Mycobacterium.pdf

Veterinary Microbiology and Mycology Practical part by Dr. Sameh Abuseir 22.10.2020 Specimens Specimens from live animals include aspirates from cavities, lymph nodes, biopsies tracheobronchial lavages and the centrifuged deposit from about 50 ml of milk in the case of suspected tuberculous mastitis...

Veterinary Microbiology and Mycology Practical part by Dr. Sameh Abuseir 22.10.2020 Specimens Specimens from live animals include aspirates from cavities, lymph nodes, biopsies tracheobronchial lavages and the centrifuged deposit from about 50 ml of milk in the case of suspected tuberculous mastitis. With dead animals, fresh and fixed (in 10% formalin for histopathology,) samples of lesions or a selection of lymph nodes from a tuberculin-reactor with no visible lesions are collected. Direct microscopy The Ziehl-Neelsen (acid-fast stain) is used to stain smears from lesions and other specimens. The mycobacteria appear as slender, often beaded, red-staining rods against a blue background (if methylene blue is the counterstain). These can be visualized only if at least 5X104 mycobacteria/ml of material is present. The numbers of M. bovis are often low in bovine specimens, but avian tuberculosis in poultry and M. bovis lesions in animals such as deer usually yield large numbers of mycobacteria. Smears stained by fluorescent dyes. such as auramine, acridine orange or fluorochrome, can be examined under a UV microscope ► mycobacteria seen more easily if relatively small numbers are present. Colonial morphology The luxuriant growth of M. tuberculosis on glycerolcontaining media, giving the characteristic ‘rough, tough and buff colonies” is known as eugonic. The growth of M. avium on media containing glycerol is also described as eugonic. M. bovis has sparse, thin growth on glycerolcontaining media that is called dysgonic. M. bovis, however, grows well on pyruvate containing media without glycerol. Pigment production response to light and The mycobacteria that produce yellowish-orange carotenoid pigments are called chromogenic. The term photochromogenic is applied to those mycobacteria that produce pigment only if exposed to light. The seotochromogenic mycobacteria produce pigment when incubated either in the light or in the dark. One procedure for isolation and cultivation from clinical material, such as nodules, is, briefly, as follows: 1. Trim fat off tissues and add 4% NaOH to digest tissue and kill contaminating bacteria. 2. Grind tissue in a sterile mortar with sterile sand, centrifuge, then add HCl to neutralize sediment. 3. Inoculate sediment onto Lowenstein-Jensen slants and egg yolk agar slants and incubate at 37°C for up to 8 weeks. 4- Stain smears from growth if the latter is characteristic of mycobacteria; identity if organisms are acid-fast. Identification is based principally on cultural, morphologic, and biochemical characteristics. This phenotypic approach is complex and timeconsuming and is being replaced by the use of specific probes. Definitive identification is usually carried out in a reference laboratory. Identification based on phenotypic characteristics can take more than a week for rapid growers and several weeks to months for slow growers. Cultural characteristics of the "classic" species of mycobacteria. To identify mycobacteria from clinical specimens in a relatively shorter period of time, commercially available, species-specific isotopic and (DNA) probes are available. Acid fast stain Purpose: to differentiate between acid-fast and non acid-fast bacteria. Principle: – some bacteria contain a waxy lipid, mycolic acid, in there cell wall. This lipid makes the cells more durable and is commonly associated with pathogens. – Acid fast cell walls are so durable that the stain (carbol fuchsin) must be driven into the cells with heat. The cells are then decolorized with acid-alcohol, all other cells will decolorize with this strong solvent, but acid fast bacteria will not. – Other cells are then counterstained with methylene blue. To the right, a mixed culture has been acid-fast stained. A. Non Acid-fast bacteria B. Acid-fast bacteria From the abscess: – make Gram-stain from the given specimes. Make Ziehl Neelsen Stain from the given plate of bacteria: