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BACTERIOLOGY > B4.2 MYCOBACTERIA LAB METHODS > Flashcards

B4.2 MYCOBACTERIA LAB METHODS Flashcards

1
Q

What are the methods used for acid-fast staining?

A

Ziehl-Neelsen/Hot Stain Procedure
Kinyoun stains/Cold Stain Procedure
Auramine-rhodamine fluorochrome stains

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2
Q

What concentration of AFB is needed to obtain a positive AFB staining?

A

5,000 to 10,000 AFB/mL

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3
Q

What are the factors affecting acid-fastness?

A

Age of colonies
Medium on which growth occurs
Exposure to UV

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4
Q

What is the minimum concentration of AFB required for detection from concentrated specimens in acid-fast staining?

A

At least 10⁴ AFB/mL

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5
Q

What is the decolorizer used in the Auramine-rhodamine fluorochrome stain method?

A

Acid Alcohol

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6
Q

What is the counterstain used in the Auramine-rhodamine fluorochrome stain method?

A

Potassium permanganate

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7
Q

At what magnification should smears be examined when using the Auramine-rhodamine fluorochrome stain?

A

250x to 400x magnification

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8
Q

What is the appearance of positive acid-fast smears when using the Auramine-rhodamine fluorochrome stain?

A

Bright yellow-orange bacilli against a dark background

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9
Q

What method confirms all positive fluorescent smears in acid-fast staining?

A

Ziehl-Neelsen stain on the same smear

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10
Q

What precautions should be taken during acid-fast staining and microscopy?

A

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

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11
Q

What are the causes of false positive acid-fast staining?

A

Changes in the cell wall
Insufficient decolorization
Laboratory contamination
Delayed processing and overgrowth of other bacteria

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12
Q

What are the causes of false negative acid-fast staining?

A

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

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13
Q

What are the incubation conditions for culture media?

A

Incubated at 35°C in the dark with 5% to 10% CO2 with humidity

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14
Q

What are the incubation conditions for tubed media?

A

Incubated at slanted position with screw caps loose for 1 week
Allows evaporation of excess fluid and entry of CO2

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15
Q

What are the incubation conditions for plated media?

A

Placed in CO2-permeable plastic bag or wrapped with CO2-permeable tape

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16
Q

How often should culture media be examined for growth?

A

Weekly

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17
Q

What is the growth time for rapid growers in culture media?

A

2-3 days

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18
Q

What is the growth time for pathogenic organisms in culture media?

A

2-6 weeks

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19
Q

What is the inhibitory agent in malachite green?

A

Inhibits nonmycobacteria

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20
Q

What is the incubation temperature for M. marinum or M. ulcerans?

A

25° to 30° C

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21
Q

What is the incubation temperature for M. haemophilum?

A

25° to 33° C (on chocolate agar plate or with X-factor disk)

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22
Q

What are the components of egg-based media?

A

Fresh whole eggs
Potato flour
Glycerol
Slight variations in defined salts, milk, and potato flour

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23
Q

What is the function of malachite green in egg-based media?

A

Suppresses gram(+) bacteria

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24
Q

What is the role of egg yolk in egg-based media?

A

Lipid source, promotes growth of mycobacteria

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25
Q

What is the shelf life of nonselective egg-based media?

A

1 year

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26
Q

What are the types of egg-based media used in mycobacteria culture?

A

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

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27
Q

What is the most commonly used egg-based medium?

A

Löwenstein-Jensen (L-J)

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28
Q

What is the concentration of malachite green in Löwenstein-Jensen (L-J) medium?

A

0.025%

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29
Q

What is the modification of L-J medium in Gruft medium?

A

+RNA
Malachite Green
Penicillin
Nalidixic acid

30
Q

What are the components of Mycobactosel medium?

A

Malachite green
Cycloheximide
Lincomycin
Nalidixic acid

31
Q

What is the modification of L-J medium for enhancing the recovery of M. bovis?

A

L-J with pyruvic acid

32
Q

What is the modification of L-J medium for enhancing the recovery of M. ulcerans?

A

L-J with glycerol

33
Q

What is the concentration of malachite green in Petragnani Medium?

A

0.052%

34
Q

What is the special feature of Wallenstein Medium?

A

Used for M. avium complex

35
Q

Löwenstein-Jensen (L-J) cannot grow which spp

A

M. genovense

36
Q

What is the modification of Petragnani Medium for heavily contaminated specimens?

A

Egg yolks
Whole milk

37
Q

What are the components of Heme-Supplemented Media?

A

Hemin
Hemoglobin
Ferric ammonium citrate

38
Q

Heme-Supplemented Media Used for

A

M. haemophilum

39
Q

What is the concentration of malachite green in American Thoracic Society medium?

A

0.02%

40
Q

What is the composition of agar-based media for mycobacteria culture?

A

Defined salts
Vitamins
Cofactors
Glycerol
Malachite green
Agar
Enrichment consisting of oleic acid, bovine albumin, glucose, and beef catalase

41
Q

Agar-Based Media

A

a. Middlebrook
b. Middlebrook 7H10
c. Middlebrook 7H11
d. Mitchison’s selective 7H11
e. Dubos Oleic Acid Albumin

42
Q

Media that enhances the growth of MAC

A

Middlebrook

43
Q

Middlebrook contains

A

2% glycerol

44
Q

Middlebrook 7H10 contains

A

Malachite green (0.00025%)

45
Q

Middlebrook 7H10 selective contains

A

carbenicillin , polymyxin B, trimethoprim lactate, and amphotericin B

46
Q

use for inhibition of pseudomonads

A

Middlebrook 7H10 selective contains -carbenicillin

47
Q

Middlebrook 7H11 components

A

Malachite green (0.0001%), 0.1% casein
hydrolysate; isoniazid

48
Q

Middlebrook 7H11 is used to isolate

A

resistant M. tuberculosis

49
Q

Middlebrook7H11 supplemented with
mycobactin J is uses to isolate

A

M. genovense

50
Q

Mitchison’s selective 7H11 components

A

casein hydrolysate,
Carbenicillin,
Amphotericin B,
Polymyxin B,
Trimethoprim lactate

51
Q

Good for AST but Middlebrook 7H10 is
preferred

A

Dubos Oleic Acid Albumin

52
Q

Liquid Media examples

A

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

53
Q
  • Part of BACTEC system
  • Requires daily agitation to enhance growth and read within 4 days of inoculation
A

BACTEC 12B medium (MB 7H12) and BACTEC
13A (MB 7H13)

54
Q

BACTEC 12B medium (MB 7H12) and BACTEC
13A (MB 7H13)

Antimicrobial agents:

A

polymyxin, B,
amphotericin B,
nalidixic acid,
trimethoprim, and
azlocillin (PANTA)

55
Q

BACTEC 12B medium (MB 7H12) and BACTEC
13A (MB 7H13)

Growth enhancer

A

polyoxyethylene stearate

56
Q

BACTEC 12B medium (MB 7H12) and BACTEC
13A (MB 7H13)

Growth indicator and differentiation between tuberculosis and NTM

A

release of CO2

✓ M. tuberculosis = 9 to 14 days:
✓ NTM = <7 days

57
Q

metabolized by mycobacteria, liberating
radioactive CO2 (14CO2] -> detected by BACTEC 460TB

substrate used for BACTEC 460TB detection of Mycobacterium

A

14C-labeled substrate (palmitic acid)

58
Q
  • Nonselective liquid media used for
    subculturing stock strains
A

Middlebrook 7H9 broth and Dubos Tween
albumin

59
Q

Septi-Chek AFB media used

A

Biphasic Media

60
Q

Biphasic Media liquid components

A

20 mL Middlebrook 7H9 broth ,20% CO2

61
Q

Biphasic Media solid phase contains

A

modified L-J, Middlebrook 7H11, and a chocolate agar slab

62
Q

Media Used in Commercially Supplied Growth and Semiautomated or Fully Automated Systems

A

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

63
Q

modified Middlebrook 7H9 broth that
incorporates fluorescencequenching- based oxygen sensor

A

Mycobacteria Growth Indicator Tube [MGIT]

64
Q

Mycobacteria Growth Indicator Tube [MGIT] semiautomated system

A

MGIT 460TB

65
Q

Mycobacteria Growth Indicator Tube [MGIT] fully automated system

A

MGIT 960

66
Q
  • modified Kirchner medium, enriched with
    additives and antibiotics, tetrazolium salt-> redox indicator
A

MB Redox

67
Q

Medium used for pressure changes in automatic machines

A

ESP Culture System II and versa TREK Culture
System II -

68
Q

Media used for colorimetric carbon dioxide sensor for automated machines

A

MB/BacT Alert 3D

69
Q

Media used to detect O2 consumption by
fluorescent sensor in automated machines

A

BACTEC 9000 MB

70
Q

Automated Radiometric System

A

BACTEC 460 TB System

BACTERIOLOGY (31 decks)

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