Antimicrobial sensitivity testing L12 Flashcards
what does clinical antibiotic-sensitivity testing tell us
Confirms the organism is sensitive to the antibiotic being used to treat the patient; or suggests an alternative antibiotic
what does epidemiological antibiotic-sensitivity testing tell us
Sensitivity data on common pathogens provides useful public health information, locally and nationally
what does pharmaceutical antibiotic-sensitivity testing tell us
Provides information on the activity of new antibiotics against a range of pathogens
what are the antibiotic-sensitivity methods
clinical
epidemiological
pharmaceutical
what are the phenotypic methods
agar diffusion assays
broth dilution assays
agar incorporation assays
what happens in an agar diffusion assays
antibiotic diffuses from an impregnated paper disc into an agar medium on which the test organism is growing
what happens in a broth dilution assay
Serial dilutions of the antibiotic in broth growth medium are inoculated with the test organism
what happens in an agar incorporated assay
antibiotic dilutions are incorporated in an agar medium and spot inoculated with a number of test organisms
how is an agar diffusion assay carried out
- Spread plate (blood agar) with lawn of test strain
- Place antibiotic discs on lawn
- Incubate overnight
- Examine plate for zones of inhibition
how can agar diffusion assays be sped up
using an antibiotic disc applicator, is automated applies all the antibiotics at once
what is the radius of the zone of inhibition determined by
- rate of antibiotic diffusion
- growth rate of the bacteria – incubation temperature
- sensitivity of the bacteria to the antibiotic
what is MIC
Minimum Inhibitory Concentration
what does the zone of inhibition relate to
size of the zone of inhibition is related to the MIC of the test organism
what controls are important in agar diffusion assays
- control organism of known sensitivity
- inoculate test organism and control organism on the same “Stokes Assay”
- standardise the methods used (EUCAST)
for staphylococcus aureus what zone size indicates it is resistant
<20mm (MRSA)
for staphylococcus aureus what zone size indicates it is sensitive
> 20mm (MSSA)
what are the problems with agar diffusion assays
- low solubility, ionic charge, high molecular weight
- growth rate of test bacteria
- antibiotic concentration in the disc
what is the problem with low solubility, ionic charge, high molecular weight in agar diffusion assay
poor diffusion = small zone of inhibition, even if the organism is sensitive
what is the problem with slow growing bacteria
slow growing organisms give rise to large zones, can diffuse out a long way before organism grows to be visible
slower growth period it can be inhibited more effectively as lower numbers of colonies on plate
how can the problem of antibiotic concentration in the disc in agar diffusion assays be solved
Commercially available discs have a tolerance of 67-150%; a 30micrograms disc could have 20-45micrograms
how can the MIC be determined
E test
broth dilution assay
agar incorporation
what is the E test
Plastic strips contain a gradient of antibiotic and are printed with the concentration
During incubation of the plate antibiotic diffuses out and sets up a gradient
higher concentration at top of strip
how is the MIC determined in the E test
E Test meniscus - the MIC corresponds to the point where the bacterial growth crosses the numbered strip.
what happens in a broth dilution assay
Doubling dilutions of antibiotic in 1ml of broth medium
Inoculate each tube with 105 cells of the test strain and incubate overnight
how is the MIC found in broth dilution assay
determine the lowest concentration which inhibits growth
what is MBC
minimum bactericidal conentration