Mycobacterium Flashcards
In M. tuberculosis lab diagnosis the diagnostic methods used
① demonstrating the bacilli by microscopy in the lesions
② isolating the bacilli in culture
③ molecular diagnostic methods: detect RNA or DNA of bacilli in clinical specimen.
④ hypersensitivity reaction to tuberculoprotien.
⑤ animal experiment: transmitting infection to experimental animals.
Why and what are methods used for decontamination concentration of specimens?
Decontamination.- as sputum can contain microorganisms other than Myco bacteria
Concentration- organic matrix in sputum that trap Mycobact
Petroffs method: sputum mix with 4% NaOh and shake, centrifuge, sediment is neutralised with N/10 HCl and used for culture etc.
NALC (N acetyl cysteine) with 2% NaOH: N acetyl cysteine used for sputum liquefaction and NaOh kills contaminating bacteria. Sample then neutralised and concentrated by centrifugation
Methods used in microscopy of M. Tuberculosis
Staining: need attest 10,000 bacilli per ml of sputum to demonstrate in direct smears
zeil Nelson technique: after conc of sputum smears are dried, heat fixedl and stained with zeil Nelson stain :
Add carbol fuschin & heat for 5-7 min.
Kinyouns modification of acid fast staining: modified cold method (no heating )
Increase conc of stain & duration of staining
Decolousise with 20% H 2 s04 & then by 95% Ethanol for 2 min
Countustains with methyl here blue, or malachite green etc for 1min.
Mycobact appear as bright red rods us blue background ( if methylene blue counter stain)
Fluorescent stains & difference btw M.tuber &-m.bovis
Auramine - rhodamine:
For several smears convenient
Smears stained with AR fluorescent dyes and seen under uv light
Bacilli seen as bright rods on dark background
M-boris stains as continuous uniformed stain not beaded
They are see n in Tapp water rubber tubes & hence can contaminate specimen.
Culture of mycobact tuber
Culture (gold standard & detect 10-100 bacilli per ml ):
Solid media: inoculate specimen into 2 l-j medium
Examine culture after 4 days of 37° incubation (look for rapid growth mycobact)
For 8-12 weeks examine twice a week for mycoba before saying negative.
A smear made from growth & stained by ZN method
Mycobact tuber- slow growing, niacin positive, non pigmented acids fast bacilli
Liquid media - middle brook, Dubos, might (mycobact growth indicater tube)
Automated systems- BACTEC MGIT,BacT/ALERT as grow rapidly than solid mediA
Molecular methods & immunodiagnons
Molecular :
PCR &LCR ( Ligase chain reaction)
Line probe assay (lpa)
GenXpert -catridge based nuclei’ acid amplification test detects bacelli & rifampin resistance.
Immoono:
Mantoux test: 0.1m1 (5IU) PPD injected intrademally on forearm of flexor aspect
If hypersensitive high only 1 IU.
Extra pulmon tb diagnosis
CSF of meningitis- PCR
B.M & liver biopsy & pus from abscess- culture & microscopy.
Pleural effusion- culture after centrifugation
Renal tb- urinary excretion is intermittent here, test 3-6 first whole voided mrng samples
vaccine for mycobact
BCG (bacille calmette- guerin): intradernal injection of live attenuated vaccine
M .bovis strain is, attenuated by serial subcultures in glycerine bile potato medium for over 13 yrs.
Advantages & disadvantages ofBCG
Disadvantage:
Local: Abcess, keloid, lupoid lesions, indolent ulcers.
Regional: enlargement & suppurations of draining lymph nodes
General: fever, mediastinal adenitis, erythema nodosum
Advantages:
Induces non specific stimulation of immune system hence protection to Leekemia & leprosy. Adjunctive therapy in some malignancies
Restoration of cellular immunity is immunodefecient patients by transfer factor.
Non tuberculous mycobact definition
Mycobact other than mammalian tubercle bacilli which cause occasional disease in humans resembling tuberculosis.
Non tuberculous mycoba runyoun classification
Group1 (photochromogens):M. Kansasii pulmo disease like tb,M.marinum (swimming pool or fish tank granuloma) Group 2 (scotochromogens):M.scrofulaceum
