Ghada Barakat

 They are obligate aerobe, rod-shaped, non-motile, non-spore forming, non- capsulated organisms.  Impermeable to various basic dyes, but once stained, resist decolorization.

 Mycobacteria are not classified as either Gram-positive or Gram-negative because they do not have the chemical characteristics of either, it stains very weakly Gram-positive or not at all.

 Mycobacterium tuberculosis complex: (M tuberculosis, M bovis, M africanum, M microtii) that cause tuberculosis in different hosts.  Non tuberculous Mycobacteria: which are classified according to their growth rate and pigmentation with and without exposure to light.  M. leprea: the causative agent of leprosy.

 M. tuberculosis is the etiologic agent of tuberculosis (TB) in humans.  Humans are the only reservoir for the bacterium.  It is a facultative intracellular organism, usually of macrophages.  The high lipids content in the cell wall of M. tuberculosis lead to impermeability to stains and dyes, resistance to many antibiotics resistance to acids and alkalis.

 Clinical specimens:  Sputum, bronchial or gastric washings.  At least 3 morning samples of sputum are required for diagnosis.

 Smear is prepared from sputum either direct or after liquefaction by N-acetyl –L cysteine.  Can be stained by carbol fuchsin (Ziehl- Neelsen) or fluorochrome stain.

Ziehl-Neelsen(ZN) stain:  Concentrated carbol fuchsin.  Sulphuric acid 20%.  Alcohol 95 %.  Methylene blue.

 By Ziehl-Neelsen(ZN) stain, acid-fast bacilli appear pink, small, straight or slightly curved, non motile, non sporuolated, occur singly, in pairs or in masses. pink in a contrasting blue background.

 By fluorochrome stain, the bacilli appear as bright yellow fluorescent against dark background.It is more sensitive than ZN stain.

 Direct smear is rapid, cheap and simple method for diagnosis but it is non sensitive, the sensitivity range is 25 to 75%.  At least ,000 bacilli per ml of sputum are needed to detect positive microscopic smear.  A positive stain and negative culture may be caused by non viable organisms, which can occur in persons receiving anti tuberculous medication

Cultural characters:  Obligate aerobe.  5 to 10% CO2, enhances growth.  Optimum temperature is 37 ºC (strict mesophile).  No growth below 30 ºC or above 39 ºC.  Very slow grower, may require 4 to 6 weeks to get visual colonies.  No growth on ordinary media.  Two media are used to grow M.TB  An egg based medium as Lowenstein-Jensen and Dorest egg media.  An agar based medium as Middlebrook's medium.

 First, Decontamination: the sputum sample is treated with NaOH.  This kills other contaminating bacteria but does not kill the MTB present because MTB is resistant to alkaline pH.

 Sputum is inoculated in selective medium containing antimicrobial agents as (Lowenstein-Jensen) as it is contaminated with normal bacterial flora.

 Cultures are incubated at 35°C to 37°C in an atmosphere of 5 to 10% CO2.  All cultures should be examined weekly for 4- 8 weeks.

Colonies on L-J medium are, raised, rough, confluent, grayish, and dry (eugenic growth). Culture is highly sensitive, specific.

 The more rapid broth systems.  The media used in the BACTEC system are broth media containing radio-labeled palmitic acid as the sole carbon source.

 As M.TB. multiplies, it utilizes the palmitic acid and release radio-labeled CO 2.  Using the BACTEC system, MTB growth can be detected in 9-16 days.

 Principle:  It is a cell-mediated hypersensitivity skin test.  Based on that individual previously infected with tubercle bacilli will develop hypersensitivity to proteins of M.TB. which develops 6-8 weeks after infection.

 Method:  The Mantoux test is the standard tuberculin test; it requires the intradermal injection of (0.1 ml) containing (5 tuberculin units) of PPD.  The injection is made into the anterior aspect of the forearm and read after 72 hours.

 Injected PPD (purified protein derivative = tuberculin) under skin  Wait hours to read test

 Reading of the test:  The arm is palpated to define an area of induration and the diameter of this induration, if present, is measured in mm transverse to the long axis of the forearm.

1) Positive reaction: (Induration >10mm) Individual has been previously exposed to M.TB. 2) False positive reaction: means positive reaction in absence of previous exposure to M.TB:  Infection with other mycobacteria.  Vaccination with BCG.

3) Negative reaction: Induration less than 5 mm. An area of surrounding erythema alone without induration Individual has never been previously exposed to M.TB The test may be repeated with higher doses of PPD e.g. 100 or 200 units

4) False negative reaction: False negatives means negative reaction in spite of previous exposure to M.TB.,e.g.  Persons with impaired CMI response as in patients with immune suppression – HIV, immunosuppressive drugs, sarcoidosis, renal failure, malignancy, malnutrition.  late stages of tuberculosis,  acute viral infections,

 DNA probes: R apid, specific and sensitive methods for identification of mycobacteria after sufficient growth is present on medium.  PCR: Is useful for direct detection of mycobacteria in clinical specimen within 24 hours or less. It is rapid, specific and sensitive.

1) Sample: early morning urine of at least 3 successive days. 2) Direct smear:  Centrifugation of urine and preparation of smear from the deposit.  Staining of the smear by Z-N stain, should be decolourized by both acid and alcohol to differentiate M.TB from M. smegmatis which is found normally in the genitalia and which is acid fast but non alcohol fast. 3) Culture: of deposit after decontamination on L-J medium.

1) Sample:  C.S.F is collected by lumbar puncture.  C.S.F is less turbid than that in meningococcal meningitis and contains excess of lymphocytes. 2) Direct smear: prepared from the deposit after centrifugation and stained with Z.N. stain. 3) Culture: the deposit is cultured directly on L-J medium or Dorest egg medium without decontamination.