EUCAST - Technical Aspects
● Disc diffusion method is based a well –known technique (Kirby-Bauer)
● The 15-15-15 minute rule:
– Use the inoculum within 15 minutes of preparation (and ALWAYS within 60 minutes)
– Apply discs within 15 minutes of inoculating plates
– Start incubation within 15 minutes of application of discs
Fiona Courtney, Pathology North, 2016
EUCAST - Technical Aspects (Media)
Mueller-Hinton II (MH) *
● Enterobacteriaceae
● Pseudomonas
● Staphylococci
● Enterococci
Mueller-Hinton with 5% horse blood and 20mg/L β-NAD (MH-F) for fastidious organisms**:
● S.pneumoniae and other streptococci
● H.influenzae
● others
* Lower concentration of thymidine than MHA
** NB- Not feasible in peripheral laboratories in PNG – probable referral function
EUCAST - Technical Aspects (Media)
Mueller-Hinton II agar
● Lower concentration of thymidine
–Trimethoprim and trimethoprim-sulfamethoxazole activity
–E.faecalis ATCC 29212 and trimethoprim/ SXT
● Controlled concentration of magnesium and calcium ions
–Aminoglycosdies (gentamicin, tobramycin, amikacin)
–P. aeruginosa ATCC 27853 and gentamicin
EUCAST - Technical Aspects (Preparation of the inoculum)
● The method requires an inoculum suspension equivalent to a 0.5 (±0.1) McFarland standard.
● Make the suspension from overnight growth on non-selective medium (try to use several morphologically similar colonies).
● Suspend the colonies in normal saline with a sterile loop or cotton swab.
● S. pneumo 0.5 McFarland from HBA plate or 1.0 McFarland from a chocolate plate.
● Use inoculum within 15 minutes of preparation.
● The surface of the agar should be dry before use
● Inoculation should result in confluent growth without being too heavy.
EUCAST - Technical Aspects (Inoculation)
● The surface of the agar should be dry before use
● Dip a sterile swab into the suspension and remove the excess fluid from the swab by turning the swab against the side of the tube
● Spread the inoculum over the entire surface of the plate by swabbing 3 ways (or using an automatic plate rotator)
● Apply discs within 15 minutes.
● Inoculation should result in confluent growth without being too heavy.
Inoculation
EUCAST - Technical Aspects (Inoculation)
EUCAST - Technical Aspects (Discs) ● Apply the discs firmly to the surface of the inoculated AND DRIED plate
● Do not move the discs once applied
● Loss of potency of antimicrobial agents in discs results in reduced zone diameters - a common source of error
– Discs (including those in dispensers) should be stored in sealed containers and protected from light.
– Store disc stocks at -20°C unless otherwise indicated by the supplier
– Store working supplies of discs at <8°C
– Allow discs to warm to room temperature before opening containers.
● Normally 6 discs are the maximum possible number on a 90mm plate
EUCAST - Technical Aspects (Incubation)
● Invert plates and incubate within 15 minutes of disc application.
● Stacking plates – uneven heating of plates.
● Incubation conditions
EUCAST - Technical Aspects (Incubation)
EUCAST - Technical Aspects (Endpoint reading)
EUCAST - Technical Aspects (Endpoint reading)
● Do not use transmitted light or a magnifying glass unless otherwise stated (e.g. S. aureus and penicillin, enterococci and vancomycin)
● Measure the zones to the nearest millimetre
● Discrete colonies growing within the zone of inhibition should be subcultured and identified and the test repeated
● Read plates at a distance of 30cm from the eye.
EUCAST - Technical Aspects (Endpoint reading)
Read zones where no obvious growth is detected by the unaided eye with the plate held at about 30cm from the eye.
EUCAST - Technical Aspects (Endpoint reading)
● S.aureus and benzylpenicillin
– Examine with transmitted light
● Disc diffusion is a reliable method for detection of penicillinase producers if zone diameter is measured correctly AND the zone edge is closely inspected
Deviations from the method - Common sources of error
● Materials used
– Mueller-Hinton agar
● Cations, thymidine, pH etc
● Manufacturing and storage
– Discs
● Compliance
● Storage
● Expiry date
Deviations from the method - Common sources of error
● Not adhering to the EUCAST methodology
– 0.5 McFarland standard – too heavy or too light
– Over inoculation of plates
– 15-15-15 minute rule
● Reading of zones – Follow the instructions!
