Conventional Methods for LaboratoryConventional Methods for Laboratory
Diagnosis of TuberculosisDiagnosis of Tuberculosis
Rapid Methods:Rapid Methods:
Demonstration of acid fastDemonstration of acid fast
bacilli in smearbacilli in smear
Demonstration of antigensDemonstration of antigens
in sample eg.CSFin sample eg.CSF
Chemical testsChemical tests
Serum- acute phaseSerum- acute phase
Slower Methods:Slower Methods:
In vivo tests-In vivo tests-
- human(TT).- human(TT).
- Animal inoculation.- Animal inoculation.
- conventional L.J.- conventional L.J.
- Rapid slide culture.- Rapid slide culture.
Sample Collection.Sample Collection.
Early morning complete sample.Early morning complete sample.
Sputum-3 consecutive samples.Sputum-3 consecutive samples.
Urine-3 consecutive samples pooled.Urine-3 consecutive samples pooled.
Concentration/Decontamination for smear.Concentration/Decontamination for smear.
Transport medium-1% cetyl pyridiumTransport medium-1% cetyl pyridium
chloride in 2% saline.chloride in 2% saline.
Ziehl-Neelsen stain.Ziehl-Neelsen stain.
Acid fastness.Acid fastness.
Hot strong carbolHot strong carbol
decolourisation bydecolourisation by
strong mineral acids.strong mineral acids.
Myth of acid andMyth of acid and
alcohol fast.alcohol fast.
Fluorescent stainFluorescent stain
Auramine O andAuramine O and
Rhodamine B.Rhodamine B.
Calcofluor white.Calcofluor white.
Less eye strain.Less eye strain.
More expensive.More expensive.
Good for labs withGood for labs with
high workload.high workload.
least count 5000-10000 bacilli per ml.(Some books-least count 5000-10000 bacilli per ml.(Some books-
Species differentiation impossible.Species differentiation impossible.
Specimen contamination.Specimen contamination.
False positive.False positive.
Saprophytic mycobacteria.Saprophytic mycobacteria.
To use sterile containers/collection pots.To use sterile containers/collection pots.
Sterile reagents,slides and equipment.Sterile reagents,slides and equipment.
Do not use tap water forDo not use tap water for
staining(saprophytic mycobacteria).staining(saprophytic mycobacteria).
New slides only-fungal spores.New slides only-fungal spores.
In children gastric aspirates-centrifuged v/sIn children gastric aspirates-centrifuged v/s
Concentration methods forConcentration methods for
1% Sodium hypochlorite.1% Sodium hypochlorite.
Sputum treated with 2%-4% NaOH.Sputum treated with 2%-4% NaOH.
Urine,C.S.F.,fluids centrifuged and depositUrine,C.S.F.,fluids centrifuged and deposit
Treated and/ or stained.Treated and/ or stained.
Reporting Smears.Reporting Smears.
?+ : 1-3 bacilli in whole smear.?+ : 1-3 bacilli in whole smear.
+ : 3-9 bacilli per 100 fields.+ : 3-9 bacilli per 100 fields.
++ : 1-9 bacilli per 10 fields.++ : 1-9 bacilli per 10 fields.
+++ : 1-9 bacilli per field.+++ : 1-9 bacilli per field.
++++ : 10 or more bacilli per field.++++ : 10 or more bacilli per field.
CDC or I.U.A.T. classification.CDC or I.U.A.T. classification.
24-48 hour detection.24-48 hour detection. Selectively targetSelectively target
Genus /speciesGenus /species
Can identify drugCan identify drug
resistant strains.resistant strains.
Back in vogue as aBack in vogue as a
rapid diagnosticrapid diagnostic
Concentration methods forConcentration methods for
4% NaOH in equal amount –incubate at4% NaOH in equal amount –incubate at
37C with shaking-neutralise with HCl/wash37C with shaking-neutralise with HCl/wash
with dist. Water/inoculate on L.J.withwith dist. Water/inoculate on L.J.with
2% NaOH with-N acetyl L-cysteine.2% NaOH with-N acetyl L-cysteine.
-Sodium citrate.-Sodium citrate.
Urine samples-oxalic acid +/- SodiumUrine samples-oxalic acid +/- Sodium
citrate as urinary saprophytes are resistantcitrate as urinary saprophytes are resistant
to alkali.to alkali.
Strict aerobe.Strict aerobe.
Growth enhanced by 5-10% CO2.Growth enhanced by 5-10% CO2.
Slow grower : doubling time 18 hours.Slow grower : doubling time 18 hours.
Conventional Lowenstein Jensen medium.Conventional Lowenstein Jensen medium.
Egg yolk,mineral salts,malachite green.Egg yolk,mineral salts,malachite green.
Colonies visible in 2-6 weeks in case ofColonies visible in 2-6 weeks in case of
May be 8 weeks or more if pt. Has receivedMay be 8 weeks or more if pt. Has received
Culture -2Culture -2
Additives to Lowenstein-Jensen mediumAdditives to Lowenstein-Jensen medium
Glycerol-prevents drying of media.Glycerol-prevents drying of media.
Pyruvate-helpful for growth of M.bovis.Pyruvate-helpful for growth of M.bovis.
P-nitro benzoic acid-differentiate betweenP-nitro benzoic acid-differentiate between
typical and atypical mycobacteria.typical and atypical mycobacteria.
Antimicrobials-for susceptibility testing.Antimicrobials-for susceptibility testing.
Slow growers-M.xenopi,M.malmoense.Slow growers-M.xenopi,M.malmoense.
By increasing incubation time from 6 to 12By increasing incubation time from 6 to 12
weeks isolation rate increases 4.1% forweeks isolation rate increases 4.1% for
M.tuberculosis,10.5% for otherM.tuberculosis,10.5% for other
mycobacteria,and 70% for M.malmoense.mycobacteria,and 70% for M.malmoense.
-33C-skin lesions.-33C-skin lesions.
Sensitivity of culture 10-100 organisms/mL.Sensitivity of culture 10-100 organisms/mL.
Increasing culture success-2Increasing culture success-2
Biopsy-homogenised by grinding withBiopsy-homogenised by grinding with
sterile phosphate buffered saline and sand insterile phosphate buffered saline and sand in
Griffith tube or mortar and pestle.Griffith tube or mortar and pestle.
Fluid-citrated sample.(2 drops 2% SodiumFluid-citrated sample.(2 drops 2% Sodium
citrate per 10 mL sample.citrate per 10 mL sample.
Urine-membrane filtration / centrifugation.Urine-membrane filtration / centrifugation.
Animal InoculationAnimal Inoculation
Guinea pig or rabbit.Guinea pig or rabbit.
Animal house.Animal house.
Animal is sacrificed.Animal is sacrificed.
Time consuming.Time consuming.
Species identificationSpecies identification
not possible.not possible.
Culture is safer,Culture is safer,
simpler, cheaper,simpler, cheaper,
Antimicrobial Susceptibility TestingAntimicrobial Susceptibility Testing
Resistance ratio method.Resistance ratio method.
Proportion method.Proportion method.
Absolute concentration method.Absolute concentration method.
Diffusion method.Diffusion method.
Radiometric method.Radiometric method.
Resistance ratio:Resistance ratio:
– Standardised suspension of culture.Standardised suspension of culture.
– LJ media incorporating doubling dilutions ofLJ media incorporating doubling dilutions of
Drug concentration inhibiting test stDrug concentration inhibiting test st
Drug concn. Inhibiting reference st.Drug concn. Inhibiting reference st.
Proportion method:- measure proportion ofProportion method:- measure proportion of
bacteria growing on drug containing mediabacteria growing on drug containing media
compared to drug free media.compared to drug free media.
Colony count required (at least 18-20).Colony count required (at least 18-20).
Expensive,time consuming,quality control.Expensive,time consuming,quality control.
On treatment – first culture negative , then smear.On treatment – first culture negative , then smear.
BACILLUS CALMETTE GUERINBACILLUS CALMETTE GUERIN
BCG : avirulent strain with capacity toBCG : avirulent strain with capacity to
induce immune response and thus resistanceinduce immune response and thus resistance
to subsequent infection with virulentto subsequent infection with virulent
tubercle bacilli. This reduces morbidity andtubercle bacilli. This reduces morbidity and
mortality among those at risk.mortality among those at risk.
1921-25 : Oral vaccine.1921-25 : Oral vaccine.
1927 : intradermal vaccine.1927 : intradermal vaccine.
1948 : globally accepted.1948 : globally accepted.
Liquid / freeze dried.Liquid / freeze dried.
WHO: Danish 1331WHO: Danish 1331
In India at GuindyIn India at Guindy
Reconstituted vaccineReconstituted vaccine
stable 3hrs.protectedstable 3hrs.protected
from light.from light.
New born: 0.05ml.New born: 0.05ml.
Adult: 0.1ml.Adult: 0.1ml.
Protection 15-20 yrs.Protection 15-20 yrs.