LICEO DE CAGAYAN UNIVERSITY

COLLEGE OF MEDICAL LABORATORY SCIENCE

CLINICAL BACTERIOLOGY LABORATORY

MYCOBACTERIA

PRESENTED BY:
JOHN PATRICK B. TUDAS, RMT
MYCOBACTERIA
• IDENTIFICATION:
1. RATE OF GROWTH
2. COLONY MORPHOLOGY
3. PIGMENTATION
4. NUTRITIONAL REQUIREMENT
5. OPTIMAL INCUBATION TEMPERATURE
6. BIOCHEMICAL TEST RESULTS
LABORATORY SAFETY CONSIDERATION
1. PERSONNEL SAFETY
• PROVIDED WITH ADEQUATE SAFETY EQUIPMENT
• TRAINED IN SAFE LABORATORY PROCEDURES
• INFORMED OF THE HAZARDS ASSOCIATED WITH
THE PROCEDURES
• PREPARED FOR ACTION FOLLOWING AN
UNEXPECTED ACCIDENT
• MONITORED REGULARYLY BY MEDICAL
PERSONNEL.
LABORATORY SAFETY CONSIDERATION

2. VENTILATION
3. BIOLOGICAL SAFETY CABINET
4. USE OF PROPER DISINFECTANT
SPECIMEN COLLECTION
• STERILE, WIDE-
MOUTHED CUP
WITH A TIGHTLY
FITTED LID.
DIGESTION AND DECONTAMINATION
1. TO LIQUEFY THE SAMPLE THROUGH
DIGESTION OF THE PROTEINACEOUS
MATERIAL
2. TO ALLOW THE CHEMICAL
DECONTAMINATING AGENT TO
CONTACT AND KILL THE
NONMYCOBACTERIAL ORGANISMS.
DIGESTION AND DECONTAMINATION
• FACTORS: CHEMICAL AGENT, EXPOSURE
TIME AND TEMPERATURE.
1. SODIUM HYDROXIDE
2. N-ACETYL-L-CYSTEINE
3. BENZAALKONIUM CHLORIDE
4. OXALIC ACID
CONCENTRATION PROCEDURE
• SG: 0.79 TO 1.07
• CONCENTRATION = CENTRIFUGATION
(3,000 X G)
STAINING FOR ACID-FAST BACILLI
• ZIEHL-NEELSEN AND KINYOUN
• AURAMINE STAIN (AURAMINE-
RHODAMINE FLUOROCHROME STAIN)
CULTURE MEDIA
CULTURE MEDIA
BIOCHEMICAL TEST
1. NIACIN ACCUMULATION
2. NITRATE REDUCTION
3. CATALASE
4. HYDROLYSIS OF TWEEN 80
5. IRON UPTAKE
6. ARYLSYLFATASE
7. PYRAZINAMIDASE
8. TELLURITE REDUCTION
9. UREASE
BIOCHEMICAL TEST
1. INHIBITORY TESTS
• THIOPHENE-2-CARBOOXYLIC ACID
HYDRAZIDE
• SODIUM CHLORIDE TOLERANCE
• GROWTH ON MACCONKEY AGAR
ADVANCED TESTING
1. CHROMATOGRAPHY
2. HYBRIDIZATION AND NUCLEIC ACID
AMPLIFICATION
3. MASS SPECTROMETRY
MYCOBACTERIA
• ACID-FAST BACILLI
• NON-MOTILE, NON-SPORE FORMING AND NON-
ENCAPSULATED.
• GROWTH PATTERN (RAPID AND SLOW GROWTH)
• MICROSCOPY: SLENDER, SLIGHTLY CURVED OR
STRAIGHT RODS THAT HAVE THE TENDENCY TO
“CLUMP”.
MYCOBACTERIA
• CULTURE: EGG-BASED AGAR – “FRIABLE
GROWTH”
• 5 – 10 % CO2
• 6.5 – 6.8 pH
• GENERATION TIME: > 12 HRS.
• 2 GROUPS:
• Mycobacterium tuberculosis Complex (MTC)
• Non-Tuberculosis Mycobacteria (NTM)
MYCOBACTERIUM TUBERCULOSIS
COMPLEX
MYCOBACTERIUM TUBERCULOSIS
• KOCH’S BACILLUS
• CULTURES:
• SLOW-GROWING EXHIBITS BUFF COLOR
• RAISED AND DRY
• “CAULIFLOWER-LIKE” APPEARANCE
• ROUGH COLONIES EXHIBITS CORDING.
• BIOCHEMICAL TEST: (+) NIACIN AND NITRATE
REDUCTION, (-) T2H INHIBITION TEST AND 68C
CATALASE TEST, (+) PYRAZINAMIDASE
MYCOBACTERIUM BOVIS
• ATTENUATED STRAINS (BACILLUS-CALMETTE-
GUERIN/ BCG VACCINE)
• CULTURE:
• SLOW-GROWING, SMALL, GRANULAR,
ROUNDED AND NON-PIGMENTED
• BIOCHEMICAL TEST: (-) NIACIN AND NITRATE
REDUCTION, (+) T2H INHIBITION TEST, (-) 68C
CATALASE TEST AND PYRAZINAMDASE
MYCOBACTERIUM AFRICANUM
• SPOLIGOTYPING (SPACER OLIGOTYPING)
MYCOBACTERIUM CANETTI
• SMOOTH STRAIN OF M. TUBERCULOSIS
• ISOLATED FROM PATIENT WITH AIDS
• CULTURE:
• RAPID GROWING COLONIES
• BIOCHEMICAL TESTING: (+) NIACIN AND
NITRATE REDUCTION
MYCOBACTERIUM MICROTI
• ISOLATED FROM TB PATIENTS IN BOTH
IMMUNOCOMPETENT AND
IMMUNOCOMPROMISED INDIVIDUALS.
NON-TUBERCULOUS
MYCOBACTERIA
MYCOBACTERIUM AVIUM COMPLEX
• MICROSCOPY: PLEOMORPHIC, SHORT.
COCCOBACILLI WITHOUT BIDDING; (+) PERIODIC
ACID SCHIFF STAIN.
• BIOCHEMICAL TESTING: (+) HEAT STABLE
CATALASE AND (-) T2H INHIBITION TEST
• SPECIES: M. AVIUM, M.INTRACELLULARE, M.
AVIUM SUBSP. PARATUBERCULOSIS AND M.
AVIUM SUBSP. SILVATICUM.
MYCOBACTERIUM KANSASII
• “YELLOW BACILLUS”
• MICROSCOPY: LONG RODS WITH DISTINCT
CROSSBANDING
• CULTURE:
1. MB 7H10 – SMOOTH TO ROUGH, DARK
CENTERS AND WAXY EDGES
2. PHOTOCHROMOGENIC (DARK RED
CRYSTALS) – 10-B-CAROTENE
MYCOBACTERIUM KANSASII
• BIOCHEMICAL TEST: (+) TWEEN 80 HYDROLYSIS
(RAPID) AND NITRATE REDUCTION; (-)
PYRAZINAMIDASE
MYCOBACTERIUM MARINUM
• “SWIMMING POOL GRANULOMA”
• MICROSCOPY: LONG RODS WITH CROSS-BARRING
• CULTURE: COLONIES ARE SMOOTH TO ROUGH,
WRINKLED AND YELLOW
(PHOTOCHROMOGENIC)
• BIOCHEMICAL TESTING: (+) TWEEN 80
HYDROYSIS, UREASE AND PYRAZIMIDASE
MYCOBACTERIUM ULCERANS
• “BURULI ULCERS”
• MICROSCOPY: MODERATELY LONG RODS
WITHOUT CROSS-BANDING
• CULTURE: SMOOTH, ROUGH, AND NON-
PIGMENTED
• BIOCHEMICAL TESTING: (+) HEAT STABLE
CATALASE
MYCOBACTERIUM GORDONAE
• “TAP WATER BACILLUS”
• CULTURE: SMOOTH AND YELLOWISH-ORANGE
(SCOTOCHROMOGEN)
• BIOCHEMICAL TEST: (+) TWEEN 80 HYDROLYSIS
AND HEAT STABLE CATALASE; (-) NITRATE
REDUCTION
MYCOBACTERIUM XENOPI
• MICROSCOPY: LONG AND FILAMENTOUS RODS
• BIOCHEMICAL TEST: HEAT-STABLE CATALASE,
PYRAZINAMIDASE AND ARYLSULFATASE
• GROWTH TEMPERATURE: 42C
• CULTURE:
1. MB 7H10 – SMALL AND FILAMENTOUS
EDGES
2. CORNMEAL GLYCEROL AGAR - ROUND
WITH BRANCHING FILAMENTS
MYCOBACTERIUM TERRAE COMPLEX
• SAPROPHYTES
• MICROSCOPY: SHORT TO MEDIUM
COCCOBACILLI
• SPECIES: M. TERRAE, M. TRIVIALE AND M.
NONCHROMOGENICUM
• BIOCHEMICAL TESTS: (+) TWEEN 80 HYDROLYSIS
AND HEAT-STABLE CATALASE; (+) GROWTH IN 5%
NACL FOR M. TRIVALE
 NON-CULTIVABLE
NON-TUBERCULOUS
MYCOBACTERIA
MYCOBACTERIUM LEPRAE
• “HANSEN’S BACIILLUS”
• MICROSCOPY: ROD-SHAPED AND EXHIBITS
“CIGAR-SHAPED” OR “POCKET-FENCE”
ARRANGEMENT
• CULTURE: COLONIES GROWTH IN LIVING TISSUES
OF THE FOOTPADS OF MICE AND ARMADILLOS
• OPTIMAL GROWTH: 30 C
• SKIN TEST: FERNANDEZ AND MITZUDA REACTION
LICEO DE CAGAYAN UNIVERSITY
COLLEGE OF MEDICAL LABORATORY SCIENCE
CLINICAL BACTERIOLOGY LABORATORY

END OF
DISCUSSION

PRESENTED BY:
JOHN PATRICK B. TUDAS, RMT