BSAC Disc Diffusion

September 2006
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 Contents
Click on main headings to move to that section

• Media
• Inoculum preparation
• Inoculation of plates
• Discs
• Incubation
• Reading zones
• Interpretation
• Control
• Methicillin susceptibility testing
• Detection of β-lactamases
– in H. influenzae, M. catarrhalis, Neisseria spp.
– AmpC
– ESBLs
– Metallo-β-lactamases
 Media
Click on main headings to move to that section

• Media
• Inoculum preparation
• Inoculation of plates
• Discs
• Incubation
• Reading zones
• Interpretation
• Control
• Methicillin susceptibility testing
• Detection of β-lactamases
– in H. influenzae, M. catarrhalis, Neisseria spp.
– AmpC
– ESBLs
– Metallo-β-lactamases
Susceptibility testing media
 Susceptibility testing media
• Use only Iso-Sensitest agar (ISA, CM471,
Oxoid, Basingstoke, UK) or media shown
to have the same performance as ISA
• Media for fastidious organisms should be
supplemented with 5% defibrinated horse
blood +/- 20 mg/L β-nicotinamide adenine
dinucleotide (NAD) (various chemical
suppliers; Mast Group sell vials in
appropriate pre-weighed amounts)
 Media for different organisms
Organisms Medium

Enterobacteriaceae Iso-Sensitest agar (Oxoid CM471)

Pseudomonas spp.
Stenotrophomonas maltophilia
Staphylococci (except oxacillin
testing)
Enterococci
Staphylococci (oxacillin testing) Columbia agar + 2% NaCl or
Mueller-Hinton agar + 2% NaCl
Streptococcus pneumoniae Iso-Sensitest agar + 5% defibrinated
Haemolytic streptococci horse blood
Moraxella catarrhalis
Neisseria spp.
Haemophilus spp. Iso-Sensitest agar + 5% defibrinated
Pasteurella multocida horse blood + 20 mg/L NAD
α-haemolytic streptococci
 Preparation of media
• Prepare media as directed by the
manufacturer.
• Do not add blood or NAD until medium has
been cooled to 42-45°C (45°C maximum-
ensure that media preparators have
accurate temperature settings), mix well and
pour plates immediately.
• Pour plates on a level surface to give a
depth of 4.0 ± 0.5 mm (25 mL in a 90 mm
Petri dish).
 Drying and storage of agar media
• Drying requirements
– there should be no visible drops of water
on the surface of the agar when the plates
are used
– plates must not be over dried.
• Storage at 4-8°C in sealed plastic bags is
most appropriate for clinical laboratories.
• Drying and storage conditions will depend on
available equipment and should be
determined by individual laboratories as part
of their quality assurance programme.
• Neither drying nor storage should have any
effect on zone sizes for control organisms.
 Inoculum preparation
Click on main headings to move to that section

• Media
• Inoculum preparation
• Inoculation of plates
• Discs
• Incubation
• Reading zones
• Interpretation
• Control
• Methicillin susceptibility testing
• Detection of β-lactamases
– in H. influenzae, M. catarrhalis, Neisseria spp.
– AmpC
– ESBLs
– Metallo-β-lactamases
 Inoculum preparation
• The method requires an inoculum giving
semi-confluent growth of colonies on
plates
– failure to achieve semi-confluent growth is
easily seen and tests should be repeated
– heavy inoculum reduces zone size and
may lead to a false resistant result
– light inoculum increases zone sizes and
may lead to a false susceptible result.
• Any method of inoculum preparation which
reliably produces semi-confluent growth
may be used.
 Inoculum preparation
• Growth method
– Touch at least 4 well-isolated colonies and
transfer to IsoSensitest broth or equivalent
– Incubate broth with shaking at 35-37°C until visib ly
turbid (turbidity at least equal to a 0.5 McFarland
standard)
• Suspension method
– Evenly suspend at least 4 well-isolated colonies in
IsoSensitest broth or equivalent, or distilled water
until visibly turbid (turbidity at least equal to a 0.5
McFarland standard)
– This is the method of choice for fastidious
organisms but with some there may be problems
with production of an even suspension
Well-isolated colonies required
Picking colonies for inoculum
Preparation of inoculum suspension
 Use of 0.5 McFarland standard
• Adjust the density of the suspension to that of
the 0.5 McFarland standard by visual
comparison. A white background with a
contrasting black line assists comparison
• Alternatively, photometers are available
commercially with a scale calibrated in
McFarland standard units (e.g. bioMerieux,
Becton Dickinson)
• Within 10 minutes of preparation of the
standard suspension dilute to required
density in distilled water
Preparation of McFarland 0.5 standard
• Add 0.5 mL 0.048M BaCl2 (1.17% w/v BaCl2.2H20) to 99.5 mL
0.18M H2SO4 (1% w/v) with constant stirring.
• Using matched cuvettes with a 1 cm light path and water as a
blank standard, measure the absorbance in a
spectrophotometer at a wavelength of 635 nm. The acceptable
range is 0.08-0.13.
• Distribute in tubes the same size as those used for test
organism suspensions and seal tubes.
• Store in the dark at room temperature.
• Vigorously vortex before use.
• Standards should be replaced after 6 months.
• Alternatively, purchase standards from a commercial source
(e.g. bioMerieux) but should be checked to ensure that
absorbance is within the acceptance range (see above).
Dilution of inoculum for comparison
with 0.5 McFarland standard
Standardisation by comparison with
0.5 McFarland standard
Dilution of 0.5 McFarland suspension
1:100 dilution 1:10 dilution No dilution
β-haemolytic streptococci Staphylococci N. gonorrhoeae

Enterococci Serratia spp

Enterobacteriaceae S. pneumoniae

Pseudomonas spp N. meningitidis

S. maltophilia
Acinetobacter spp M. catarrhalis

Haemophilus spp α-haemolytic

P. Multocida streptococci

Use suspensions within 15 min of preparation

Photometric standardisation of inoculum
• For the method described the photometer must
have a cell holder for 100 x 12 mm tubes.
• Prepare suspension in 3mL volumes in 100 x 12
mm new glass tubes (do not reuse).
• Zero spectrophotometer at 500 nm with
suspension fluid blank and measure the
absorbance of the bacterial suspension.
• Dilute the suspension by adding volumes as
indicated in the table to 5 mL distilled water.
• Mix the diluted suspension and use within 15
minutes of preparation.
Reading turbidity with spectrophotometer
 Suspension dilution (may need
adjustment for different photometers)
Organisms Absorbance Vol. (µ
µL) in 5
mL water

Enterobacteriaceae 0.01 – 0.05 250

Acinetobacter spp >0.05 – 0.1 125
Pseudomonas >0.1 – 0.3 40
Staphylococci >0.3 – 0.6 20
Enterococci >0.6 – 1.0 10

Haemophilus 0.01 – 0.05 500

Streptococci >0.05 – 0.1 250
Other fastidious organisms >0.1 – 0.3 125
>0.3 – 0.6 80
>0.6 – 1.0 40
 Inoculation of plates
Click on main headings to move to that section

• Media
• Inoculum preparation
• Inoculation of plates
• Discs
• Incubation
• Reading zones
• Interpretation
• Control
• Methicillin susceptibility testing
• Detection of β-lactamases
– in H. influenzae, M. catarrhalis, Neisseria spp.
– AmpC
– ESBLs
– Metallo-β-lactamases
 Inoculation of plates
• Use the adjusted suspension within 15
minutes.
• Dip a cotton wool swab in the suspension and
remove excess by turning swab against the
side of the tube.
• Spread inoculum evenly over the entire
surface by streaking in three directions or use
a rotary plater, taking care not to leave gaps
between streaks.
• Allow the plate to dry before applying discs.
Inoculation of plates on rotary plater
Inoculation of plates by streaking
in three directions
 Acceptable range of inoculum
Lightest acceptable Ideal Heaviest acceptable
 Discs
Click on main headings to move to that section

• Media
• Inoculum preparation
• Inoculation of plates
• Discs
• Incubation
• Reading zones
• Interpretation
• Control
• Methicillin susceptibility testing
• Detection of β-lactamases
– in H. influenzae, M. catarrhalis, Neisseria spp.
– AmpC
– ESBLs
– Metallo-β-lactamases
 Storage of antimicrobial discs
• Store discs (including those in dispensers) in sealed
containers with an indicating desiccant, protected
from light.
• Store stocks of discs as recommended by the maker.
• Store working supplies at 4-8°C.
• To prevent condensation, allow discs to warm to
room temperature before opening containers. It is
better to keep discs at room temperature during the
day than to transfer repeatedly to and from cold
storage.
• Discard discs on the expiry date shown on the
container.
 Application of antimicrobial discs
• Inoculated plates must be dry before discs
are applied.
• Firmly apply discs to the surface of the
medium within 15 min of inoculation.
• No more than 6 discs should be applied to
9 cm plates.
Application of antimicrobial discs
 Incubation
Click on main headings to move to that section

• Media
• Inoculum preparation
• Inoculation of plates
• Discs
• Incubation
• Reading zones
• Interpretation
• Control
• Methicillin susceptibility testing
• Detection of β-lactamases
– in H. influenzae, M. catarrhalis, Neisseria spp.
– AmpC
– ESBLs
– Metallo-β-lactamases
 Incubation
• Incubate plates within 15 min of disc
application to avoid increased zone sizes.
• Do not stack plates too high as uneven
heating of plates may affect zone sizes
(depends on efficiency of incubator).
• Note that oxacillin susceptibility tests on
staphylococci are incubated at 30°C for 24 h.
• Note that cefoxitin susceptibility tests on
staphylococci are incubated at 35°C.
• Note that enterococci should be incubated for
24 h before reporting strains susceptible to
vancomycin or teicoplanin.
 Incubation of plates
Organism Incubation conditions
Enterobacteriaceae 35-37oC in air for 18-20h
Pseudomonas spp. 35-37oC in air for 18-20h
S. maltophilia 30oC in air for 18-20h
Staphylococci 35-37oC in air for 18-20h
30oC in air for 24h for oxacillin/methicillin
35oC in air for 18-20h for cefoxitin
M. catarrhalis 35-37oC in air for 18-20h
α-haemolytic streptococci 35-37oC in 4-6% CO2 for 18-20h
β-haemolytic streptococci 35-37oC in air for 18-20h
Enterococci 35-37oC in air for 24h
N. meningitidis 35-37oC in 4-6% CO2 for 18-20h
S. pneumoniae 35-37oC in 4-6% CO2 for 18-20h
Haemophilus spp. 35-37oC in 4-6% CO2 for 18-20h
N. gonorrhoeae 35-37oC in 4-6% CO2 for 18-20h
P. multocida 35-37oC in 4-6% CO2 for 18-20h
 Reading zones
Click on main headings to move to that section

• Media
• Inoculum preparation
• Inoculation of plates
• Discs
• Incubation
• Reading zones
• Interpretation
• Control
• Methicillin susceptibility testing
• Detection of β-lactamases
– in H. influenzae, M. catarrhalis, Neisseria spp.
– AmpC
– ESBLs
– Metallo-β-lactamases
 Reading zones
• Use reflected light except for staphylococci with
oxacillin and enterococci with vancomycin
(reflected and transmitted light).
• Zone edges should be taken as the point of
complete inhibition as judged by the naked eye.
• Ignore tiny colonies within obvious zone edges,
swarming of Proteus spp and faint growth in
sulphonamide and trimethoprim zones.
• Subculture colonies growing within zones and
repeat tests if necessary.
• Measure zone diameters with a ruler, calipers or
automated zone reader; alternatively, a template
may be used.
Measuring zones with digital caliper
Measuring zones with a ruler
 Interpretation
Click on main headings to move to that section

• Media
• Inoculum preparation
• Inoculation of plates
• Discs
• Incubation
• Reading zones
• Interpretation
• Control
• Methicillin susceptibility testing
• Detection of β-lactamases
– in H. influenzae, M. catarrhalis, Neisseria spp.
– AmpC
– ESBLs
– Metallo-β-lactamases
 Interpreting zones
• If zones are measured, interpret into
categories of susceptibility according to
published tables.
• If templates are used, read the category
of susceptibility from the template.
• Confirm that control zones are within
acceptable limits before interpreting
tests.
 Interpreting zones with templates

A template program is available at www.bsac.org.uk

 Control
Click on main headings to move to that section

• Media
• Inoculum preparation
• Inoculation of plates
• Discs
• Incubation
• Reading zones
• Interpretation
• Control
• Methicillin susceptibility testing
• Detection of β-lactamases
– in H. influenzae, M. catarrhalis, Neisseria spp.
– AmpC
– ESBLs
– Metallo-β-lactamases
 Control strains
Organism NCTC (ATCC) strain Characteristics
E. coli 12241 (25922) or Wild type
10418
E. coli 11560 β-lactamase producer
S. aureus 12981 (25923) or Wild type
6571
S. aureus 12493 Oxacillin resistant
P. aeruginosa 12934 (27853) or Wild type
10662
E. faecalis 12697 (29212) Wild type
H. influenzae or 11931 Wild type
H. influenzae 12699 (49247) Ampicillin-resistant, β-
lactamase negative
S. pneumoniae 12977 (49619) Penicillin intermediate
N. gonorrhoeae 12700 (49226) Wild type
 Control of susceptibility testing

• Use susceptible strains to monitor test

performance
• Resistant strains may be used to confirm the
ability to detect mechanisms of resistance.
• Strains may be purchased from the NCTC or
from commercial sources, eg. Oxoid, Mast
Laboratories, Becton Dickinson or TCS
Biosciences.
 Use of control strains
• Use a reading frame of 20 consecutive days
• Acceptable if not more than 1 in 20 results is
out of range
• Two or more results out of range requires
investigation
• Also look for trends (+ or -) and for zones
falling consistently above or below the mean
• Test daily until results are acceptable then
move to weekly testing
 Monitoring test performance
Single results outside
control limits
25
Upper limit
Zone diam eter (mm)

20 Mean
Lower limit
15
Ten results within Consecutive results
10
limits but on one outside limits on same
side of the mean side of the mean
5

0
0 5 10 15 20 25 30 35 40
Day
Response to QC results out of control
• Investigate but report:
– Single control zone size above or below
acceptable range but frequency of susceptible
test results no greater than normal
– Single control zone above or below acceptable
range but frequency of resistant test results no
greater than normal
• Suppress, investigate and retest:
– More than one in 20 control zone sizes above or
below acceptable range
– failure to recognise resistance in resistant control
 Additional QC measures
• Investigate and retest:
– detection of resistance not previously
observed
– detection of resistance rare locally
– detection of any atypical result
– errors indicated by UK NEQAS
 Potential sources of error
Test conditions Controls Medium Discs
Non-susceptibility Wrong agent or
Pre-incubation Contamination
testing agar content
Not prepared to
Pre-diffusion Mutation manufacturer’s Labile agent
instructions
Batch to batch
Incubation temperature Inoculum density Light sensitive agent
variation

Incubation atmosphere Uneven inoculation Antagonists Incorrect storage

Discs not at room

Incubation time Age of culture pH
temperature

Illumination Divalent cations Labelling

Reading zone edges Depth Expiry date passed

Expiry date Number of discs
Storage & subculture of control strains
• Store strains at -70 °C in glycerol broth on
beads: one “in-use” vial, one “archive”.
• Subculture weekly from the in-use vial onto
appropriate non-selective media and check for
purity.
• Subculture from the purity plate each day for up
to 7 days.
• Fastidious organisms only may be serially sub-
cultured for 6 days.
• When the in-use vial is depleted, subculture
from the archive vial and prepare another in-
use vial from the subculture.
Methicillin susceptibility testing
Click on main headings to move to that section

• Media
• Inoculum preparation
• Inoculation of plates
• Discs
• Incubation
• Reading zones
• Interpretation
• Control
• Methicillin susceptibility testing
• Detection of β-lactamases
– in H. influenzae, M. catarrhalis, Neisseria spp.
– AmpC
– ESBLs
– Metallo-β-lactamases
Methicillin susceptibility testing of staphylococci
• Susceptibility testing can be difficult
– expression of resistance is affected by test
conditions
– resistance is often heterogeneous.
• Lower incubation temperatures and
increased NaCl concentration improve
reliability of tests.
• Coagulase-negative staphylococci may not
grow well on media + NaCl or at 30°C and
may be slower growing than S. aureus.
Methicillin susceptibility testing of staphylococci
Methicillin or oxacillin discs
• Prepare Columbia or Mueller-Hinton agar to manufacturer’s instructions and add
2% NaCl. Autoclave, mix well and pour.
• Use controls ATCC 25923 (or NCTC 6571) to test disc content and NCTC
12493 as a representative MRSA.
• Use methicillin 5 µg, oxacillin 1 µg discs.
• Incubate at 30°C for 18-20h. Detection of resistan ce in CNS may require
incubation for 48 h.
• Interpretive criteria
– susceptible ≥ 15 mm diameter
– resistant ≤ 14 mm diameter.
• Examine zones carefully in good light, both reflected and transmitted, to detect
colonies, which may be minute, in zones. If contamination is suspected, these
should be identified and re-tested.
• Increase in oxacillin zone size in the presence of clavulanate is not a reliable
test for detection of hyper-producers of β-lactamase. PCR or latex agglutination
tests for mecA are required.
Methicillin/oxacillin zones with MRSA
Methicillin susceptibility testing of S. aureus
Cefoxitin discs
• Prepare Iso-Sensitest agar according to manufacturer’s instructions.
• Use controls ATCC 25923 (or NCTC 6571) to test disc content and
NCTC 12493 as a representative MRSA.
• Use cefoxitin 10µg discs.
• Incubate at 35°C for 18-20h.
• Interpretive criteria
– susceptible ≥ 22 mm diameter
– resistant ≤ 21 mm diameter.
• Detection of resistance in CNS is not reliable under these conditions.
• Examine zones carefully in good light, both reflected and transmitted, to
detect colonies, which may be minute, in zones. If contamination is
suspected, these should be identified and re-tested.
• Hyper-producers of β-lactamase give zones within the range of the
susceptible population
Reading cefoxitin zones with S. aureus

“Obvious” outer zone

Inner zone with small colonies

 Detection of β-lactamases
Click on main headings to move to that section

• Media
• Inoculum preparation
• Inoculation of plates
• Discs
• Incubation
• Reading zones
• Interpretation
• Control
• Methicillin susceptibility testing
• Detection of β-lactamases
– in H. influenzae, M. catarrhalis, Neisseria spp.
– AmpC
– ESBLs
– Metallo-β-lactamases
 Detection of β-lactamase in
Haemophilus influenzae, Moraxella
catarrhalis and Neisseria spp.
• Nitrocefin test
• Acidometric test (not for M. catarrhalis)
 Inducible AmpC β-lactamase
• Chromosomal and inducible
– Enterobacter spp., Citrobacter freundii, Serratia spp.,
Morganella morganii, Providencia spp.,
Pseudomonas aeruginosa
– May appear susceptible to 2nd & 3rd generation
cephalosporins (poor inducers)
– Best recognised by speciation
– Enterobacter spp. and C. freundii are cefoxitin
resistant
– Demonstrated by cefoxitin/cefotaxime disc
antagonism
Cefoxitin/cefotaxime antagonism

Cefoxitin (a labile
agent and a good
inducer of AmpC)
blunts the zone of
cefotaxime (also a
labile agent but a
poor inducer).
 Derepressed AmpC
• AmpC-inducible strains may segregate derepressed
mutants
• This resistance is obvious in the BSAC disc diffusion test
• Susceptible inducible strains may segregate resistant
mutants during therapy (20% infections) and these may
result in clinical failure
• Report all inducible strains as resistant to 2nd & 3rd
generation cephalosporins
• AmpC may be carried on plasmids in Klebsiella spp and
E. coli. This may be distinguished from ESBL-mediated
resistance by resistance to cefoxitin in AmpC producers
Derepressed AmpC

Derepressed
colonies within
cephalosporin
zones (inducible
AmpC-producing
strains can become
permanently
derepressed)
 Exended spectrum β-lactamases
(ESBLs)
• Mainly in klebsiellae and E.coli
• Mostly mutants of TEM and SHV enzymes or CTX-M
variants
• Hydrolyse oxyimino-aminothiazolyl cephalosporins
• Resistance is not always obvious in disc diffusion tests
• Associated with clinical failure
 Detection of ESBLs
• Infer from resistance of klebsiellae or E. coli to
cefotaxime or ceftazidime (test both), or to
cefpodoxime
• Confirm with:
– Double-disc tests
– Combined disc methods
– Etest ESBL strips
– Automated susceptibility methods
• Cefepime/clavulanate may be useful for genera
where clavulanate induces AmpC or AmpC is
derepressed
 Combination disc tests for ESBLs
(E coli with CTX-M)
Cefpodoxime +
clavulanate 10+1µg
Cefpodoxime 10µg

Discs contain the cephalosporin alone and in combination with clavulanate. Difference of
> 5 mm in zone diameter (Oxoid) or > 50% increase in zone diameter (Mast, BD)
indicates ESBL
 Combination disc tests for ESBLs
(E coli with CTX-M)
Cefotaxime + Cefotaxime 30µg Ceftazidime + Ceftazidime 30µg
clavulanate 30+10µg clavulanate 30+10µg

Discs contain the cephalosporin alone and in combination with clavulanate. Difference of
> 5 mm in zone diameter (Oxoid) or > 50% increase in zone diameter (Mast, BD)
indicates ESBL
 Double-disc tests for ESBLs
(E coli with TEM-5)
Co-amoxiclav 20+10µg

Ceftazidime 30µg Cefotaxime 30µg

Use clavulanate (co-amoxiclav) discs in proximity to cephalosporin discs. The distance

between discs is critical. Any distortion of zones indicates ESBL
 Etest for ESBLs
(E coli withTEM-5)
ceftazidime + clavulanate ceftazidime

Etests (cefotaxime or ceftazidime +/- clavulanate) may be used to confirm ESBL

production. > eightfold decrease in MIC in presence of clavulanate indicates ESBL
 Metallo-β-lactamases

• Confer resistance to carbapenems

• Mainly Acinetobacter spp and Pseudomonas spp
• Detection methods involve testing for synergy between
imipenem and EDTA (Etest) but note limitations:
– Potentiation may not relate to inhibition of β-lactamases
– Some metallo-β-lactamase producers are susceptible in vitro
(permeability defect may also be required)
 Detection of metallo-β-lactamase
with the Etest

A metallo-β-lactamase in Ps.aeruginosa confers resistance to

imipenem. The Zn-dependent enzyme is inactivated by the
chelating agent EDTA, reducing the MIC by 8-fold or greater.