens such as cerebrospinal fluid and tissue biopsies, which are unlikely to be contaminated, are inoculated directly onto culture media.
  • Dry, rough, raised, wrinkled, off white to buff-colored colonies on LJ medium, commonly called as rough, tough, and buff colonies.
  • The use of the liquid medium with radiometric growth detection such as BACTEC 460 TB has simplified the culture method.

3. Biochemical analysis

  • Niacin accumulation test: positive
  • Arylsulphatase test: positive
  • Neutral red test: positive
  • Catalase peroxidase test: peroxidase positive and weakly catalase-positive
  • Amidase test: positive
  • Nitrate reduction test: positive
  • Tween 80 hydrolysis: variable result
  • Susceptibility to pyrazinamide: sensitive
  • Susceptible to thiophene -2-carboxylic acid hydrazide (TCH): not susceptible

Molecular techniques

  • Subsequent to the introduction of commercially available hybridization assays, commercially available and inhouse– developed nucleic acid amplification tests were used successfully for early identification of M. tuberculosis complex grown in liquid cultures.
  • PCR-based sequencing for mycobacterial identification consists of PCR amplification of mycobacterial DNA with genus-specific primers and sequencing of the amplicons.
  • The organism is identified by comparison of the nucleotide sequence with reference sequences.
  • The most reliable sequence for the identification of mycobacteria is the approximately 1500 bp 16S rRNA gene.

Indirect detection methods

Tuberculin test

  • A purified protein derivative (PPD) is obtained by chemical fractionation of old tuberculin.
  • PPD is standardized in terms of its biologic reactivity as tuberculin units (TU).
  • A large amount of tuberculin injected into a hypersensitive host may give rise to severe local reactions and a flare-up of inflammation and necrosis at the main sites of infection (focal reactions).
  • For this reason, tuberculin tests in surveys use 5 TU in 0.1 mL solution; in persons suspected of extreme hypersensitivity.
  • After the tuberculin skin test is placed, the area is examined for the presence of induration no later than 72 hours after placement.

Treatment of tuberculosis

The first line of anti-TB agents that form the core of treatment regimens are

  • Isoniazid (INH)
  • Rifampin (RIF)
  • Pyrazinamide (PZA)
  • Ethambutol (EMB)
  • The current World Health Organization recommendations are that all new patients with tuberculosis, irrespective of site or severity of the disease, and in the absence of evidence of drug resistance, should receive a 6-month course of therapy, consisting of a 2-month intensive phase of rifampicin, isoniazid, pyrazinamide, and ethambutol followed by a 4-month phase of rifampicin and isoniazid.
  • Isoniazid may cause mild psychiatric disturbances and peripheral neuropathy, particularly in alcoholics, but these are usually preventable by prescribing pyridoxine (vitamin B6).
  • Resistance may develop during therapy (acquired resistance) with poor-quality drugs or inadequate supervision, or patients may be infected with resistant strains (initial or primary resistance).
  • Multidrug resistant strains are defined as those resistant to isoniazid and rifampicin, with or without resistance to additional drugs.
  • The newer category of extensively drug-resistant tuberculosis (XDR-TB) is defined as resistance to, at least, isoniazid, rifampicin, fluoroquinolone, and any injectable agent.

The second line drugs include

  • A later generation of fluoroquinolones such as moxifloxacin, levofloxacin, or gatifloxacin.
  • An injectable agent such as amikacin, kanamycin, or capreomycin.
  • Two or more core second-line agents include ethionamide, prothionamide, cycloserine, terizidone, clofazimine, or linezolid.

Prevention of tuberculosis

Human tuberculosis is preventable:

  • by the early detection and effective therapy of the open or infectious individuals in a community
  • by lowering the chance of infection by reducing overcrowding as the most important factors affecting the incidence of tuberculosis are socio-economic ones, particularly those leading to a reduction of overcrowding in homes and workplaces.
  • to a limited extent, by vaccination which includes BCG vaccination that is given by intracutaneous injection after the birth.
  • neonatal vaccination is recommended as prior exposure of the human population to environmental mycobacteria confer some protection, but in others induce inappropriate immune reactions that antagonize protection.


Possible References Used

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