Mycobacteriology Part I

Question 1

Objective 6: List the strain of M. bovis which is used to produce the M. tuberculosis vaccine

Answer 1

Bacille Calmette-Guerin (BCG)

Question 2

Objective 7: List two methods used to immunologically screen for possible MTB exposure

Answer 2

1. Tuberculin Skin Test (PPD)
2. Interferon-gamma release assay

Question 3

Objective 9: **List the three main members of the M. tuberculosis complex which will be identified with the TB complex DNA probe

Answer 3

Mycobacterium tuberculosis
Mycobacterium bovis
Mycobacterium africanum

Question 4

Objective 10: List the species of Mycobacterium which cannot be cultured in the laboratory routinely (requires living cells)

Answer 4

Mycobacterium leprae

Question 5

Objective 1: Explain why acid-fast bacteria stain acid-fast

Answer 5

Cell wall contains mycolic acid which is resistant to decolorization with acids/ethanol

Question 6

Objective 2: Discuss the possible appearance of mycobacteria when performing a Gram Stain

Answer 6

Some might stain GPRs — “Beaded or “ghosted”

Question 7

Objective 4: **Define “rapid grower” and “slow grower”

Answer 7

Rapid grower: Visible growth in < 7 days (subculture)

Slow grower: Visible growth in > 7 days (subculture)

Question 8

Objective 5: **Define “scotochromogen”, “photochromogen”, and “non-chromogen”

Answer 8

Non-chromogen: no pigment production
Photochromogen: pigment produced only when exposed to light
Scotochromogen: pigment produced in either light or dark

Question 9

Objective 11: Discuss the BSL level and laboratory containment mechanisms required when working with mycobacterial cultures

Answer 9

• Biosafety Level 3 (BSL-3)
• Restricted lab access
• Directional airflow — Negative air pressure
• Specimen/culture manipulations performed in
  biosafety cabinets (BSCs)
• PPE: N95 masks or PAPR

Question 10

Objective 12: Outline the specimen collection recommendations for respiratory mycobacteria
cultures (time of day, number of specimens to collect, time over which to collect them, etc.)

Answer 10

Three (3) separate specimens collected at least eight (8) hours apart [early morning preferred]

Question 11

Objective 13: Recognize the situation in which it is appropriate to submit a stool sample for mycobacterial culture

Answer 11

Detection of Mycobacterium avium complex in AIDS patients

Question 12

Objective 14: **Outline sputum processing for mycobacteria including the purpose and method
for decontamination, the method for digestion, the purpose of concentration and force/time at which it is done, and the stain which should be used to screen
specimens.

Answer 12

Decontamination: kill or reduce the amount of contaminated flora

• 2% NaOH (up to 5%) — decontamination
• NALC — digest mucolytic agent
• Oxalic acid (optional) — P. aeruginosa
• Phosphate buffer — stop/neutralize reaction

Concentration: increase recovery of mycobacteria
Force/Time: 3000 - 3800 x g for 15 min.

Stain: Auromine Rhodamine

Question 13

Objective 15: Identify the “ideal” contamination rate that mycobacteria labs should strive for when assessing their decontamination process

Answer 13

2-5% contamination

Question 14

Objective 3: Recognize the appearance of mycobacteria and the background when using the
Ziehl-Neelsen or Kinyoun methods

Answer 14

Positive: Red/Pink
Negative: Blue

Background: White

Question 15

Objective 16: Classify the Ziehl-Neelson, Kinyoun, and Auromine Rhodamine stains as Carbolfuchsin based or fluorescent.

Answer 15

Carbolfuchsin-based: Ziehl-Neelson & Kinyoun
Fluorescent: Auromine Rhodamine

Question 16

Objective 17: Compare and contrast microscopic examination of direct specimens using Carbol-fuchsin based stains vs. fluorescent stains.

Answer 16

Carbol-fuchsin based stains:
- Positive: Red/Pink
- Negative: Blue

Fluorescent stains:
- Positive: Yellow/orange fluorescence
- Negative: Dark, no fluorescence

Question 17

Objective 18: **Recognize the fluorescent stain that is specific for acid-fast organisms and is the best choice for screening specimens

Answer 17

Auromine Rhodamine

Question 18

Objective 20: Recognize the mycobacterium species and antimicrobial susceptibility that are identified with the GeneXpert (Xpert MTB/RIF) system

Answer 18

• Detects MTB complex organisms
• Detects rifampin resistance

Question 19

Objective 22: Identify whether broth or solid media is able to recover organisms more quickly

Answer 19

Broth media

Question 20

Objective 23: Recognize the 2 categories of solid media as well as the specific examples of each mentioned in lecture which are used for the isolation of mycobacteria

Answer 20

Egg based
- i.e. Lowenstein Jensen (LJ)

Agar based
- i.e. Middlebrook 7H10 & 7H11, Chocolate agar

Question 21

Objective 24: List the special requirements for isolation of M. haemophilum

Answer 21

• Chocolate agar at 30 deg. C

Question 22

Objective 25: Recognize the broth media used in broth media systems such as MGIT, BacT/ALERT, and BACTEC Myco/F Lytic system

Answer 22

Middlebrook 7H9 broth

Question 23

Objective 26: Differentiate the temperature(s) at which plates are incubated for skin cultures and sputum (respiratory) cultures

Answer 23

Skin/superficial tissue culture: 30 deg. C
Respiratory culture: 35-37 deg. C

Question 24

Objective 28: **List the mycobacteria species that grow best at a temperature of 30°C or less

Answer 24

1. M. chelonae
2. M. haemophilum
3. M. ulcerans
4. M. marinum

Question 25

Objective 29: List the mycobacteria species which grows best at a temperature greater than 35-37°C

Answer 25

M. xenopi

Question 26

Objective 30: Recognize how long mycobacteria cultures should be incubated before reporting
final results

Answer 26

6-8 weeks