Home›Microbiology›Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format
MicrobiologyJoVE (Open Access)Citable · DOI
Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format
DOI: 10.3791/3094-v
What you'll learn
✓Perform broth microdilution antimicrobial susceptibility testing for M. tuberculosis complex
✓Interpret minimum inhibitory concentration (MIC) values for first and second-line drugs
✓Prepare inocula and evaluate antibiotic resistance using manual or automated platforms
Protocol
Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.
Difficulty
advanced
Total time
~3–4 weeks (culture growth and incubation period)
Biosafety
BSL-2
Steps
1
Execute experimental preparation and biosafety procedures
Establish biosafety protocols and prepare workspace for handling M. tuberculosis complex. Review containment requirements and safety procedures for pathogenic mycobacterial work.
▶ 01:47
2
Prepare standardized inoculum suspension
Generate inoculum from M. tuberculosis culture adjusted to appropriate optical density for susceptibility testing. Ensure consistency and viability of bacterial suspension.
▶ 03:13
3
Dispense inoculum into microtiter plate wells
Aliquot standardized inoculum into microtiter plates containing serial dilutions of antimycobacterial drugs. Distribute volume uniformly across wells.
▶ 05:05
4
Determine antibiotic resistance by manual inspection
Visually assess well turbidity and growth inhibition to identify resistance patterns. Compare results against control wells.
▶ 08:07
5
Determine antibiotic resistance using Vizion platform
Use automated platform to objectively measure optical density and calculate MIC values. Generate resistance profiles for first and second-line drugs.
▶ 09:12
6
Verify sample purity and perform colony counts
Confirm culture purity through microscopy or subculture and enumerate viable bacterial cells to validate inoculum quality.