WEEK 11: ANTIMICROBIAL SUSCEPTIBILITY TESTING Flashcards
reviewed the susceptibility testing
literature.
Kirby and Bauer
standardized procedure for the disk diffusion
henceforth called the
Kirby-Bauer disk diffusion test.
` is responsible for updating the original
procedure of Kirby Bauer disk diffusion through global
consensus process to ensure uniformity and reproducibility
CLSI
A. Conventional Methods IN ANTIMICROBIAL SUSCEPTIBILITY
- Broth dilution (gold standard)
- Agar dilution
- Disk diffusion
COMMERCIAL SYSTEM IN ANTIMICROBIAL SUSCEPTIBILITY
Commercial Systems
1. Antibiotic gradient diffusion or E-test
2. Vitek 2
Doubling dilution is incorporated into agar
Multiple isolates tested on each plate
AGAR DILUTION
Visually examine for growth, determine MIC (minimum
inhibitory concentration)
AGAR DILUTION
Final amount of organism spotted IN AGAR DILUTION
Final amount of organism spotted: 1 x 104 CFU
Agent is applied in gradient to a test strip
Plate is seeded with organism as in K-B
Agent diffuses away from strip to inhibit growth
Antibiotic Gradient Diffusion (E-Test)
Surface of agar plate seeded with lawn of test
organism
Inoculum: swan from 0.5 McFarland
Disk containing known concentration of agent placed
on surface of plate
Measure if diameter of zone of inhibitioN
Disk-Diffusion (Kirby-Bauer)
INOCULUM FOR Disk-Diffusion (Kirby-Bauer)
Inoculum: swan from 0.5 McFarland
ginal method of determining susceptibility to
antimicrobials was based on
broth dilution methods
proposed a single disk method for
antimicrobial susceptibility testing.
University of Washington School of Medicine and the King
County Hospital
form a committee in 1961 to lay the
groundwork for the development of a standardized
procedure for single antimicrobial disk susceptibility
testing.
WHO
represents the standard for clinical
laboratories performing susceptibility testing today
Performance Standards for
Antimicrobial Disk Susceptibility Tests; Approved
Standard 9th Edition,
determine the sensitivity or
resistance of pathogenic aerobic and facultative
anaerobic bacteria t
Kirby-Bauer disk diffusion
viable alternative to broth
dilution methods for laboratories without the resources
to utilize the newer automated methods for broth
microdilution testing.
e disk diffusion method of Kirby and Bauer
FILTER PAPER SIZE
6-mm filter paper disk impregnated with a
knownconcentration of an antimicrobial compound is
placed on a Mueller-Hinton (MH) agar plate,
The rate of diffusion of the antimicrobial through the agar is dependent on
the diffusion and solubility properties
of the drug in MH agar and the molecular weight of the antimicrobial compound.
r the inhibitory effects of the antimicrobial
compound. The estimated time of a bacterial
suspensionto reach critical mass is
4 to 10 hours
The size of the zone of inhibition of growth is
influenced by the depth of the agar, since the
antimicrobial diffuses in three dimensions, thus a shallow layer of agar will produce a larger zone of inhibition thana deeper layeR
The concentration of
antimicrobial compound at this margin is called the
critical concentration
The current interpretation standards can be found in
the Clinical Laboratory Standards Institute
Performance Standards for Antimicrobial Disk
Susceptibility Tests: Approved Standards 9th Edition
MH agar is considered the best medium to use for
routine susceptibility testing of non-fastidious
bacteria
MH AGAR
Mueller-Hinton agar is stable for approximately
70 days
f you prepare the MH agar plates from dehydrated media, the plates must be poured to a depth of
4 mm (approximately 25 ml of liquid agar for 100-mm plates and 60 ml of liquid agar for 150-mm plates, but in any case, to a measured depth of 4 mm).
pH of the MH agar should fall
between 7.2 and 7.4 at
room temperature
______________ can reverse the
inhibitory effects of sulfonamides and trimethoprim resulting in ___________
Excessive thymidine or thymine can reverse the
inhibitory effects of sulfonamides and trimethoprim
resulting in smaller and less distinct zones of inhibition,
or no zones at al
incorrect concentration of divalent cations (calcium
and magnesium) will affect the
results of aminoglycoside
and tetracycline tests against Pseudomonas aeruginosa.
Excess cation concentration will result in
reduced zone sizes and low concentration will increase zone size
Excess calcium will increase the zone size of P.
aeruginosa against daptomycin. Excess zinc ions may
reduce the zone size of carbapenems against P. aeruginosa
spring-loaded cartridges contain
25 or 50 disks
ealed cartridges containing commercially prepared paper disks should be stored at room temperature
either 8°C or frozen at - 14°C in a non-self-defrosting freezer
are suspensions of either
barium sulfate or latex particles that allow visual
comparison of bacterial density
mcFarland standards
, which is a small card
containing parallelblack lines.
Wickerham card
nasa macfarland standard siya
preparing mh agar
. Allow a MH agar plate (one for each organism to be
tested) to come to room temperature. It is preferable to
allow the plates to remain in the plastic sleeve while they
warm to minimize condensation.
2. If the surface of the agar has visible liquid present, set the
plate inverted, ajar on its lid to allow the excess liquid to
drain from the agar surface and evaporate. Plates may be
placed in a 35°C incubator or in a laminar flow hood at
room temperature until dry (usually 10 to 30 minutes).
3. Appropriately label each MH agar plate for each
organismto be tested
preparation of incolum
- Using a sterile inoculating loop or needle, touch four or
five isolated colonies of the organism to be tested. - Suspend the organism in 2 ml of sterile saline.
- Vortex the saline tube to create a smooth suspension.
- Adjust the turbidity of this suspension to a 0.5 McFarland
standard by adding more organism if the suspension is
too light or diluting with sterile saline if the suspension is
too heavy. - Use this suspension within 15 minutes of preparation.
Antibiotic Susceptibility Disks
Mcfarland Standard
Protocol
Preparation of Mueller-Hinton (MH) Plate
P
inc
