Antimicrobial Susceptibility Testing PART 3 Flashcards
Broth Dilution:
- Inoculum prep
3-5 colonies and make a McFarland standard
Broth Dilution:
- McFarland Standard
Regular 0.5 McFarland diluted down to a concentration of 5x10^5 CFU/ml
Broth Dilution Mirotiter Tray:
- Incoculation
- Deliver inoculum, via delivery prongs, into sterility tray with sterility well (neg) and growth control well (pos)
- make a PURITY PLATE
Broth Dilution Mirotiter Tray:
- Incubation
- ambient air
- 16-20 hours
- 35C
Broth Dilution Mirotiter Tray:
- Quantitative or Qualitative
Quantitative because you report a # AND you look as CLSI table to interpret S, I or R
Broth Dilution:
- Reading results
MIC is lowest concentration that inhibits growth
- needs to be reproducible with one well margin of error
SXT is read at what % inhibition
80%
MIC/MBC procedure
Take clear tubes from MIC and incubate again on a plate. If < 50 colonies can grow on a plate thats considered killed!
Minimal Bactericidal Concentration
killing capability, 99.9% of original inoculum
Minimal Inhibitory Dilution (MID) procedure
patients serum is serially diluted and a standard inoculum (5x10^5 CFU/ml) of patient organism is added. Incubate and read highest dilution which inhibits growth.
Minimal Bactericidal Dilution (MBD)
- What is MBD
- What are the results
- Highest dilution of patient serum that kills 99.9% of an original inoculum of patients organism
- results are dilutions 1:8 to 1:32 is adequate
What is the reference method for susceptibility testing?
Agar Dilution
Agar dilution set up
Antibiotic diluted into agar in two-fold concentrations
Agar Dilution used for what type of organisms
Fastidious organisms (Neisseria gonorrhoeae)
Agar Dilution, Steers replicator:
- standard inoculum per spot =
10^4 CFU/ml
Agar Dilution, Steers replicator:
- incubation requirements
- ambient air
- 35C
- 18-20 hours
Automated Methods: DADE MICROSCAN
- three methods
- Traditional MIC methods
- Photometric Reading (turbidity)
- Fluormetric Reading (best method)
Automated Methods: BD PHOENIX SYSTEM
- 3 methods
- Gravity-Based inoculation process
- Redox indicator system
- Data management system
Automated Methods: VITEK
- 3 methods
- Computer assisted analysis of growth
- Algorithm derived MIC
- Calculated MIC
Three methods for beta-lactamase detection
- Chromogenic cephalosporin: Cefinase, Nitrocefin
- Acidometric
- Iodometric
Results of the Beta Lactamase Test
Positive = organism produces enzyme to degrade penicilin, ampicillin, and amoxicillin Negative = does not produce enzyme but it may have some other mechanism of resistance to the same drug
What is the value of measuring serum levels ?
Want to know how much antibiotic is at site of infection so that therapeutic range can be determined
