ANTIMICROBIAL SUSCEPTIBILITY TESTING Reading of Plates & Interpretation of Results

Disk Diffusion Method (KIRBY-BAUER Test) • The lawn must be confluent or almost confluent – if
the growth is poor & nonconfluent, the test should be
• For Aerobes and Facultative microorganisms
repeated.
• Principle: the larger the zone of inhibition, the lower
• Provided growth is satisfactory, the diameter of each
the MIC (Minimum Inhibition Conc.)
inhibition zone is measured using a caliper or ruler
• Medium: Mueller-Hinton Agar (MHA)
• Appearance of individual colonies is unacceptable.
• Standard: 0.5 McFarland / Barium Sulfate Suspension (clear dapat ang zone of inihibition)
(99.5ml of 1% H2SO4 & 0.5ml of 1.175% BaCl2
• Standard inoculum: 1.5 x 10^8 organisms/ml Read:
• pH: 7.2-7.4
• Susceptible: microorganism should respond to
• Depth: 4mm
therapy with that antimicrobial agent.
• Condition: Aerobic, No CO2 (nangamatay ang bacteria na nandon sa plate)
• Temp: 35-37°C • Intermediate: microorganism falls into a range of
• Incubation Time: 16-18 hrs susceptibility in which the MIC approaches or
• Antibiotic disc: 6mm exceed the level of antimicrobial agent that can
• Petroff-Hausser: bacterial count be achieved and for which clinical response is
• 15mm: Disk distance likely to be less than with a susceptible strain
• 150mm = 12 discs (hindi masyado gina tanggap/consider)
• 100mm = 5-6 discs • Resistant: no zone or small zone inhibition –
antibiotic is not the appropriate choice for
False Resistant False Sensitive
treatment.
15 mins (delay of disc 15 mins (delay of
application) incubation) Instances:
Increase moisture Increase drying
Thick medium Thin medium Bakit naga delay ang False Resistant?
Increase organisms = Decrease organisms = - konte lang or maliit ang inoculum time or the
decrease ZOI Increase ZOI bacteria with the antimicrobial agent
Increase moisture
Procedure: - it will dissolve your antimicrobial agent
- pag ma dissolve ang microbial agent mas madami
1. (To make a standard inoculum) Pure inocula are obtained by na ang concentration ng organism compared sa
selecting 4-5 colonies of the same morphology antimicrobial agent that’s why merong RESISTANCE
2. Colonies are suspended into 0.85% NSS to achieve a turbid If agar is too thick
suspension - smaller zones will appear because effectiveness of
the antibiotic conc. has been decreased
3. Bacterial suspension & McFarland solution are compared by - di ganon kadami ang ma disperse na antimicrobial
matching the turbidity if the tubes against a dark background
agent
4. Turn the plate 60 degrees between each streaking
(Overlapping Streaking) = 3x mag streak False Sensitive
- delay of incubation
5. Within 15mins. of inoculation, antimicrobial agents are
Increase drying
applied onto MHA. (wag tagalan)
- di na humid ang atmosphere ng bacteria
6. Within 15 mins of disk placement, plates are inverted & - organism cannot function well
incubated at 35 degree C for 16-18hrs If agar is too thin
- ma sobrahan ng disperse sa antimicrobial agent
7. Diameter of each inhibition zone is measured using a caliper
within the medium, then mag karoon ng SENSITIVITY
or ruler.