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!