Professional Documents
Culture Documents
Objectives
1. Isolate, identify and analyze the bacteria that cause disease in humans.
2. Prediction and interpretation of antimicrobial susceptibility patterns
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c. Radiation
X rays, gamma rays and UV disposables: syringes, catheters or gloves
2. Chemical Methods
a. Antiseptics b. Disinfectant (Sterilizers)
Type Agents Type Agents
Alcohol (50%-70%) ethanol, isopropanol Aldehydes formaldeyde (1-8%), glutaraldehyde (2%)
Halogens iodophors Halogen chlorine and chlorine compounds (Bleach)
Heavy Metals AgNO3 & HgCl2 Detergents quaternary ammonium compounds
Phenolics chlorhexidine, triclosan Phenolics hexachlorophene
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b. Biosafety Level-2 - For handling common or likely encountered pathogens in a routine clinical laboratory. Use BSC
I or II. Trained personnel, presence of biosafety manual and sharps containers.
c. Biosafety Level-3 - For handling organisms that can be transmitted by aerosols. Controlled access. Ducted air
ventilation, special lab clothing and personal respirator.
d. Biosafety Level-4. Research facilities handling exotic viruses and potential bioterrorist agents. Personnel and all
materials are decontaminated before leaving the facility. Non Circulating ventilation system. Maximum
containment and use of class II or III BSCs.
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D. Microscopes
Microscope Magnification Application
Bright Field 10-1000 Stained cells
Dark Field 10-400 For cells not readily stained
Phase-contrast 10-400 Living or unstained cells
Fluorescence 10-400 Cells stained w/ fluorochromes
Review Questions
1. All of the following sites contain normal flora, EXCEPT:
A) Trachea B) Urethra C) Small intestine D) Groin area
2. The biosafety level practice for handling blood samples suspected of containing HIV and HBV:
A) BSL I B) BSL II C) BSL III D) BSL IV
3. Which of the following specimen preparation procedure is commonly applied to pus discharge?
A) Homogenization B) Concentration C) Decontamination D) None of these
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A. Introduction
1. To grow (cultivate) and isolate all bacteria in the specimen
2. To determine which bacteria that grow are most likely causing the infection
3. To obtain sufficient growth of clinically important bacteria and allow identification
B. Nutritional Requirements
1. Types of Bacteria by Nutrient Requirements
a. Fastidious - Complex nutritional requirement b. Non fastidious - Basic nutritional requirement
2. Media Classifications
a. Supportive (general isolation) media - support growth of most non fastidious organisms
b. Enriched (nonselective) media - supplement added to supportive media for growth of fastidious microbes
i. Sheeps Blood Agar - trypticase soy agar w/ 5% sheep's blood
Enriched & differential. Determines hemolytic patterns
Beta- complete clearing of RBC, Alpha - greenish discoloration around the colony, Gama - no effect
ii. Chocolate Agar - blood are lysed when added to molten base releasing hemin & NAD
Can support for N. gonorrhoeae & Haemophilus spp.
c. Enrichment Broth - permits growth of certain bacteria while inhibitory to others
d. Selective Media - permits growth of certain bacteria while inhibitory to others.
e. Differential Media - provides a distinct cultural appearance of microorganisms.
f. Antibiotic Media - Selective for a certain group of bacteria through addition of antibiotics
g. Back-up Broth - Broth w/ agar (0.075% ) & thioglycolic acid (reducing agent) creating anaerobiosis
C. Environmental Requirements
1. O2 and CO2 Availability
a. Obligate aerobe - requires oxygen for growth
b. Facultative anaerobe - can grow either with or without oxygen
c. Obligate anaerobe - cannot grow in the presence of oxygen
d. Aerotolerant anaerobe - can survive in the presence of oxygen but do not use oxygen for metabolism
e. Microaerophile - requires a reduced level of oxygen for growth
f. Capnophilic - requires extra carbon dioxide (5% to 10%)
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B. Staphylococcus epidermidis
1. Virulence Factor: Exopolysaccharide or biofilms
2. Infections: Hospital acquired UTI, prosthetic valve endocarditis
C. Staphylococcus saprophyticus
1. Virulence Factor: Adherent to Urogenital tract epithelium
2. Infections: UTI in sexually active young females and in older women with indwelling catheters
D. Staphylococcus lugdunensis; E. Staphylococcus haemolyticus
III. Laboratory Diagnosis
A. Isolation
1. Specimen: Aspirate or Swabs
2. Culture Media:
a. Sheeps Blood Agar (SBA): Enriched and Differential (5% Sheeps blood)
S. aureus: Medium to large. Pigmented yellow. -hemolytic
S. epidermidis: Small to medium. Gray-white. -hemolytic
S. saprophyticus: Small to medium. White-yellow or orange. -hemolytic
b. Mannitol Salt Agar (MSA): Selective (7.5% NaCl) and Differential (D-mannitol, phenol red)
S. aureus: Growth w/ fermentation (yellow halos)
S. epidermidis: Growth w/o fermentation (remains pink )
c. Laboratory Diagnosis
Organism Slide Coagulase Tube Coagulase DNase MSA Fermentation Novobiocin (5 g)
S. aureus + + + +
S. epidermidis S
S. saprophyticus R
a. Coagulase Test- Test for the ability to convert fibrinogen into fibrin. Differentiate S. aureus from Coagulase (-)
Staphylococci. 2 types: Bound and Free coagulase
a1. Coagulase Slide Test- D .
Positive (+): Macroscopic clumping
Negative (-): No clumping;
a2. Coagulase Tube Test - Detects free coagulase.
Positive (+): Clot of any size
Negative (-): No clot
b. DNase Test - Test for DNA hydrolysis.
Positive (+): clear zone
Negative (-): No clearing
c. Novobiocin Susceptibility - Test for susceptibility to 5 g Novobiocin.
Susceptible(S): zone diameter >16 mm
Resistant (R):
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B. Bacillus cereus
1. Virulence Factor and Clinical Infections:
Characteristics a) Diarrheal toxin b) Emetic toxin
Incubation period 8-16 hrs 1-5 hrs
Symptoms Diarrhea Vomiting
Duration of illness 24 hrs 9 hrs
Food Implicated Meat producers Fried & Boiled rice
Stability to heat Negative Positive
2. Laboratory Diagnosis:
a. Microscopy: Large, G+ bacilli
b. Colony: Large, feathery, spreading, w
c. Identification: Lecithinase Test, Motility test, Penicillin Susceptibility, Presensence of Parasporal crystals
Organism Lecithinase Motility Penicillin Sensitivity Parasporal Crystals
B. anthracis (+) (-) (+) (-)
B. cereus (+) (+) (-) (-)
B. thuringiensis (+) (+) (-) (+)
B. mycoides (+) (-) (-) (-)
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F. Identification
Blood MTM, ML, Superoxol
Organisms Glucose Maltose Lactose DNase / TH
Agar NYC (30% H2O2 )
N. gonorrhoeae (-) (+) (+) (+) (-) (-) (-)
N. meningitidis (+) (+) (-) (+) (+) (-) (-)
M. catarrhalis (+) (-) (-) (-) (-) (-) (+)
4a. Fastidious Gram Negative Bacilli
General Characteristics
Pleomorphic, small G(-) bacilli, MacConkey (-)
I. Haemophilus spp.
Facultative anaerobes, ferment carbohydrates (exp. H. ducreyi), oxidase & catalase (+)
Requires growth factors: Hemin/hematin (X Factor) and nicotinamide adenine dinucleotide (NAD or V Factor)
II. Clinically significant species
1. H. influenzae ( )
a. Virulence factor: Capsule [ a-f (b, most common), lacks adherent capability, associated with systemic & invasive
infections], IgA protease and LPS
b. Clinical Manifestations of Infections:
Encapsulated Strains Nonencapsulated Strains
Systemic & RT Infections Otitis Media
Septicemia, Septic arthritis Conjunctivitis
Tracheitis, Meningitis, Pericarditis Bacteremia
Pneumonia, Epiglotitis Sinusitis, Pneumonia
2. H. aegyptius (Koch-Weeks bacillus) or H. influenzae bio. aegyptius: & Brazilian Purpuric Fever
3. H. ducreyi: Chancroid (soft chancre)
III. Laboratory Diagnosis
1. Specimen Processing & Isolation
a. Specimen: Premoised/transport swab, blood & body fluids
b. Culture Media: Chocolate Agar (w/ Bacitracin or 1 % IsoVitaleX for H. ducreyi or H. aegypticus)
c. Incubation: i. Most Haemophilus spp.: 5-10% CO2 at 35-37°C (2-3 days),
ii. Haemophilus ducreyi: 5-10% CO2
2. Microscopy
a. Coccobacilli & filamentous
b. H. ducreyi - school of fish railroad tracks finger prints
3. Culture
a. H. influenzae: Translucent, smooth and convex; mousy / bleach like odor; encapsulated (larger & mucoid)
b. H. ducreyi - Small, flat, smooth, transparent to opaque; colonies can pushed intact; clumpy in saline
4. Identification
a. Neufeld Quellung Rxn: antisera is reacted with the antigens in the capsule making the capsule more prominent
b. Staphylococcus Streak: Haemophilus is streaked w/ S. aureus, S. pneumoniae, Neisseria and certain yeasts
Positive: Satellitism dewdrop colonies surrounding S. aureus
c. X and V requirement; d. Hemolytic patterns
e. Porphyrin Test: Test for the ability to produce ALA (ALA porphobilinogen/porphyrin Hemin)
Porphobilinogen (add emit with 360nm UV, red-orange)
Species X factor V factor Hemolysis ALA
H. influenzae (+) (+) (-) (-)
H. aegyptius (+) (+) (-) (-)
H. haemolyticus (+) (+) (+) (-)
H. parahaemolyticus (-) (+) (+) (+)
H. parainfluenzae (-) (+) (-) (+)
H. ducreyi (+) (-) (-) (-)
A. aphrophilus (-) (-) (-) (+)
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B. Acinetobacter spp.
1. Clinical significance: 2nd most common nonfermenter
2. Identifying characteristics: Plump, paired G(-) coccobacilli; A. baumanii (saccharolytic) - purplish (MAC) or
cornflower blue (EMB) ; A. iwoffi (assaccharolytic)
C. Stenotrophomonas maltophilia
1. Clinical significance: 3rd most common nonfermenter
2. Identifying characteristic: lavender green (BAP), Ammonia-like smell, oxidizes glucose W(+), oxidizes maltose S(+)
a. Burkholderia cepacia
1. Clinical significance: Pneumonia in patients with cystic fibrosis or chronic granulomatous dse.
2. Identifying characteristics: Dirtlike odor in BAP, ONPG +; dark pink in Mac in 4-7 d
b. Burkholderia pseudomallei
1. Clinical significance: Melioidosis
2. Identifying characteristics: Bipolar staining (G/S), w
c. Burkholderia glandioli: Associated w/ patients w/ CF & CGD,
d. Burkholderia mallei: Glanders, Nonmotile
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C. Exogenous
Contamination of wound or puncture by objects. E.g. C. tetani (tetanus) and C. perfringens (gas gangrene)
Ingestion of preformed toxins in vegetable or meat. E.g. C. botulinum (botulism), C. perfringens (food poisoning)
Colonization of GI tract of toxin-producing organism. E.g. C. botulinum (infant botulism)
D. Endogenous
Gain access to normally sterile site
Skin: Propionibacterium acnes
RT: Prevotella, Porphyromonas, Fusobacterium, Anaerobic Cocci
GIT: Bacteroides fragilis, Clostridium difficile
GUT: Bacteroides, Prevotella, Fusobacterium
II. Specimen Selection, Collection, Transport and Processing
A. Specimen Quality (Selection)
1. Suitable specimens: Tissue biopsy (necrotic tissues), needle and syringe aspiration (exudates, abscess)
2. Unsuitable Specimens: Swabs, voided urine, feces, coughed sputum, bronchial washings.
3. Fecal specimens for Clostridial illness: Food poisoning (C. perfringens), botulism (C. botulinum),
pseudomembranous enterocolitis (C. difficile), neutropenic enterocolitis (C. septicum)
B. Specimen Transport
1. Rubber-stoppered collection vial: For liquid specimens (i.e. pus & body fluids)
2. Oxygen free transport tubes and anaerobic pouch: For swab specimens and tissue specimens, respectively
3. Contents: i. Reducing agent (thioglycolic acid, Na thioglycolate); ii. redox indicator (resazurin, methylene blue)
C. Processing Clinical Specimens
1. Macroscopic Examination: Foul odor (Anaerobic G- bacilli), brick-red fluorescence and necrotic tissue black
exudates (Porphyromonas, Prevotella), sulfur granules (anaerobic G+ bacilli)
2. Microscopic Examination of the Specimen: To determine a polymicrobic infection, guide for media selection,
provide a presumptive identification and reveal leukocytes and squamous epithelial cells
3. Inoculation to Plated or Tubed Media
a. Anaerobic blood agar: Enriched media
b. PEA & CNA blood Agar: Selective for G(+) anaerobes
c. Anaerobic broth: i . Thioglycollate broth ; ii. Cooked meat broth and iii. peptone-yeast extract glucose (PYG) -
analysis of metabolic end products by GLC
d. Kanamycin-vancomycin-laked blood (KVLB):Selective for Bacteroides & Prevotella
e. Bacteroides bile esculin agar (BBE): Selective & differential for B. fragilis
f. Cycloserine cefoxitin fructose agar (CCFA): Selective & differential for C. difficile
g. Egg-yolk Agar (EYA): For determination of lecithinase and lipase production
C. perfringens: Lecithinase + (white opaque zone) ; F. necrophorum, C. botulinum: Lipase + (iridescent sheen)
4. Anaerobic Incubation
a. Anaerobe Chamber: Storage & inoculation under anaerobic condition
b. Anaerobic Jars and Bags: Anaerobiosis produced by gas generator envelope
c. Holding Jars: During processing, for inoculated plates pending incubation & examination of cultures
Contents: gas generator (85-90% N2 , 5% H2, 5-10% CO2), catalyst (palladium pellets, iron powder),
desiccant (silica gel, blue pink), redox indicator (methylene blue and resazurin)
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II. Gram Positive Bacilli: Outline: (1) Disease, (2) Cellular Morphology, (3) Colony Morphology, ( 4) Other Characteristics
A. Actinomyces israelli
1. sulfur granules ),
2. Branching filamentous rods
3. White, opaque and r molar tooth
B. Propionibacterium acnes
1. Actinomycosis, acne, subcute bacterial endocarditis, contaminants of blood culture bottles
2. Anaerobic diphtheroids
3. Small & white to large & yellowish tan
C. Bifidobacterium
1. Actinomycosis, in mixed infections of abdomens and GUT
2. dog bones
3. Small, white, convex, shiny with irregular edge
Branched bacilli Catalase/Indole Comments
A. israelii + - Molar tooth colony
P. acnes - + Diptheroid
Bifidobacterium - - Rods w/ forked ends
III. Gram Negative Bacilli
1. Virulence Factor: Polysaccharide capsules, adherence factors
2. Spectrum of Disease: In mixed infection, brain abscess and peritoneal infections
3. Cellular Morphology; 4. Colonial Characteristics; 5. Other Characteristics
A. Bacteroides fragilis
3. Coccobacili or pleomorphic
4. Gray black colonies on BBE Agar, Grow in KVLB agar
5. Tolerates and hydrolyze 20% bile, saccharolytic and resistant to kanamycin, vancomycin & colistin
B. Prevotella spp.
3. Tiny coccobacilli
4. Fluoresces brick red, Black pigment in KVLB agar.
5. Inhibited by 20% bile, saccharolytic, susceptible to colisten
C. Porphyromonas spp.
3. Tiny coccobacilli
4. Fluoresces brick red, No growth in KVLB agar.
5. Inhibited by 20% bile, asaccharolytic, susceptible to vancomycin
D. Fusobacterium nucleatum
3. Spindle-shaped w/ pointed ends
4. Crumblike or Ground glass, Greening on air exposure, Fluoresces chartreuse, Lipase positive (EYA)
5. Asaccharolytic, susceptible to kanamycin and colistin
Bile Esculin Growth on Brick Red Chartreuse Iridescent
Species V K Co
Resistance Hydrolysis KVLB Fluorescence fluorescence sheen (EYA)
B. fragilis R R R + + + - - -
Prevotella R R S - - + + - -
Porphyromonas S R R - - - + - -
Fusobacterium R S S - - - - + +
IV. Cocci
A. Spectrum of Disease: In mixed infection, brain abscess and peritoneal infections
B. Cellular Morphology:
1. G(-) diplococci / in chains -> Veillonella parvula
2. G(+) cocci -> Peptococcus & Peptostreptococcus (Finegoldia magna)
C. Colony Morphology: Red fluorescence -> Veillonella parvula
V. Confirmatory Test
Gas-liquid chromatography: Metabolic ends products from glucose metabolism
Gas chromatography: Whole-cell long chain fatty acid methyl ester (FAME) analysis
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9a. Spirochetes
I. Clinically Signifant Species
A. Leptospires
1. Characteristics:
Tightly coiled, thin, spirochetes w/ hooked ends
C Stuart, EMJH
Visible by dark-field, phase contrast & immunoflourescence microscopy
2. Leptospira interrogans:
Acquired through contact with urine of animals (e.g. rodents) who carry the organism.
Leptospirosis and Weil disease (systemic disease with intravascular disease, renal & hepatic failure)
B. Borreliae
1. Characteristics:
Less tightly coiled (3 -10 coils); culturable to Kelly medium; visualized by bright-field microscopy
2. Borrelia recurrentis:
Endemic (tickborne) and epidemic relapsing fever (louse-borne)
3. Borrelia burgdorferi:
Lyme disease
1. Localized (Erythema chronicum migrans),
2. Early disseminated
3. Late persistent
C. Treponemes
1. Characteristics
Coiled (4-14); non culturable, motile with graceful flexuous movements; visible by dark-field
2. Treponema pallidium subsp. pallidum:
i. Syphilis manifests as
a. Primary (chancre)
b. Secondary (condylomas)
c. Tertiary (gummas, neurosyphilis)
ii. Congenital syphilis
iii. Serologic Tests: VDRL & RPR, FTA-ABS, TP-PA and EIA
3. Treponema pallidum subsp. pertenue - Yaws
4. Treponema pallidum subsp. endemicum - Endemic Syphilis or Bejel
5. Treponema carateum - Pinta
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9b. Chlamydiaceae
I. Introduction: Obligate Intracellular Bacteria
A. General Characteristics
C. trachomatis C. pneumoniae C. psittaci
EB Morphology Round Pear-shaped Round
Glycogen in inclusions (+) (-) (-)
Sulfa drug sensitivity (+) (-) (-)
Natural Hosts Humans Humans Birds
B. Chlamydia trachomatis
1. Clinical Infections
Biovars Clinical Syndromes Route of Transmission
A,B,Ba,C Ocular (Endemic) Trachoma Hand to eye from fomites
L1,L2,L3 Lymphogranuloma venereum
Sexual
Urogenital Disease (PID , Reiter Syndrome)
D-K
Inclusion conjunctivitis Hand to eye
2. Laboratory Diagnosis
Cell Culture, serology , molecular (PCR)
(intradermal skin test of LGV Ag): Positive: erythema (redness) and induration (firmness)
C. Chlamydophilia pneumoniae:
Pneumonia & pharyngitis
Guillain-Barre syndrome
D. Chlamydophilia psitacci:
Psittacosis & ornithosis (parrot fever)
acquired from birds by inhalation of aerosols
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10a. Mycobacteria
General Characteristics
Slender, slightly curved rods, high lipid content (mycolic acid), acid fast (stained by basic fuchsin dye but resist
decolorization by 3% HCl), slow growth (2-6 weeks)
1. Mycobacterium tuberculosis complex
A. Mycobacterium tuberculosis
a. Epidemiology: >1 billion persons are infected, 8-10 million new cases / year, 15-20% develops disease
b. Transmission: Inhalation of aerosols or close contact
c. Spectrum of disease: Tuberculosis
i. Primary / Reactivation: Tubercles or granuloma (granulomatous lesions from multinucleated cells) and
Caseation (cheese like masses from break down of tubercles) in the lungs
ii. Extrapulmonary (Miliary): Kidney, joints, CNS, GUT, body cavities, larynx, etc.
d. Culture: Raised, dry, rough, buff (2-3 weeks in LJ, 5-10 days in Middlebrook)
B. Mycobacterium bovis and BCG
a. Transmission: M. bovis (Ingestion of milk from infected cattle)
M. bovis / Bacille Calmette-Guerin (Immunization of immunocompromised individuals)
b. Culture: water droplets
c. ScreeningTest: Tuberculin Skin Testing (Mantoux test, Pirquet test, PPD test)
Positive: erythema (redness) and induration (firmness) in 48-72 hours
2.
Runyon Classification of NTM: Based on the ability to produce carotenoids
A. Photochromogens Unpigmented when grown in the dark and develop pigment after light
a. M. asiaticum
nd
b. M. kansasii: Yellow bacillus most common NTM in lungs
c. M. marinum: of the sea -32°C
d. M. simiae
B. Scotochromogens Pigmented when grown in the dark but may intensify with exposure to light
a. M. gordonae: Tap water bacillus
b. M. szulgai: Photochromogen (22°C) & scotochromogen (37°C)
c. M. scrofulaceum: Cervical lymphadenitis in children
d. M. xenopi:
C. Nonphotochromogens Nonpigmented when grown in the dark and exposed to light
a. M. avium complex Battey bacillus Most commonly isolated NTM;
Most common cause of systemic bacterial infection in AIDS patients
b. M. avium subs. paratuberculosis:
c. M. celatum: Frequent isolate in respiratory specimen
d. M. genavense: Associated with infections of AIDS patients
e. M. haemophilium: Requires hemoglobin and hemin
f. M. malmoense: Coccobacilli without cross-bands
g. M. terrae complex: Radish bacillus
h. M. ulcerans: Grows best 42°C, 3rd most common Mycobacteria
D. Rapid-growers
a. M. fortuitum and M. chelonae abscessus group: Non photochromogen, Arylsulfatase (+) in 3 d, MacConkey (+)
b. M. smegmatis Schotochromogen
E. Noncultivatable
a. M. leprae - e
i. Tuberculoid Leprosy - Skin lesions and peripheral nerve involvement
ii. Lepromatous leprosy - Extensive skin lesions and symmetric nerve damage
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3. Laboratory Diagnosis
A. Laboratory Safety Considerations
1. Specimen is collected in sterile, leak proof container.
2. BSL 2 - for preparing AFB smears and culture, BSL 3 - For propagation
3. Aerosol - generating procedures is performed in Class II or III BSC
B. Specimen Collection
1. Sputum and gastric lavage - early morning specimens in 3 consecutive days
2. Urine - early morning urine in 3 consecutive days, midstream clean catch
3. Stool - for isolation of M. avium in AIDS patients
4. Blood - for isolation of M. avium complex, can be recovered by BACTEC or by Isolator lysis centrifugation
5. Tissue and body Fluids - homogenized and concentrated), respectively
C. Specimen Preparation
1. n-Acetyl-L-cysteine (NALC) or dithioreitol: liquefy specimen (splits disulfide bonds)
2. 2-4% NaOH: both digestant (mucolytic) and decontaminating (antibacterial) agent
3. Trisodium phosphate & Benzalkonium chloride: acts as digestant and as decontaminating agent, respectively.
4. Oxalic Acid: for Pseudomonas contaminated samples
D. Staining of Acid Fast Bacilli: Acid-Fast Stain (AFB Stain)
1. Ziehl-Neelsen (hot stain) & Kinyuon (cold)
2. Auramine-Rhodamine fluorochrome stain. Examined at (250-400x). More sensitive
AFB (1000x) Flourochrome (450x) Report
0 0 No AFB seen
1-2/300 fields 1-2/70 fields Repeat test
1-9/100 fields 2-18/50 fields 1+
1-9/10 fields 4-36/10 fields 2+
1-9/field 4-36/field 3+
>9/field >36 field 4+
E. Media & Isolation Methods
1. Solid Media: 35°C, 5-10% CO2
i. Egg Based (18-24 d) - Lowenstein-Jensen and Petragnani
ii. Agar based (10-12 d) - Middlebrook 7H10 & 7H11 and (Selective Middlebrook)
2. Liquid Media: Middlebrook broth (10 d)
F. Laboratory Identification
1. Niacin (Accumulation) Test
Test for the ability to produce and accumulate Niacin (niacin niacin ribonucleotide)
Positive (Formation of yellow liquid w/ addition of cyanogen bromide); Negative (Liquid remains milky white)
2. Nitrate Reduction
Test for the ability of to reduce nitrate (nitrates nitroreductase nitrite)
Positive (Development of pink to red color); Negative (No color development)
3. Semiquantitative Catalase Test : >45 mm of bubbles or <45 mm of bubbles
4. Heat stable (68ºC) Catalase Test
Test for the ability of catalase enzyme to remain active after heating
Stable or inactivated at 68°C for 20 mins.
5. Growth Inhibition by T2H (TCH)
Test for the ability for tolerance to Thiophene-2-Carboxylic Acid Hydrazide
Positive (No Growth); Negative (Growth)
Species Niacin Growth on T2H Nitrate reduction 68° & SQ Catalase
M. tuberculosis + R (-) + (-)
M. bovis (-) S (+) (-) (-)
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Incubation N
Atmosphere Humidified ambient air
Temperature 35°C
Length Disk diffusion: 16-18 hr
Others
Age of the Colony 24 hours
Colonies Sampled
4-5
from isolates
Distance 20 mm apart
Maximum stacks 5
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