Antimicrobial Susceptibility Testing

AST STANDARDIZATION

  • McFarland Turbidity Standards

    • 1% H2SO4
    • 1.175% BaCl2
  • 0.5 McFarland – most commonly used
  • Growth Medium

    • pH – 7.2 – 7.4
    • cation concentration
    • blood and serum components
    • thymidine content
Testing Medium for Different Organism
OrganismMediaIncubationNotes
Enterobacteriace ae P. aeruginosa EnterococciMueller Hinton

35°C; air
16 – 20 hrs.

 
StaphylocociMH w/ 2% NaClMH w/ 2% NaCl 
StreptococcusMHA w/ 5% Sheep’s Blood

35°C
5-10% CO2

Not needed for S. pneumoniae
H. influenzaeHTM20 – 24 hrs 
N. meningitidisMH w/ 2-5% lysed horse blood35°C;
5- 7% CO2;
24 hrs
 
N. gonorrhoeaeGC agar + supplements35°C;
5% CO2;
24 hrs
No broth dilution
AnaerobesBrucella BA w/ HeminAnaerobic,
35- 37°C;
48 hrs

Broth dilution: 1 x 10^6 cfu/ml

Agar Dilution: 1 x 10^5 cfu/spot

AUTOMATED ANTIMICROBIAL SUSCEPTIBILITY SYSTEMS
Vitek 2
  •  64-well; has specific concentration of antibiotics
  • Advanced Expert System (AES)
MicroScan WalkAway
  •  Microdilution manullary inoculated with multiprong device
Phoenix System
  • Convenient, albeit manual, gravity-based inoculation process
  • Growth monitoring – based on redox indicator system
SUPPLEMENTAL METHODS FOR ANTIMICROBIAL RESISTANCE DETECTION
Oxacillin Agar screen For staphylococcal resistance to penicillinase-resistant penicillin
Vancomycin agar screen For enterococcal resistance to vancomycin
Aminoglycoside screens For acquired enterococcal high level resistance to aminoglycosides that would compromise synergy with a cell wall-active agent
Oxacillin disk screen For streptococcus resistance among S. aureus resulting from efflux
Cefoxitin disk test 30 ug To improve detection of oxacillin resistant CoNS
Aminoglycosides For serious enterococcal infections, and acquired high-level resistance