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MYCOBACTERIA. Dr. Sudheer Kher. Acid Fast Ziehl-Neelsen Stain Mycolic acids Tuberculosis (Koch’s disease) M. tuberculosis M. bovis M. leprae Tubercle Lowenstein Jensen medium. PPD Tuberculin BCG Polymerase chain reaction Runyon groups. KEYWORDS. Tubercle bacilli Human – MTB

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Dr. Sudheer Kher

Acid Fast

Ziehl-Neelsen Stain

Mycolic acids

Tuberculosis (Koch’s disease)

M. tuberculosis

M. bovis

M. leprae


Lowenstein Jensen medium




Polymerase chain reaction

Runyon groups

classification of mycobacteria
Tubercle bacilli

Human – MTB

Bovine – M. bovis

Murine – M. microti

Avian – M. avium

Cold blooded – M. marinum

Lepra bacilli

Human – M. leprae

Rat – M. leprae murium

Mycobacteria causing skin ulcers

M. ulcerans

M. belnei

Atypical Mycobacteria (Runyon Groups)




Rapid growers

Johne’s bacillus

M. paratuberculosis

Saprophytic mycobacteria

M. butyricum

M. phlei

M. stercoralis

M. smegmatis


Classification of Mycobacteria

(TB, Consumption, Koch’s Disease)

  • M. tuberculosis
  • major human disease
    • healthy people
  • problems
      • association with AIDS
      • multiple drug-resistance
      • Chronic disease
      • Prolonged treatment
general characters of the genus
Slender rods

Resist staining but once stained, resist decolorization by dilute mineral acids; hence called ACID FAST BACILLI (AFB)

Aerobic, Non-motile, Non-sporing, Non-capsulated.

Growth generally slow

Genus includes

Obligate parasites

Opportunist pathogens


General characters of the genus
mycobacterium tuberculosis
Mycobacterium tuberculosis
  • One of the most serious infectious diseases in the developing world
  • One third of world’s population infected with M. tuberculosis
  • Thirty million people have active disease
  • Nine million new cases occur
  • Three million people die of the disease, each year.
mycobacterium tuberculosis mtb
Mycobacterium tuberculosis (MTB)
  • Morphology –
    • Ziehl – Neelsen stain – Once stained by Carbol fuchsin, resist decolorization by 20% Sulphuric acide and absolute alcohol. Acid & Alcohol Fast (AFB)
    • Fluorescent dyes like Auramine O or Rhodamine also stain and the decolorization is resisted.
    • Reason for Acid & Alcohol fastness –
      • Presence of unsaponifiable wax Mycolic acid
      • Semi permeable membrane around the cell
      • Property of cell wall and related to integrity of the cell wall
    • Staining may be uniform or granular
mtb cultural characters
Grow slowly. Generation time 14-15 hrs

Colonies appear after 2 weeks or at 6-8 weeks

MTB - Obligate aerobe

MTB grows more luxuriantly (eugonic) than M. bovis (dysgonic).

Addition of 0.5% Glycerol supports growth of human strains. No effect or inhibitory effect on bovine strains.

MTB : Cultural characters
mtb culture media
Solid media –

Egg containing

Lowenstein-Jensen Medium

Petragnini medium


Blood containing –


Serum containing –

Loeffler’s serum slope

Potato containing –


Liquid media –



Proskauer & Beck’s



MTB : Culture media
  • Not specifically resistant to heat. 60 C x 20 min destroys.
  • In sputum can survive 20-30 hrs
  • Relatively resistant to disinfectants. Survives exposure to
    • 5 % Phenol
    • 15 % Sulphuric acid
    • 3 % Nitric acid
    • 5 % Oxalic acid
    • 4 % NaOH
biochemical reactions
Biochemical reactions
  • Niacin test – Human MTB produces niacin when grown in egg medium.
  • Aryl Sulphatase test – Enzyme Aryl sulphatase formed by only atypical mycobacteria.
biochemical reactions1
Biochemical reactions
  • Neutral red test – Virulent strains of tubercle bacilli bind neutral red in alkaline solution while avirulent strains can not.
  • Catalase–paroxidase test – Most atypical mycobacteria are strongly catalase positive while MTB is only weakly positive. MTB is strongly peroxidase positive while atypical mycobacteria are negative.
  • Nitrate reduction test – Positive in MTB and negative in M. bovis
mycobacterium tuberculosis mtb1
Morphology –

Straight or slightly curved rods

Mycobacterium tuberculosis (MTB)
Modes of infection

1- Droplet infection

Person to person by inhalation aerosols

Mycobacterium tuberculosis (Pulmonary tuberculosis)

2- Ingestion of milk

Infected cattle

Mycobacterium bovis (Intestinal tuberculosis)

3- Contamination of abrasion

Laboratory workers (Skin infection)

pathogenesis of tuberculosis
Pathogenesis of tuberculosis
  • infects lung
  • distributed within macrophages
  • facultative intracellular pathogen
      • inhibits phagosome-lysosome fusion
      • resists lysosomal enzymes
Other minor pathogenesis factors
  • tuberculosis
  • mycobactin
    • siderophore
  • cord factor
    • damages mitochondria
cell mediated immunity tuberculosis
Cell-mediated immunity -tuberculosis
  • infiltration
    • macrophages
    • lymphocytes
  • granulomas
  • tubercles
Classical tubercular lesion – Granuloma with typical Langhan’s giant cells, epithelioid cells, lymphocytes and fibrosis.
There are multiple light areas (opacities) of varying size that run together (coalesce). Arrows indicate the location of cavities within these light areas. The appearance is typical for chronic pulmonary tuberculosis.

Clinical picture :

* Low grade fever

* Weight loss

* Night sweats

* Fatigue

* Cough & haemoptysis

laboratory diagnosis m tuberculosis
Laboratory diagnosis M. tuberculosis
  • Acid fast bacteria in sputum
  • Culture on L J media
  • Biochemical identification
  • Antibiotic sensitivity test
  • Tuberculin test
  • PCR
Lowenstein Jensen Medium –

Selective. Always in screw capped bottle. Bluish Green.

Contains – Egg protein – Solidifying agent

Mineral salts – Mg sulphate, Mg citrate


Malachite Green – Selective agent

Sterilized by - Inspissation

laboratory diagnosis tuberculosis
Laboratory diagnosis - tuberculosis
  • skin testing
    • delayed hypersensitivity
    • tuberculin
    • protein purified derivative, PPD
  • X-ray
tuberculin test mantoux test
Tuberculin Test (Mantoux test)
  • Delayed hypersensitivity skin test to assay: cell mediated immunity to tubercle bacillius Material: A purified protein derivative (PPD)Dose : 0.1 ml of (PPD) is injected intradermalReading : Positive test is defined as - Induration equal or greater than 10 mm - Develop 48-72 hours after injection
positive skin test tuberculosis
Positive skin test -tuberculosis
  • indicates exposure to organism
  • does not indicate active disease
tuberculin test
Tuberculin Test


* A positive test indicates previous exposure and carriage of T.B.

* A negative tuberculin test excludes infection in suspected persons

* Tuberculin positive persons may develop reactivation type of T.B.

* Tuberculin negative persons are at risk of gaining new infection

* False positive reactions are mainly due to:

- Infection with nontuberculous mycobacteria

* False negative reactions may be due to:

- Sever tuberculosis infection (Miliary T.B.) - Hodgkin’s disease

- Corticosteroid therapy - Malnutrition - AIDS

* Children below 5 years of age with no exposure history:

- Positive test must be regarded suspicious

laboratory diagnosis
Laboratory Diagnosis
  • Demonstration of bacilli
  • Culture & isolation or Animal inoculation
  • Demonstration of hypersensitivity to tubercular protein
  • Serological tests limited value


* According to site of infection :

- Sputum - Urine - Body fluids

- Gastric lavage - Blood - Tissue biobsy

* Specimens need appropriate processing

Liquefaction with N-acetyl-L- cysteine

Sputum Decontamination with NaOH


laboratory diagnosis1
Laboratory Diagnosis
  • Pulmonary TB –
    • Specimen –
      • Sputum – Early morning, if scanty 24 hrs, three consecutive day samples. Laryngeal swabs or gastric lavage in children.
    • Microscopy –See at least 100 field / 10 minutes.
      • Grading –
        • 1+ -> 3-9 bacilli in entire smear
        • 2+ -> 10 or more in entire smear
        • 3+ -> 10 or more bacilli seen in most oil immersion fields
    • Culture & AST
    • Animal inoculation – Animal of choice Guinea Pig.
laboratory diagnosis2
Laboratory Diagnosis
  • Extra -Pulmonary TB –
    • Specimen –
      • CSF – in suspected meningitis
      • Pleural fluid & other exudates
      • 2-3 days urine in renal TB
      • Biopsy material.
concentration methods
Concentration methods
  • Purpose – Homogenization & Concentration in sputum & other specimens.
  • Methods –
concentration methods1
Concentration methods
  • Useful for microscopy, culture & animal inoculation
    • Petroff’s method
      • Most widely used
      • Equal volumes of Sputum + 4% NaOH incubated at 37C X 20 min.Centrifuge at 3000 rpm X 30 min. Sediment neutralized by N/10 HCl.
      • Can be used for smear, culture, animal inoculation
    • Simpler method
      • To avoid centrifugation & Neutralization
      • Equal volumes of sputum + solution of Cetrimoniom bromide 20 g + NaOH 40 g in one litre. Allow to stand for five minutes. Inoculate Acid Buffered LJ Medium with swab.
laboratory diagnosis m tuberculosis culture
Laboratory diagnosis M. tuberculosis (culture)
  • grows very slowly
    • several weeks
    • non-pigmented colonies
    • niacin production
      • differentiates from other mycobacteria
  • polymerase chain amplification
    • rapid diagnosis


* Inhalation of tubercle bacilli

* They multiply in the alveolar macrophages

* An early tubercle (granuloma) is formed



* Lesions, healing or progression of infection depend upon

1- Dose of infecting mycobacteria

2- Resistance and hypersensitivity of host

* Virulence :

Glycolipids on the outer surface of bacteria

- Enhance granuloma formation

- Inhibit migration of polymorphnuclear leucocytes

- Help survival of tubercle bacilli inside macrophages



A- Primary infection:

* An exudative lesion :

- spread to regional lymph nodes

- A scar of healing may later calcify (Ghon’ focus)

- Lymph nodes caseate and then calcify

- Bacilli in the lesion slowly die

- Tuberculin test becomes positive

- The person immune & hypersensitive



B- Reactivation type :

* Activation of tubercle bacilli due to immunity

* Formation of tubercles that caseate

- fibrosis

- open into a bronchus (open tuberculosis)

* Tubercle bacilli erode a blood vessels

- Infect any organ (Miliary T.B.)

  • Chemoprophylaxis – INH for one year
  • Domicilliary treatment preferred
  • Drugs –
    • Rifampicin
    • Isoniazide Bactericidal
    • Pyrazinamide
    • Streptomycin
    • Ethambutol
    • Ethionamide
    • Thiacetazone Bacteriostatic
    • Paraminosalicylic acid
    • Cycloserine
  • Short term chemotherapy of six months is sufficient
  • Problem area – Development of resistance by mutant selection
    • Solution – Treatment by two to three drug combination, adequate treatment.

Use multiple drug therapy to prevent emergence of resistant mutants

* Long duration treatment (6-18 months)

* Four drugs are usually started in initial therapy due to:

- Intracellular location of bacilli

- Slow growth rate of bacilli

- Caseous material blocks penetration of drugs

- Some bacilli persist in a metabolically inactive state

* Sputum becomes non-infective 2-3 weeks after starting therapy


Drugs used :

1- First line drugs :

- Isoniazid - Rifampicin - Pyrazinamide

- Ethambutol - Streptomycin

2- Second line drugs (more toxic and less effective):

- Kanamycin - capreomycin - Cycloserin

- ethionamide - ciprofloxacin - Ofloxacin

* Noncompliance (failure to complete the course):

Directly observed therapy (DOT)

Health care workers observe the medication

immuno prophylaxis
  • Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG).
  • The strain causes self limited lesion and induces hypersensitivity & immunity.
  • Coverts tuberculin negative person to positive reactor.
  • Immunity lasts for 10-15 years. Immunity 60-80%
  • Given at birth without tuberculin testing
  • Protects against TB, the disease runs milder course in protected, prevents skeletal, meningeal & miliary forms.
  • Also found useful in leprosy, leukaemias and other malignancies by non-specific stimulation of RE system.