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Sunday, October 16, 2011

Phenotypic Antimicrobial Assay: Broth Dilution

Similar to agar dilution method, broth dilution methods test the organisms in medium containing antimicrobial agents in serials of twofold dilutions. Instead of growing bacteria on solidified medium, bacteria are grown in liquid medium during susceptibility test process, and at the end of the test, bacterial growth is evaluated by the turbidity of broth. Macrodilution testing is performed in serials of
13 × 100 mm tubes with each one containing 2 mL of broth. Microdilution testing uses multiwell microdilution trays with each well containing 0.1 mL of broth.
Because the microdilution trays with prepared panels of antimicrobial dilutions either frozen or freeze-dried are commercially available, allowing testing of multiple organisms simultaneously, the method has replaced macrodilution and has been widely used in clinical microbiology laboratories. Cation-adjusted Mueller–Hinton broth (CAMHB) is recommended for standardized broth dilution methods (NCCLS, 2003). The cations Ca2+(20 to 25 mg/L) and Mg2+(10 to 12.5 mg/L) in the broth are critical for the activity of aminoglycosides tested againstP. aeruginosa
as well as for tetracycline tested against other bacteria (D’Amato et al., 1975). For convenience, CAMHB is adopted as the standardized testing medium. The final inoculum for microdilution broth methods is 5 × 105 CFU/mL. As the first step,the turbidity equal to 0.5 McFarland standard (approximately 108 CFU/mL) of bacteria suspension containing tested isolate is made either by growing in broth or direct suspension. Bacterial suspension prepared by direct inoculation is required
for testing staphylococci (CLSI, 2005). To make bacteria suspension directly, only the colonies from overnight growth on a nonselective agar plate should be used.The standard suspension is diluted 1:10 with sterile saline or broth to 107 CFU/mL,and 5 μL of the diluted suspension is added to each well containing 100 μL of broth with tested drug. As the inoculum volume is less than 10% of the total volume,the change in drug concentration after inoculation is minimal, and there is no need to increase the final drug concentration. The bacteria suspension has to be
inoculated within 30 min of preparation to maintain the desired inoculation size.
The control well without antibiotics should also be inoculated to determine the viability of the tested organism. To confirm the inoculum density and purity, 5 μL of bacteria suspension should be plated on a nonselective agar plate. All tubes and plates are incubated at 35◦C for 16 to 20 h before the MICs are determined. The recommended incubation temperature for testing staphylococci in agar dilution should be used in broth dilution. The incubation time should be extended to 24 h in order to detect vancomycin-resistant enterococci and oxacillin-resistant staphylococci (NCCLS, 2003). As with agar dilution, the drug concentration in the first well showing no bacterial growth indicated by broth turbidity is the MIC value.
For bacteriostatic agents, the drug concentration that inhibits 80% of growth is regarded as the MIC.
Both agar dilution and broth dilution are well standardized methods. They are reliable and have served as gold standards for antimicrobial susceptibility testing methods. They allow testing multiple isolates simultaneously and allow flexibility in selecting the drug combinations for testing to best fit the institution formulary.
When fastidious organisms are tested, necessary supplement can be added into the agar or broth to provide better support for bacteria growth. However, both methods are labor intensive and require certain levels of experience to read the MIC results consistently. These methods are no longer used routinely in most clinical laboratories.
For rapid detction of antimycobacterial drug susceptibility tests read:
Mycobacterium Tuberculosis, Current status in Rapid Laboratory Diagnosis [Kindle Edition]

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