AST PT.2 Flashcards
Kirby Bauer Method Test Performance/Procedure
Disk Storage:
Working Supply (1week): 1.
Stocks (Long Term): 2.
Store in a tightly sealed container with dessicant
* Culture Medium: 3
May be supplemented with 4. or other nutrients
- 2-8C
- -20C in a non frost freezer
- Mueller Hinton
- 5% sheep blood
Kirby Bauer Method Test Inoculum and Inoculation:
0.5 McFarland Standard Inoculum
* 1. is dipped onto the inoculum and swabbed evenly on the culture medium
* 2. mm diameter 3. plate is used
It can accommodate up to 4. antibiotic disks
* After __ minutes, the disks are then placed
- Sterile Cotton Swab
- 150
- MHA (Mueller hinton agar)
- 12
- 15 mins
Advantages of Kirby Bauer Method
convenient and user friendly
Disadvantages of Kirby Bauer Method
- Not all organisms have interpretative criteria
- Unable to provide more precise data about the level of resistance
Determine Minimum Inhibitory Concentration (MIC)
* Use of serial 2-fold dilutions of the antimicrobial agent (expressed as ug or mcg/ml)
* Once MIC is determined, it is interpreted as S, I, R
DILUTION SUSCEPTIBILITY TESTING
DILUTION SUSCEPTIBILITY TESTING Principle
* Standard Culture: _
* Final Standard Bacterial Inoculum: ___
MHA( Mueller Hinton agar), 5x10^5 CFU/ml
- Challenging the organism with antimicrobial agents in a liquid environment
- Use of different range of antimicrobial agent concentrations
Determination of MIC (Minimal Inhibitory Concentration)
DILUTION SUSCEPTIBILITY TESTING
Prepared from reference standard antimicrobial powders
Stock Solutions
What is the storage for DST: Stock solutions?
-60C
Use of test tubes
Volume: 1-2 ml
Macrodilution
Use of wells
Volume: 0.05-0.1ml
Microdilution
- The first tube or well showing no visible bacterial growth is the MIC
- Maximum of Number of Disks per 150mm diameter plate: 12 disks
- Culture Plates should not be stacked more than 5 plates high
Broth Dilution
Antimicrobial Concentrations and organisms to be tested are placed together on an agar medium (Spot inoculation)
Series of dilutions, with one dilution per plate
Standard Inoculum: 1x104 CFU
MIC: lowest concentration of an antimicrobial agent in agar that completely inhibits visible growth
Agar Dilution
Broth Microdilution Methods
BBL Sceptor, MicroScan touch SCAN-SR
Agar Dilution Derivations
Spiral Biotech
Diffusion in Agar Derivatio
E-test: Gradient Diffusion Susceptibility Test
- Establishment of an antimicrobial density gradient
Uses thin plastic strips (with gradations) placed in a radial fashion on an inoculated plate.
Interpretation: MIC is read wherein the growth ellipse intersects the Etest strip
Useful for fastidious organisms
E-test
Test for the presence of Beta-lactamas
Phenotypic Methods
Use of filter paper disks impregnated with nitrocefin
Moisten with water or NSS and add a loopful of the colony onto the disk
(+) Result: RED COLORATION
Cephalosporinase test (Cefinase Test)
other methods for Beta-lactamse
Iodometric
Acidometric Methods
Determination of chloramphenicol resistance
Chloramphenicol Acetyltranferase Detection
Detection of Methicillin (Oxacillin) resistant S. aureus
Use of chromogenic agar (ex. CHROM Agar)
For Reliable Detection: MIC
MIC of >/= 4ug/ml (Oxacillin) = MRSA
VRSA
VISA
Vancomycin Resistance in Staphylococcus
Inducible Clindamycin Resistance Test)
D-zone test – for Staphylococcus
Molecular Methods to determine presence of resistant genes
*Detection of mecA gene and Detection of vanA gene
Genotypic Methods
– gold standard for confirmation of phenotypic oxacillin resistance
* Latex Agglutination Test
*Molecular Methods
Detection of mecA gene
Tests that Predict Effectiveness of Therapy
- MIC
- Disk Diffusion
- Etest
- Growth Curve Calculation
