U2.1.2 AST Flashcards
Performedonbacteriaisolatedfromclinical specimens that are clinically significant
AST
T/F The goal of AST is to provide STANDARDIZED in-vitro testing of a bacterial pathogen to a set of available antibiotics to determine its “antibiogram” in order to predict the in vivo effectiveness of a particular
antibiotic or antibiotic regimen
True
T/F In AST, the normal flora and the implicated/probable bacteria causing the infection should be tested.
False, Implicated/probable bacteria causing the infection should only be tested !
T/F All organisms are subject to AST
False
Odd One Out
Organisms that are not subject to AST
a. S. pyogenes
b. H. influenzae
c. N. meningitidis
d. L. monocytogenes
b. H. influenzae
- Testing occasionally required
Odd One Out
Organisms that are subject to AST
a. S. pyogenes
b. S. pneumoniae
c. Enterococci
d. P. aeruginosa
a. S. pyogenes
- not subject to AST
Odd One Out
Organisms that are subject to AST
a. N. gonorrhoeae
b. H. influenzae
c. N. meningitidis
d. M. cattarhalis
c. N. meningitidis
- not subject to AST
T/F
Standardization of AST is important to optimize bacterial growth conditions to ensure that the inhibition of growth can be attributed to the antimicrobial agent.
T
T/F
Standardization of AST is important to optimize conditions for maintaining antimicrobial integrity and activity, attributing the failure to inhibit bacterial growth to organism-associated resistance.
T
T/F
Standardization of AST is important to maintain reproducibility and consistency in the resistance profile of an organism, regardless of what laboratory performs the test.
T
T/F
Methods used in AST directly measure the activitv of only one antimicrobial agents
F; one or more
T/F
Methods of AST directly detect the absence of a specific resistance mechanism
F; directly detect the presence
T/F
Methods of AST use special methods that measure complex antimicrobial-organism interactions
T
Standardization of AST
Bacterial inoculum size
1.5x10^8 CFU/mL
Standardization of AST
Colonies
should not be > 1 day old
Standardization of AST
If colony is too thin, _____
If colony is too thick, ____
too thin = incubate
too thick = add broth, sterile NSS
Standardization of AST
Growth medium : pH
7.2-7.4
Standardization of AST
Growth medium : Cation concentration
Ca : 25 mg/L
Mg : 12.5 mg/L
Standardization of AST
Incubation atmosphere
humidified, ambient air; no CO2
Standardization of AST
Incubation temperature
35-37 C
Standardization of AST
Incubation duration
16-18 hrs
Standardization of AST
Antimicrobial concentrations
Short term : 2-8C
Long term : -70C
Standardization of AST
Plate Diameter
100 mm = # of disks
10 disks
Standardization of AST
Plate Diameter
150 mm = # of disks
12 disks
3 Methods that directly measure the activity of one or more antimicrobial agents
- Traditional AST Methods
- Commercially Available Methods
- Special Screens & Indicator Tests
Traditional AST Methods
Preparation of the Standardized Inoculum : Picking of #-# similar looking colonies from a non-inhibitory medium
4-5 similar looking colonies
Traditional AST Methods
Preparation of the Standardized Inoculum : Picked colonies are then transferred to a broth medium
and allowed to grow to ____ phase (___ hrs)
Log phase; 3-5 hrs
Traditional AST Methods
Preparation of the Standardized Inoculum : Alternative Method for Fastidious Organisms
- Pick 4-5 colonies from a fresh
(16-24hr) culture - Suspend in broth or NSS diluted to
proper density
Traditional AST Methods
Preparation of Standardized Inoculum : Tubirdity of broth is standardized by comparing it with ______
0.5 MacFarland Standard
Traditional AST Methods
Preparation of MacFarland Standard
0.5mL 1.175% Barium Chloride
99.5 mL 1.5% Sulfuric Acid
Traditional AST Methods
Inoculum Standardization : Standardized Inoculum must be used within ____ minutes
15 minutes
Traditional AST Methods
Comparison is done ____. Methods done for precision are ___
comparison : visually
methods : nephelometric or spectrophotometric
Traditional AST Methods
Commercially available and widely used standard
0.5 McFarland Standard
Traditional AST Methods
QC : McFarland Standard should be checked _____. At _____nm
checked montly; 625nm
Traditional AST Methods: Choice of Antimicrobials
Selection of Test Batteries/ Panel depends on :
- Protocol of hospital
- ID of organism
- any known resistance patterns
- method of AST
- availability of antimicrobial agents
Traditional AST Methods
Other name for disk diffusion Method
Kirby Bauer
Traditional AST Methods
Commonly used in vitro tests in the laboratory
Disk Diffusion Method
Traditional AST Methods
Agar used in Kirby Bauer
Mueller Hinton Agar
Traditional AST Methods
Measured in Kirby Bauer
Diameters of zones of inhibitions
Traditional AST Methods
Findings in Kirby Bauer is reported as
SIR
Susceptible, Intermediate, Resistant
T/F Kirby Bauer Method : As the agent diffuses farther, the concentration increases until it reaches a point when the bacterial growth is not anymore inhibited.
F, concentration decreases
Traditional AST Methods
Based on the inverse linear relationship between the zone of inhibition diameter and the logarithm of the minimum inhibitory concentration (MIC)
Kirby Bauer Method
Kirby Bauer Method
Incubation
35C for 16-18 hours at ambient atmosphere
Kirby Bauer Method
For Fastidious Organisms
5-10% CO2
Kirby Bauer Method
Characteristic of reading plates
should be confluent, no individual colonies should be present
Kirby Bauer Method
Factors ignored during reading (3)
- Proteus : hazy/swarming growth within the zone
- Sulfonamides & TMP : hazing
- Beta-hemolytic bacteria : hemolysis produced
Traditional AST Methods
Determine Minimum
Inhibitory Concentration (MIC)
Dilution Susceptibility Testing
Traditional AST Methods
- Use of serial 2-fold dilutions
of the antimicrobial agent (expressed as ug or mcg/ml) - Once MIC is determined, ti is interpreted as S, I, R
Dilution Susceptibility Testing
Dilution Susceptibility Testing
2 Types of Broth Dilution
Macrodilution & Microdilution
Dilution Susceptibility Testing : Broth Dilution
Use of test tubes, Volume: 1-2 ml
Macrodilution
Dilution Susceptibility Testing : Broth Dilution
Use of wells, Volume: 0.05-0.1mL
Microdilution
Broth Dilution
Report the MIC and is translated into the 3 general interpretative categories :
SIR
Susceptible, Intermediate, Resistant
Broth Dilution
NB: Recently added categories include
- Susceptible Dose Dependent
- Non susceptible
Broth Dilution
specific concentrations that separate or define the different
categories
Breakpoints
Dilution Susceptibility Testing
Antimicrobial Concentrations and organisms to be tested are placed together on an agar medium
Spot Inoculation
Dilution Susceptibility Testing
Series of dilutions, with one dilution per plate
Agar Dilution
Dilution Susceptibility Testing
Standard Inoculum for Agar Dilution
1x10^4 CFU/spot
Dilution Susceptibility Testing
lowest concentration of an antimicrobial agent ni agar that completely inhibits visible growth
MIC
Dilution Susceptibility Testing
Advantage: Anaerobic OrganismsUseful for determining the MIC of N. gonorrhoeae
Agar Plate Dilution
Commercially Available Methods (2)
- Diffusion in Agar Derivation
- E-test
Commercially Available Methods
also known as Gradient Diffusion Susceptibility Test
E-test
Commercially Available Methods
Principle: Establishment of an antimicrobial density gradient
E-test
E-test
Uses _____________ placed in a radial fashion on an inoculated plate.
thin plastic strips (with gradations)
E-test
Interpretation : MIC is read wherein the ____________-
growth ellipse intersects the Etest strip
E-test
used for ____ organisms
fastidious organisms
