B4.2 MYCOBACTERIA LAB METHODS Flashcards
What are the methods used for acid-fast staining?
Ziehl-Neelsen/Hot Stain Procedure
Kinyoun stains/Cold Stain Procedure
Auramine-rhodamine fluorochrome stains
What concentration of AFB is needed to obtain a positive AFB staining?
5,000 to 10,000 AFB/mL
What are the factors affecting acid-fastness?
Age of colonies
Medium on which growth occurs
Exposure to UV
What is the minimum concentration of AFB required for detection from concentrated specimens in acid-fast staining?
At least 10⁴ AFB/mL
What is the decolorizer used in the Auramine-rhodamine fluorochrome stain method?
Acid Alcohol
What is the counterstain used in the Auramine-rhodamine fluorochrome stain method?
Potassium permanganate
At what magnification should smears be examined when using the Auramine-rhodamine fluorochrome stain?
250x to 400x magnification
What is the appearance of positive acid-fast smears when using the Auramine-rhodamine fluorochrome stain?
Bright yellow-orange bacilli against a dark background
What method confirms all positive fluorescent smears in acid-fast staining?
Ziehl-Neelsen stain on the same smear
What precautions should be taken during acid-fast staining and microscopy?
Smears should not come in contact with one another during staining
Staining jars should not be used
Wipe the oil immersion lens (OlO) before reading the next smear slide
What are the causes of false positive acid-fast staining?
Changes in the cell wall
Insufficient decolorization
Laboratory contamination
Delayed processing and overgrowth of other bacteria
What are the causes of false negative acid-fast staining?
Overzealous decontamination
Loss from concentration technique
Organism obscured by a very thick smear
Over-decolorization of the smear
Poor counterstaining
Lack of observer proficiency in reading stains
What are the incubation conditions for culture media?
Incubated at 35°C in the dark with 5% to 10% CO2 with humidity
What are the incubation conditions for tubed media?
Incubated at slanted position with screw caps loose for 1 week
Allows evaporation of excess fluid and entry of CO2
What are the incubation conditions for plated media?
Placed in CO2-permeable plastic bag or wrapped with CO2-permeable tape
How often should culture media be examined for growth?
Weekly
What is the growth time for rapid growers in culture media?
2-3 days
What is the growth time for pathogenic organisms in culture media?
2-6 weeks
What is the inhibitory agent in malachite green?
Inhibits nonmycobacteria
What is the incubation temperature for M. marinum or M. ulcerans?
25° to 30° C
What is the incubation temperature for M. haemophilum?
25° to 33° C (on chocolate agar plate or with X-factor disk)
What are the components of egg-based media?
Fresh whole eggs
Potato flour
Glycerol
Slight variations in defined salts, milk, and potato flour
What is the function of malachite green in egg-based media?
Suppresses gram(+) bacteria
What is the role of egg yolk in egg-based media?
Lipid source, promotes growth of mycobacteria
What is the shelf life of nonselective egg-based media?
1 year
What are the types of egg-based media used in mycobacteria culture?
Löwenstein-Jensen (L-J)
Gruft (Modification of L-J)
Mycobactosel
L-J with pyruvic acid
L-J with glycerol
Petragnani Medium
Heme-Supplemented Media
American Thoracic Society
Wallenstein Medium
What is the most commonly used egg-based medium?
Löwenstein-Jensen (L-J)
What is the concentration of malachite green in Löwenstein-Jensen (L-J) medium?
0.025%
What is the modification of L-J medium in Gruft medium?
+RNA
Malachite Green
Penicillin
Nalidixic acid
What are the components of Mycobactosel medium?
Malachite green
Cycloheximide
Lincomycin
Nalidixic acid
What is the modification of L-J medium for enhancing the recovery of M. bovis?
L-J with pyruvic acid
What is the modification of L-J medium for enhancing the recovery of M. ulcerans?
L-J with glycerol
What is the concentration of malachite green in Petragnani Medium?
0.052%
What is the special feature of Wallenstein Medium?
Used for M. avium complex
Löwenstein-Jensen (L-J) cannot grow which spp
M. genovense
What is the modification of Petragnani Medium for heavily contaminated specimens?
Egg yolks
Whole milk
What are the components of Heme-Supplemented Media?
Hemin
Hemoglobin
Ferric ammonium citrate
Heme-Supplemented Media Used for
M. haemophilum
What is the concentration of malachite green in American Thoracic Society medium?
0.02%
What is the composition of agar-based media for mycobacteria culture?
Defined salts
Vitamins
Cofactors
Glycerol
Malachite green
Agar
Enrichment consisting of oleic acid, bovine albumin, glucose, and beef catalase
Agar-Based Media
a. Middlebrook
b. Middlebrook 7H10
c. Middlebrook 7H11
d. Mitchison’s selective 7H11
e. Dubos Oleic Acid Albumin
Media that enhances the growth of MAC
Middlebrook
Middlebrook contains
2% glycerol
Middlebrook 7H10 contains
Malachite green (0.00025%)
Middlebrook 7H10 selective contains
carbenicillin , polymyxin B, trimethoprim lactate, and amphotericin B
use for inhibition of pseudomonads
Middlebrook 7H10 selective contains -carbenicillin
Middlebrook 7H11 components
Malachite green (0.0001%), 0.1% casein
hydrolysate; isoniazid
Middlebrook 7H11 is used to isolate
resistant M. tuberculosis
Middlebrook7H11 supplemented with
mycobactin J is uses to isolate
M. genovense
Mitchison’s selective 7H11 components
casein hydrolysate,
Carbenicillin,
Amphotericin B,
Polymyxin B,
Trimethoprim lactate
Good for AST but Middlebrook 7H10 is
preferred
Dubos Oleic Acid Albumin
Liquid Media examples
BACTEC 12B medium (MB 7H12) and BACTEC
13A (MB 7H13)
Middlebrook 7H9 broth and Dubos Tween
albumin
Septi-Chek AFB
BBL Mycobacteria Growth Indicator Tubes
- Part of BACTEC system
- Requires daily agitation to enhance growth and read within 4 days of inoculation
BACTEC 12B medium (MB 7H12) and BACTEC
13A (MB 7H13)
BACTEC 12B medium (MB 7H12) and BACTEC
13A (MB 7H13)
Antimicrobial agents:
polymyxin, B,
amphotericin B,
nalidixic acid,
trimethoprim, and
azlocillin (PANTA)
BACTEC 12B medium (MB 7H12) and BACTEC
13A (MB 7H13)
Growth enhancer
polyoxyethylene stearate
BACTEC 12B medium (MB 7H12) and BACTEC
13A (MB 7H13)
Growth indicator and differentiation between tuberculosis and NTM
release of CO2
✓ M. tuberculosis = 9 to 14 days:
✓ NTM = <7 days
metabolized by mycobacteria, liberating
radioactive CO2 (14CO2] -> detected by BACTEC 460TB
substrate used for BACTEC 460TB detection of Mycobacterium
14C-labeled substrate (palmitic acid)
- Nonselective liquid media used for
subculturing stock strains
Middlebrook 7H9 broth and Dubos Tween
albumin
Septi-Chek AFB media used
Biphasic Media
Biphasic Media liquid components
20 mL Middlebrook 7H9 broth ,20% CO2
Biphasic Media solid phase contains
modified L-J, Middlebrook 7H11, and a chocolate agar slab
Media Used in Commercially Supplied Growth and Semiautomated or Fully Automated Systems
a. Mycobacteria Growth Indicator Tube [MGIT]
b. MB Redox
c. ESP Culture System II and versa TREK Culture
System II
d. MB/BacT Alert 3D
e. BACTEC 9000 MB
modified Middlebrook 7H9 broth that
incorporates fluorescencequenching- based oxygen sensor
Mycobacteria Growth Indicator Tube [MGIT]
Mycobacteria Growth Indicator Tube [MGIT] semiautomated system
MGIT 460TB
Mycobacteria Growth Indicator Tube [MGIT] fully automated system
MGIT 960
- modified Kirchner medium, enriched with
additives and antibiotics, tetrazolium salt-> redox indicator
MB Redox
Medium used for pressure changes in automatic machines
ESP Culture System II and versa TREK Culture
System II -
Media used for colorimetric carbon dioxide sensor for automated machines
MB/BacT Alert 3D
Media used to detect O2 consumption by
fluorescent sensor in automated machines
BACTEC 9000 MB
Automated Radiometric System
BACTEC 460 TB System
