tb biochemical test Flashcards
(30 cards)
Biochemical testing
growth rate, morphology, pigment, biochem profiles
arylsulfatase
ability to produce detectable level of phenolphthalein after cleaving sulfate group (from aromatic ring
three-day- m. fortuitum+ M.phlei-
M. xenopi produced at 42, MAC doesn’t
14 day- arylsulfatase M.triviale + / M.terrae -
catalase 4 groups
1) devoid
2) produce 45 mm
4) lose activity when heated at 68 for 20 min
semi-quantitaivie catalase
incubate 2 weeks at 37
1 ml diluted h202 is added
measure heightof effervescence
MTB is negative
Heat-stable catalase
heating at 68 c for 20 minutes inactivates
mtb, m. gastri, m. haemophilum
distinguish mtb from other slow growers when niacin is weakly positive
iron uptake
capable of converting ferric ammonium citrate to iron oxide
key for rapid growers
M. fortuitum + vs. M. chelonae -
5% NaCl
most rapid growers and M. triviale
two LJ slants one regular one with 5% Nacl
M. fortuitum, M. abscessus, M. flavescens +
M. gordonae, M. chelonae -
Niacin
mtb accumulates a large amount of niacin
differentiate M. tb+ / M. bovis -
Cyanogen bromide HAZARDOUS neutralize with 10% NaOH
NItrate
ability of some mycobacteria to reduce nitrate
most rapid growers have the enzyme except M. chelonae and M. abscessus
M. bovis is nitrate negative vs. M. tb
incubate culture with 2 ml of buffer for 2 hrs
Tween Hydrolysis
hydrolysis of tween 80 differentiate pathogens from saprophytic scoto and photochromogens M. kansasii + M. gordonae +w M. bovis and M. avium - enzyme cleaves oleic acid from tween 80 freeing the indicator dye and turning the solution red
Tween Opacity
oleic acid are released from tween80
differentiates M. flavescens + from other slow growers
Urease
results in alkalinzation of the medium
all scoto except m. gordonae, m. kansasii, MTBC, m. gastri
mac, m. gordonae, m. xenopi are negative
tellurite reduction
potassium tellurite can be reduced to metallic tellurium
7H9 broth is inoculated with test culture, incubate for 10 days, 0.2% KTel solution added, incubate an additional 3 days
mac and most rapid growers are positive
MTB is negative
growth on macconkey w/o crystal violet
pathogen (+) vs. saprophytes (-)
Carbon utilization
sodium citrate, inositol, mannitol
sole source of carbon in presence of ammoniacal nitrogen
m. chelonae and m. fortuitum
Pyrazinamide Hydrolysis
PZA deaminated in some mycobacteria by pyrazinamidase
ferrous ammonium sulfate is added at days 4 and 7
results in pink band after incubation for 30 min
MTB v. M. bovus
M. kansasii - 4 / + 7 vs. M. marinum +4
HPLC
ID of mycobacteria based on mycolic acid analysis
HPLC principle
1) extraction of fatty acids
2) separation of esters by column
3) detection by UV or FL detector
4) Visual or mathematical analysis
Saponification reagent
potassium hydroxide, methanol, water
HPLC componens
sample in mobile phase injector column detector recorder
column
sorbet(active component of the column
granular material made of solid particles
separate due to different degrees of interaction with sorbent particles
Gen-Probe Accuprobe steps
Saponification Heat Killing Hybridization Selection Detection POSITIVE & NEGATIVE WITH EACH ASSAY
Gen-Probe organisms
MTB complex
MAC
M. kansasii
M. gordonae
PCR
nucleic acid amplification- amply a single or few copies of a piece of DNA generating millions of copies of a particular sequence
denaturation (94)
annearling (50-65)
extension (72)
