Journal of Antimicrobial Chemotherapy (2001) 48, Suppl.

S1, 43–57
JAC
BSAC standardized disc susceptibility testing method

Jennifer M. Andrews* for the BSAC Working Party on Susceptibility Testing

Department of Microbiology, City Hospital NHS Trust, Birmingham B18 7QH, UK

For nearly a decade microbiologists have used the MIC breakpoints published in the BSAC
Guide to Susceptibility Testing to interpret susceptibility. Historically, and unlike the rest of
Europe, the UK and Ireland have used a comparative method of disc testing to interpret suscept-
ibility rather than one based on a correlation between MIC and zone of inhibition. Although
innovative when introduced in the 1970s, Stokes’ comparative method has evolved ad hoc and
it has become increasingly apparent that there is a need for a standardized method of disc
testing that is correlated with BSAC MIC breakpoints. The method described here, like all other
standardized methods of disc testing, cannot be adapted by the user, and interpretative
criteria are only applicable if the method is adhered to fully. A major advantage of this approach
to susceptibility testing is that data from several sources can be combined for surveillance
of resistance, a task that has been made much easier by the introduction of this method and
coincides with the availability of automated zone measuring devices. It is hoped that the
method described here will provide the core document for standard operating procedures;
however, changes will necessarily occur over time as the method is developed and refined.

Introduction Fortuitously, the introduction of the standardized method

The BSAC Guide to Sensitivity Testing1 was published in
1991 and one of its most important sections was that deal-
ing with breakpoints for clinically relevant bacteria. These
breakpoints have been used extensively to interpret MIC
results and for single concentration ‘breakpoint’ tests.
However, a criticism of the guidelines was that they did not
provide a standardized method of disc diffusion testing
with zone limits that correlated with these MIC break-
points. The limitations of the widely used Stokes’ compara-
tive method were also a cause for concern.
The task of developing such a method of disc testing is
immense, and the Working Party and the Council of the
BSAC needed evidence that there was sufficient interest to
warrant the investment required not only in the short term,
but also for continuing support and development. This
necessary confirmation was obtained from a questionnaire
survey,2 which indicated that 90.6% of UK laboratories
would be prepared to switch to an upgraded disc test, and
the development and ‘field testing’ of the standardized
method was consequently undertaken [see elsewhere in this
Supplement (Andrews3)].
coincides with the availability of automated zone measur-
ing devices, which aid measuring and interpretation con-
siderably. With laboratories using the same method there is
a real opportunity to combine zone diameter data so that
levels of resistance in the UK and Ireland can be surveyed,
and subtle changes in susceptibility detected.
The method, like all standardized disc tests, cannot be
adapted by the user, with the exception that various
methods of inoculum preparation can be used to achieve
semi-confluent growth.
For microorganisms not included in this section, work is
either ongoing (e.g. anaerobes) or reported elsewhere (e.g.
mycobacteria4).
1. Preparation of plates
1.1 Prepare IsoSensitest agar (ISA; Oxoid, Basingstoke,
UK), or media shown to have the same performance as
ISA, according to the manufacturer’s instructions. Media
for fastidious organisms are supplemented with 5% defib-
rinated horse blood or 5% defibrinated horse blood 

*Tel: 44-121-507-5693; Fax: +44-121-551-7763; E-mail: Jenny.Andrews@cityhospbham.wmids.nhs.uk

43
© 2001 The British Society for Antimicrobial Chemotherapy
 J. M. Andrews for the BSAC Working Party on Susceptibility Testing

20 mg/L β-nicotinamide adenine dinucleotide (NAD, e.g. 2. Selection of control organisms

from Sigma Diagnostics, Poole, UK or Merck BDH, Poole,
UK) as in Table I.
1.2 Pour sufficient molten agar into sterile Petri dishes to
give a depth of 4 mm (25 mL in a 90 mm Petri dish).
1.3 Dry the surface of the agar to remove excess moisture.
The length of time needed to dry the surface of the agar
depends on whether a fan-assisted drying cabinet (approxi-
mately 5–10 min) or a ‘still air’ incubator is used. Important:
do not overdry plates.
1.4 Ideally, the plates should be stored in vented plastic
boxes at 8–10C before use. Storage can extend for up to
1 week. Alternatively the plates may be stored at 4–8C in
sealed plastic bags. Acceptable methods of drying plates
and durations of storage should be validated by individual
laboratories as part of their quality assurance programme.
In particular, tests should confirm that excess moisture is
not produced in a sealed environment and that plates are
not overdried in an unsealed environment.
2.1 The control strains listed in Table II should be
included, as appropriate, with every batch of susceptibility
tests.
3. Preparation of inoculum
An inoculum giving semi-confluent growth of colonies after
overnight incubation should be used. A denser inoculum
will result in reduced zones of inhibition and a decreased
inoculum will have the opposite effect. Use of an inoculum
that yields semi-confluent growth has the advantage that
an incorrect inoculum can readily be seen. Figure 1 shows
the acceptable range of inoculum densities. The following
method reliably gives semi-confluent growth. Other methods,
such as photometric measurement of turbidity of suspen-
sions, may be adequate but must be shown to be equivalent
to the following.

Table I. A summary of media for testing depending on organism type

Organisms Medium to be used

Enterobacteriaceae ISA
Pseudomonas spp. ISA
Staphylococci (other than methicillin/oxacillin) ISA
Staphylococci (methicillin and oxacillin)a Columbia agar (Oxoid CM331 or equivalent) with 2% NaCl
Enterococci ISA
Streptococcus pneumoniae ISA  5% defibrinated horse blood
Haemolytic streptococci ISA  5% defibrinated horse blood
Moxarella catarrhalis ISA  5% defibrinated horse blood
Neisseria meningitidis ISA  5% defibrinated horse blood
Haemophilus spp. ISA  5% defibrinated horse blood  20 mg/L NAD
Neisseria gonorrhoeae ISA  5% defibrinated horse blood
a
See elsewhere in this Supplement.5

Table II. Recommended control strains for inclusion, as appropriate, with every
batch of susceptibility tests

Strain

Organism either or

Escherichia coli NCTC 12241 (ATCC 25922) NCTC 10418

Staphylococcus aureus
Pseudomonas aeruginosa
Enterococcus faecalis
Haemophilus influenzae
S. pneumoniae
N. gonorrhoeae
NCTC 12981 (ATCC 25923) NCTC 6571
NCTC 12934 (ATCC 27853) NCTC 10662
NCTC 12697 (ATCC 29212)
NCTC 12699 (ATCC 49247) NCTC 11931
NCTC 12977 (ATCC 49619)
NCTC 12700 (ATCC 49226)

44
 BSAC disc susceptibility testing method

Figure 1. The acceptable range of inoculum densities.

Table III. The suspension should be diluted in sterile 3.1 Comparison with 0.5 McFarland standard
distilled water as follows:
3.1.1 Preparation of the McFarland standard
Add 0.5 mL of 0.048 M BaCl2 (1.17% w/v BaCl2·2H2O) to
1:100 1:10 No dilution 99.5 mL of 0.18 M H2SO4 (1% w/v) with constant stirring.
Distribute the standard into screw cap tubes of the same
Haemolytic streptococci staphylococci N. gonorrhoeae size and volume as those used to prepare the test inoculum.
Enterococci S. pneumoniae Seal the tubes tightly to prevent loss by evaporation. Store
Enterobacteriaceae N. meningitidis protected from light at room temperature. Vigorously agi-
Pseudomonas M. catarrhalis tate the turbidity standard on a vortex mixer before use.
Acinetobacter Standards may be stored for up to 6 months, after which
Haemophilus time they should be discarded. Alternatively, prepared
standards can be purchased (e.g. from bioMérieux, Basing-
These suspensions should be used within 15 min of preparation.
stoke, UK).
3.1.2 Inoculum preparation by the growth method (for non-
fastidious organisms, e.g. Enterobacteriaceae, Pseudo-
Table IV. Conditions of incubation depending on genera monas spp. and staphylococci)
Touch at least four morphologically similar colonies with a
Organisms Incubation conditions sterile loop. Transfer the growth into IsoSensitest broth or
an equivalent that has been shown to have no adverse
effect on the test. Incubate the broth with shaking at
Enterobacteriaceae 35–37C in air for 18–20 h
35–37C, until the visible turbidity is equal to or greater
Pseudomonas spp. 35–37C in air for 18–20 h
than the 0.5 McFarland standard.
Staphylococci (other than 35–37C in air for 18–20 h
methicillin/oxacillin) 3.1.3 Inoculum preparation by the direct colony suspension
Staphylococci (methicillin/ 30C in air for 24 h method (the method of choice for fastidious organisms, e.g.
oxacillin) Haemophilus spp., Neisseria gonorrhoeae and Strepto-
M. catarrhalis 35–37C in air for 18–20 h coccus pneumoniae)
Haemolytic streptococci 35–37C in air for 18–20 h Colonies are taken directly from the plate into IsoSensitest
Enterococci 35–37C in air for 24 ha broth (or equivalent) or distilled water. The suspension
N. meningitidis 35–37C in 4–6% CO2 in air should match or exceed the density of the 0.5 McFarland
for 18–20 h standard. Note that with some organisms, production of an
S. pneumoniae 35–37C in 4–6% CO2 in air even suspension of the required turbidity is difficult, and
for 18–20 h growth in broth is a more satisfactory option.
Haemophilus 35–37C in 4–6% CO2 in air
3.1.4 Adjustment of the organism suspension to the density
for 18–20 h
of the 0.5 McFarland standard
N. gonorrhoeae 35–37C in 4–6% CO2 in air
Adjust the density of the organism suspension prepared, as
for 18–20 h
in 3.1.2 or 3.1.3, to equal that of the 0.5 McFarland standard
a
It is essential that plates are incubated for at least 24 h before reporting by adding sterile distilled water. To aid comparison, com-
a strain as susceptible to vancomycin or teicoplanin. pare the test and standard against a white background with

45
 Table V. MIC and zone breakpoints for Enterobacteriaceae and Acinetobacter

MIC breakpoint (mg/L) Interpretation of zone diameters (mm)

Acceptable zone range Acceptable zone range
Antibiotic R I S Disc content (g) R I S for NCTC 10418 (mm) for ATCC 25922 (mm)

Ampicillina 16 – 8 10 17 – 18 21–26 16–22

Cefuroxime (axetil) 2 – 1 30 24 – 25 25–32 24–29
Cefuroxime (parenteral) 16 – 8 30 19 – 20 25–32 24–29
Ceftazidime 4 – 2 30 27 – 28 32–40 31–39
Ceftazidimeb 4 – 2 30 21 – 22 32–40 31–39
E. coli and Klebsiella spp.
Ciprofloxacinc 2 – 1 1 17 – 18 31–40 31–37
Gentamicin 2 – 1 10 19 – 20 21–27 21–27
Amikacin 8 – 4 30 19 – 20 24–27 23–27
Aztreonam 16 – 8 30 23 – 24 39–44 36–40
Cefaclor 2 – 1 30 34 – 35 – –
Cefamandoled 16 – 8 30 19 – 20 – –
Cefepime 4 – 2 30 31 – 32 – –
Cefixime 2 – 1 5 19 – 20 32–36 27–30
Cefoperazoned 2 – 1 30 24 – 25 – –
Cefotaxime 2 – 1 30 29 – 30 36–45 34–44
Cefotetand 8 – 4 30 23 – 24 – –
Cefoxitin – 30 – 28–33 26–30

46
8 4 19 20
Cefpirome 2 – 1 20 24 – 25 – –
Cefpodoxime 2 – 1 5 33 – 34 – –
Ceftibuten 2 – 1 10 27 – 28 – –
Ceftizoxime 2 – 1 30 29 – 30 – –
Ceftriaxone 2 – 1 30 27 – 28 – –
Cephalothin 2 – 1 30 26 – 27 – –
Cephradine 4 – 2 30 11 – 12 – –
Chloramphenicol 16 – 8 30 20 – 21 – 20–29
Co-amoxiclav 16 – 8 20/10 17 – 18 18–31 20–26
Colistine 8 – 4 25 14 – 15 – 16–20
Co-trimoxazolef 64 – 32 25 15 – 16 – –
Doxycycline 2 – 1 30 28 – 29 – –
Gatifloxacin 2 – 1 2 19 – 20 – –
J. M. Andrews for the BSAC Working Party on Susceptibility Testing

Gemifloxacin 0.5 – 0.25 1 19 – 20 – –

Imipenem 8 – 4 10 22 – 23 32–37 33–37
Levofloxacin 4 – 2 1 19 – 20 30–33 –
Meropenem 8 – 4 10 22 – 23 38–42 27–39
Mezlocillin 32 – 16 75 21 – 22 – –
Moxifloxacin 2 – 1 1 19 – 20 – –
Netilmicin 2 – 1 10 24 – 25 22–27 22–26
Ofloxacin 4 – 2 1 23 – 24 – –
Piperacillin/tazobactam 32 – 16 75/10 23 – 24 – –
Piperacillin 32 – 16 75 23 – 24 – –
Streptomycind 16 – 8 10 12 – 13 – –
 Sulphamethoxazole 64 – 32 100 13 – 14 – –
Tetracycline 2 – 1 30 33 – 34 – –
Timentin 32 – 16 85 20 – 21 33–37 27–31
Tobramycin 2 – 1 10 17 – 18 24–27 –
Trimethoprim 4 1–2 0.5 2.5 14 15–19 20 28–34 20–26
The information in bold is tentative. Tentative breakpoints are based on information that is currently available to the Working Party, and for some antimicrobial/organism combinations few
data are available or breakpoints are based on information from a single laboratory. Breakpoints will remain tentative for 1 year from when first published.
Problems with reporting the susceptibility of Acinetobacter and Serratia spp. were observed in the field trial (related to obtaining the correct inoculum). Once a strain of Acinetobacter or
Serratia spp. considered clinically significant has been identified, it might be prudent to determine the susceptibility by either an MIC method, or to repeat the disc testing if the inoculum size is
outside the limits.
a
Amoxycillin—report as for ampicillin.
b
Strains of E. coli and Klebsiella spp. have been identified with MICs to ceftazidime of 1 mg/L. These MICs are higher than for the ‘wild susceptible’ population (c. 0.12 mg/L). These strains do
not possess extended-spectrum β-lactamases and until a mechanism of resistance has been identified this tentative zone diameter BP is suggested.
c
Strains of E. coli and Klebsiella spp. with ciprofloxacin MICs of 0.25 and 0.5 mg/L may be reported as resistant. These MICs are higher than the ‘wild susceptible’ population for these species
and may indicate a mechanism of resistance with clinical significance.
d
Zone breakpoints only valid for E. coli, Klebsiella spp. and P. mirabilis.
e
Colistin—some strains of Enterobacteriaceae (particularly Serratia, Providencia, Citrobacter and Enterobacter spp.) have been shown to produce clear zones of inhibition with small colonies
around the disc. These strains should be regarded as resistant as the MICs typically exceed 128 mg/L.
f
Relative to sulphonamide.

Table VI. MIC and zone breakpoints for Pseudomonas

MIC breakpoint (mg/L) Interpretation of zone diameters (mm)

Acceptable zone range Acceptable zone range
Antibiotic R I S Disc content (g) R I S for NCTC 10662 (mm) for ATCC 27853 (mm)

Gentamicin 8 2–4 1 10 14 15–21 22 20–26 22–28

47
Amikacin 32 8–16 4 30 17 18–21 22 21–30 26–32
Ceftazidime 16 – 8 30 23 – 24 29–37 27–35
Imipenem 8 – 4 10 21 – 22 20–27 23–28
Meropenem 8 – 4 10 21 – 22 32–39 30–38
Piperacillin 32 – 16 75 23 – 24 27–35 27–34
Piperacillin/tazobactam 32 – 16 75/10 23 – 24 28–35 29–37
Ciprofloxacin 8 2–4 1 1 9 – 10 21–28 24–30
BSAC disc susceptibility testing method

Ciprofloxacin 8 2–4 1 5 15 16–19 20 30–38 30–39

Aztreonam 16 – 8 30 19 – 34 27–30 26–30
Carbenicillin 256 – 128 100 12 – 13 – –
Cefotaxime 2 – 1 30 26 – 27 20–29 20–24
Cefpirome 2 – 1 20 19 20–24 25 – –
Ceftriaxone 2 – 1 30 29 – 15 – –
Colistin 8 – 4 25 14 – 20 – –
Gatifloxacin 2 – 1 2 19 – 20 – –
Gemifloxacin 2 – 1 5 19 – 20 – –
Levofloxacin 4 – 2 5 17 – 18 – –
Moxifloxacin 8 2–4 1 5 17 18–24 25 –
Netilmicin 8 2–4 1 30 15 16–18 19 17–20 20–24
Ofloxacin 16 4–8 2 5 12 13–19 20 18–26 18–25
Timentin 128 32–64 16 85 19 – 20 25–29 24–27
Tobramycin 8 2–4 1 10 16 17–19 20 23–30 26–32

The information in bold is tentative.

 Table VII. MIC and zone breakpoints for staphylococci

MIC breakpoint (mg/L) Interpretation of zone diameters (mm)

Acceptable zone range Acceptable zone range
Antibiotic R I S Disc content (g) R I S for NCTC 6571 (mm) for ATCC 25932 (mm)

Gentamicin 2 – 1 10 19 – 20 24–30 20–26

Penicillina 0.25 – 0.12 1 unit 24 – 25 32–40 29–36
Methicillinb 8 – 4 5 13 – 14 18–30 18–27
Vancomycinc 8 – 4 5 11 – 12 14–20 13–17
Teicoplaninc,d 8 – 4 30 14 – 15 17–23 16–20
Rifampicin 0.12 – 0.06 2 29 – 30 27–39 29–36
Erythromycin 1 – 0.5 5 19 – 20 22–31 22–29
Quinupristin/dalfopristine 4 – 2 15 19 – 20 27–31 –
Amikacin, coagulase- 32 8–16 4 30 21 22–24 25 – –
negative staphylococci
Amikacin, S. aureus 32 8–16 4 30 18 – 19 – –
Azithromycin 2 – 1 15 19 – 20 – –
Chloramphenicol 16 – 8 10 19 – 20 20–26 19–27
Ciprofloxacin 2 – 1 1 17 – 18 25–32 17–22

48
Clarithromycin 1 – 0.5 2 19 – 20 – –
Clindamycin 1 – 0.5 2 25 – 26 30–35 26–33
Co-amoxiclavb 2 – 1 3 17 – 18 – 27–32
Co-trimoxazolef 64 – 32 25 16 – 17 – –
Fusidic acid 2 – 1 10 29 – 30 32–40 30–37
Gatifloxacin 2 – 1 2 19 – 20 – –
Gemifloxacin 0.5 – 0.25 1 19 – 20 – –
Linezolidg 8 – 4 10 19 – 20 – –
Moxifloxacin 2 – 1 1 19 – 20 – –
Mupirocinh 8 – 4 5 21 – 22 26–35 24–34
Neomycin – – – 10 16 – 17 – 21–27
J. M. Andrews for the BSAC Working Party on Susceptibility Testing

Netilmicin, S. aureus 2 – 1 10 23 – 24 – 22–28

Netilmicin, 2 – 1 10 29 – 30 – 22–28
coagulase-negative
staphylococci
Ofloxacin 4 – 2 1 23 – 24 – –
Tetracycline 2 – 1 10 19 – 20 31–40 26–35
Tobramycin, S. aureus 2 – 1 10 20 – 21 – 29–35
 BSAC disc susceptibility testing method

a contrasting black line. Note that the suspension should be

Mupirocin: an Etest or other MIC method should be performed on any strain designated resistant by the disc method. This will identify whether the strain has low-level resistance (MIC 8–256
The presence of blood has a marked effect on the activity of quinupristin/dalfopristin. On the rare occasions when blood needs to be added to enhance the growth of staphylococci, 15 mm
used within 15 min.
The information in bold is tentative. Tentative breakpoints are based on information that is currently available to the Working Party, and for some antimicrobial/organism combinations few

Staphylococci exhibiting resistance to methicillin/oxacillin should be regarded as resistant to other penicillins, cephalosporins, carbapenems and combinations of β-lactam and β-lactamase 3.2 Dilution of suspension adjusted to the turbidity of a 0.5

Linezolid. This is a tentative breakpoint. The BSAC is awaiting further information on patients with serious staphylococcal infections. In such patients an MIC determination might be
McFarland standard. See Table III.

heterogeneous resistance to vancomycin. If, on clinical grounds, resistance to vancomycin is suspected, it is recommended that the organism be sent to a specialist laboratory, such as
–

Glycopeptide intermediate S. aureus (VISA) cannot be detected by this method or any other method of disc testing. Population studies are the most reliable method for detecting

4. Inoculation of agar plates

Dip a sterile cotton-wool swab into the suspension and
remove the excess liquid by turning the swab against the
side of the tube. Spread the inoculum evenly over the entire
data are available or breakpoints are based on information from a single laboratory. Breakpoints will remain tentative for 1 year from when first published.

surface of the plate by swabbing in three directions. Allow

Teicoplanin: disc testing not recommended for coagulase-negative staphylococci. An MIC method of testing should be used to determine susceptibility.
24–34

the plate to dry before applying discs. Note that if inocu-

–

lated plates are left at room temperature for any length of

Southmead Hospital in Bristol or the Antibiotic Resistance Monitoring and Reference Laboratory at PHLS Colindale for further investigation.

time before the discs are applied, the organism may begin
to grow, and this will result in reduced zones of inhibition.
Discs should therefore be applied to the surface of the agar
within 15 min of inoculation.
30

28

Use of rotary platers for susceptibility testing

Rotary platers can be used for inoculating susceptibility
tests but care must be taken. The swab must be moved at an
even pace to ensure that the inoculum is semi-confluent
and that no gaps are present between the swab streaks.
–

mg/L) or high-level resistance (MIC 512 mg/L). The latter is clinically significant but the former is not.

5. Antimicrobial discs
29

27

5.1 Disc contents are given in Tables V–XVI.

5.2 Application of discs. Discs should be firmly applied to
the surface of an agar plate that has been dried previously.
The contact with the agar should be even. A 90 mm plate
will accommodate six discs without unacceptable over-
10

lapping of zones.
5.3 Storage and handling of discs. Loss of potency from
discs will result in reduced zones of inhibition. To avoid loss
appropriate using comparison with NCTC or ATCC controls.

of potency due to inadequate handling, the following pro-

0.5
1

cedures are essential.

5.3.1 Store discs in sealed containers with a desiccant and
Penicillin: check for heaped zone edge (resistant).

protected from light (this is particularly important for some

–

light-susceptible agents such as metronidazole, chloram-

phenicol and the quinolones).
Based on the MIC of sulphamethoxazole.

5.3.2 Store stocks at –20C, except for drugs known to be

2

1

unstable at this temperature. If this is not possible, store

discs at 8C.
5.3.3 Store working supplies of discs at 8C.
zones imply susceptibility.
coagulase-negative

5.3.4 To prevent condensation, allow discs to warm to

staphylococci

room temperature before opening containers.

Trimethoprim
Tobramycin,

5.3.5 Store disc dispensers in sealed containers with an

indicating desiccant.
inhibitors

5.3.6 Discard any discs past the expiry date shown on the
side of the container.
b

h
a

g
c

49
 J. M. Andrews for the BSAC Working Party on Susceptibility Testing

Table VIII. MIC and zone breakpoints for S. pneumoniae

Interpretation of zone
MIC breakpoint (mg/L) diameters (mm)
Acceptable zone range
Antibiotic R I S Disc content (g) R I S for ATCC 49619 (mm)

Penicillina,b,c 2 0.12–1 0.06 oxacillin 1 19 – 20 8–16

Erythromycin 1 – 0.5 5 19 – 20 23–36
Tetracycline 2 – 1 10 19 – 20 26–36
Chloramphenicol 16 – 8 10 17 – 18 21–29
Ciprofloxacind 4 2 – 1 9 10 – 14–21
Azithromycin 2 – 1 15 19 – 20 –
Cefaclorb 2 – 1 30 24 – 25 –
Cefiximeb 2 – 1 5 19 – 20 –
Cefotaximeb 2 – 1 5 29 – 30 –
Cefpodoximeb 2 – 1 1 21 – 22 –
Ceftibutenb 2 – 1 10 27 – 28 –
Ceftizoxime b
2 – 1 30 29 – 30 –
Ceftriaxoneb 2 – 1 30 27 – 28 –
Cefuroximeb 2 – 1 5 24 – 25 –
Cephadroxilb 2 – 1 30 24 – 25 –
Cephalexin b
4 – 2 30 24 – 25 –
Clarithromycin 1 – 0.5 2 19 – 20 –
Gatifloxacin 2 – 1 2 19 – 20 –
Gemifloxacin 0.5 – 0.25 1 19 – 20 –
Imipenem b
8 – 4 10 24 – 25 –
Levofloxacin 4 – 2 1 19 – 20 –
Linezolid 8 – 4 10 19 – 20 –
Meropenemb 8 – 4 10 27 – 28 –
Moxifloxacin 2 – 1 1 17 – 18 –
Ofloxacin 4 – 2 1 19 – 20 –
Quinupristin/
dalfopristin 4 – 2 15 19 – 20 21–29
Rifampicin 2 – 1 5 21 – 22 32–37

The information shown in bold is tentative. Tentative breakpoints are based on information that is currently available to the Working Party,
and for some antimicrobial/organism combinations few data are available or breakpoints are based on information from a single laboratory.
Breakpoints will remain tentative for 1 year from when first published.
a
Organisms with reduced susceptibility to penicillin: confirm resistance with a test for penicillin MIC. Organisms with an MIC  1 mg/L are
considered susceptible to β-lactam antibiotics except in infections of the central nervous system. In addition, an MIC of cefotaxime for strains
isolated from meningitis or other invasive infections is advised.
b
Penicillin resistance in S. pneumoniae is detected with an oxacillin 1 g disc.
c
The MIC BPs for penicillin have been changed to reflect international standards; however, the Working Party is considering a further
alteration.
d
The Working Party has amended the in vitro breakpoint concentration of ciprofloxacin for pneumococci. Isolates with MICs of ciprofloxacin
2 mg/L are considered as having intermediate susceptibility.

50
 BSAC disc susceptibility testing method

6. Incubation
6.1 If the plates are left at room temperature after discs
have been applied, larger zones of inhibition may be
obtained compared with the zones produced when plates are
incubated immediately. Plates therefore should be incu-
bated within 15 min of disc application.
6.2 Conditions of incubation. Conditions of incubation
depending on genera are summarized in Table IV.
In general, avoid stacking plates more than six high in
the incubator, as this may affect results due to uneven heat-
ing of plates. However, incubators differ in their efficiency,
and higher stacks are acceptable if it can be shown that they
do not affect zone diameters.

7. Measuring zones and interpretation

7.1 Measure the diameters of zones of inhibition to the
nearest mm (zone edge should be taken as the point of inhi-
bition as judged by the naked eye) with a ruler, callipers or
an automated zone reader. A template (Figure 2) can also
be used for interpreting susceptibility. Tiny colonies at the
Figure 2. Template for interpreting susceptibility.
edge of the zone, films of growth due to the swarming of
Proteus spp. and slight growth within sulphonamide or
trimethoprim zones should be ignored. Colonies growing
within the zone of inhibition should be subcultured and
is available from the BSAC (http://www.bsac.org.uk). The
identified, and the test repeated if necessary.
test plate is placed over the template and the zones of inhi-
7.2 Confirm that the zone of inhibition for the control bition are examined in relationship to the template zones.
strain falls within the acceptable ranges shown in Tables If the zone of inhibition of the test strain is within the area
V–XVI before interpreting the test. marked with an ‘R’ the organism is resistant. If the zone of
7.3 A template can also be used for interpreting zone inhibition is equal to or larger than the marked area the
diameters (Figure 2). A programme for preparing templates organism is susceptible.

Table IX. MIC and zone breakpoints for enterococci

Interpretation of zone
MIC breakpoint (mg/L) diameters (mm)
Acceptable zone range
Antibiotic R I S Disc content (g) R I S for ATCC 29212 (mm)

Gentamicin 1024 – 512 200 9 – 10 22–27

Ampicillin 16 – 8 10 19 – 20 26–35
Vancomycin 8 – 4 5 12 – 13 13–19
Teicoplanin 8 – 4 30 19 – 20 19–25
Quinupristin/
dalfopristina 4 – 2 15 19 – 20 –
Azithromycin 2 – 1 15 29 – 30 –
Imipenem 8 – 4 10 19 – 20 –
Meropenem 8 – 4 10 19 – 20 –
Linezolid 8 – 4 10 19 – 20 –

The information shown in bold is tentative. Tentative breakpoints are based on information that is currently available to the Working Party,
and for some antimicrobial/organism combinations few data are available or breakpoints are based on information from a single laboratory.
Breakpoints will remain tentative for 1 year from when first published.
a
Although not fully explained, the presence of blood has a marked effect on the activity of quinupristin/dalfopristin. On the rare occasions
when blood needs to be added to enhance the growth of enterococci, a zone diameter 15 mm implies susceptibility.

51
 Table X. MIC and zone breakpoints for haemolytic streptococci

MIC breakpoint (mg/L) Interpretation of zone diameters (mm)

Disc content Acceptable zone range Acceptable zone range
Antibiotic R I S (g unless stated) R I S for NCTC 6571 (mm)a for ATCC 25923 (mm)a

Penicillin 0.25 – 0.12 1 unit 19 – 20 32–40 27–35

Tetracycline 2 – 1 10 19 – 20 30–38 28–36
Erythromycin 1 – 0.5 5 19 – 20 22–29 23–29
Clarithromycinb 1 – 0.5 2 19 20 –
Azithromycin 2 – 1 15 19 – 20 –
Cefadroxil 2 – 1 30 24 – 25 –
Cefixime 2 – 1 5 19 – 20 –
Cefotaxime 2 – 1 5 27 – 28 –
Cephalexin 4 – 2 30 24 – 25 –
Cephalothin 2 – 1 30 28 – 29 –
Co-trimoxazolec 64 – 32 25 16 – 17 –
Linezolid 8 – 4 10 19 – 20 –

The information shown in bold is tentative. Tentative breakpoints are based on information that is currently available to the Working Party, and for some antimicrobial/organism combinations
few data are available or breakpoints are based on information from a single laboratory. Breakpoints will remain tentative for 1 year from when first published.
a
Values obtained using media supplemented with 5% defibrinated horse blood.
b
Active metabolite not taken into consideration.
c
Based on the MIC of sulphamethoxazole.

52
Table XI. MIC and zone breakpoints for M. catarrhalis

MIC breakpoint (mg/L) Interpretation of zone diameters (mm)

Acceptable zone range Acceptable zone range
Antibiotic R I S Disc content (g) R I S for NCTC 6571 (mm) for ATCC 25923 (mm)

Ampicillina 2 – 1 2 29 – 30

Cefaclor 2 – 1 30 22 – 23
Cefuroxime 2 – 1 5 19 – 20
Chloramphenicol 4 – 2 10 22 – 23
Ciprofloxacin 2 – 1 1 17 – 18
Clarithromycinb 1 – 0.5 2 19 – 20
J. M. Andrews for the BSAC Working Party on Susceptibility Testing

Co-amoxiclav 2 – 1 2/1 18 – 19

Erythromycin 1 – 0.5 5 27 – 28 22–29 23–29
Gatifloxacin 2 – 1 2 19 – 20
Gemifloxacin 0.5 – 0.25 1 19 – 20
Levofloxacin 4 – 2 1 19 – 20
Linezolid 8 – 4 10 19 – 20
Moxifloxacin 2 – 1 1 17 – 18
Tetracycline 2 – 1 10 21 – 22 30–38 28–36

The information shown in bold is tentative. Tentative breakpoints are based on information that is currently available to the Working Party, and for some antimicrobial/organism combinations
few data are available or breakpoints are based on information from a single laboratory. Breakpoints will remain tentative for 1 year from when first published.
a
Test for β-lactamase.
b
Active metabolite not taken into consideration.
 BSAC disc susceptibility testing method

Table XII. MIC and zone breakpoints for N. gonorrhoeae

Interpretation of zone
MIC breakpoint (mg/L) diameters (mm)
Acceptable zone range
Antibiotic R I S Disc content (g) R I S for NCTC 6571 (mm)a

Spectinomycin 128 – 64 25 13 – 14

Nalidixic acidb – – – 30 – – –
Penicillinc,d 2 0.12–1 0.06 1 unit 17 18–25 26 39–43
Cefuroxime 2 – 1 5 19 – 20
Tetracycline 2 – 1 10 19 – 20
Rifampicin 2 – 1 2 20 – 21 32–37

The information shown in bold is tentative. Tentative breakpoints are based on information that is currently available to the Working Party,
and for some antimicrobial/organism combinations few data are available or breakpoints are based on information from a single laboratory.
Breakpoints will remain tentative for 1 year from when first published.
a
Values obtained using media supplemented with 5% defibrinated horse blood and incubated in 4–6% CO2.
b
Quinolone resistance is most reliably detected with nalidixic acid. Strains with reduced susceptibility to fluoroquinolones have no zone of
inhibition to nalidixic acid.
c
Test for β-lactamase.
d
Confirm resistance by MIC if β-lactamase negative.

Table XIII. MIC and zone breakpoints for N. meningitidis

Interpretation of zone
MIC breakpoint (mg/L) diameters (mm)
Acceptable zone range
Antibiotic R I S Disc content (g) R I S for NCTC 6571 (mm)a

Penicillin 0.12 – 0.06 1 unit 24 – 25 39–43

Cefotaxime 2 – 1 5 29 – 30
Chloramphenicol 4 – 2 10 19 – 20 21–26
Rifampicin 2 – 1 2 29 – 30 32–37
Erythromycin 1 – 0.5 5 26 – 27 25–29
Tetracycline 2 – 1 10 21 – 22
Ciprofloxacin 2 – 1 1 31 – 32
The information shown in bold is tentative. Tentative breakpoints are based on information that is currently available to the Working Party,
and for some antimicrobial/organism combinations few data are available or breakpoints are based on information from a single laboratory.
Breakpoints will remain tentative for 1 year from when first published.
a
Values obtained using media supplemented with 5% defibrinated horse blood and incubated in 4–6% CO2.

8. Direct susceptibility testing
8.1 Direct susceptibility testing of urines
The Working Party does not advocate direct susceptibility
testing, as the control of inoculum is impossible. However,
we are aware that this is a common practice in many labor-
atories and therefore we are suggesting methods that will
achieve the correct inoculum size for a reasonable propor-
tion of infected urines. The following methods have been
developed and recommended by laboratories that use the
BSAC method and we suggest adopting whichever method
best suits individual laboratory working practice. If the
inoculum is not correct and growth is not semi-confluent or
the culture is mixed the test must be repeated.
(i) Method 1: thoroughly mix the urine, then place a 10 L
loop of urine in the centre of the susceptibility plate and
spread with a dry swab.
(ii) Method 2: thoroughly mix the urine, then dip a sterile
cotton-wool swab in the urine and remove excess. Make a
cross in the centre of the susceptibility plate then spread
with a sterile dry swab. If only small numbers of organisms
are seen in microscopy, the initial cotton-wool swab may be
used to inoculate and spread the susceptibility plate.
8.2 Direct susceptibility testing of positive blood
cultures
The Working Party does not recommend direct suscept-
ibility testing of positive blood culture. However, we are

53
 Table XIV. MIC and zone breakpoints for H. influenzae

MIC breakpoint (mg/L) Interpretation of zone diameters (mm)

Acceptable zone range Acceptable zone range
Antibiotic R I S Disc content (g) R I S for NCTC 11931 (mm) for ATCC 49247 (mm)

Ampicillina 2 – 1 2 19 – 20 22–30 6–13

Cefuroxime 2 – 1 5 19 – 20 22–28 6–16
Cefotaxime 2 – 1 5 24 – 25 33–45 27–38
Tetracycline 2 – 1 10 21 – 22 27–35 9–14
Chloramphenicol 4 – 2 10 24 – 25 30–40 30–38
Azithromycinb 8 0.5–4 0.25 15 19 20–34 35 24–36 20–30
Cefaclor 2 – 1 30 36 – 37 –
Ceftazidime 4 – 2 30 29 – 30 –
Ceftriaxone 2 – 1 30 34 – 35 –
Ciprofloxacinc 2 – 1 1 27 – 28 32–40 33–44
Clarithromycind 32 1–16 0.5 5 9 10–24 25 –

54
Co-amoxiclav 2 – 1 2/1 19 – 20 20–27 10–20
Co-trimoxazole 64 – 32 25 21 – 22 –
Erythromycin 16 1–8 0.5 5 14 15–27 28 12–23 9–16
Gatifloxacinc 2 – 1 2 19 – 20 –
Gemifloxacinc 0.5 – 0.25 1 19 – 20 –
Imipenem 8 – 4 10 19 – 20 –
Levofloxacinc 4 – 2 1 19 – 20 –
Meropenem 8 – 4 10 27 – 28
Trimethoprim 1 – 0.5 2.5 20 – 21
Nalidixic acidc – – – 30 – – – 33–38

The information shown in bold is tentative. Tentative breakpoints are based on information that is currently available to the Working Party, and for some antimicrobial/organism combinations
J. M. Andrews for the BSAC Working Party on Susceptibility Testing

few data are available or breakpoints are based on information from a single laboratory. Breakpoints will remain tentative for 1 year from when first published.
a
Test for β-lactamase.
b
No resistant strains yet described.
c
Quinolone resistance is most reliably detected with nalidixic acid. Strains with reduced susceptibility to fluoroquinolones give no zone of inhibition with a 30 g nalidixic acid disc.
d
Active metabolite not taken into consideration.
 Table XV. MIC and zone breakpoints for urinary tract isolates (Gram-negative bacilli)

Interpretation of zone diameters (mm)

Acceptable zone range
MIC breakpoint (mg/L) Coliforms E. coli P. mirabilis
Disc NCTC ATCC NCTC
Antibiotic R I S content (g) R I S R I S R I S 10418 25922 11560a

Ampicillin 64 – 32 25 15 – 16 24 – 25 24–30

Cephalexinb 64 – 32 30 15 – 16 11 – 12 21–28 16–21
Ciprofloxacin 8 – 4 1 19 – 20 31–40 31–37
Co-amoxiclav 64 – 32 20/10 17 – 18 17 – 18 18–31 20–26 18–23
Fosfomycin/ 256 128 200/50 19 – 20 33 – 34
glucose-6-phosphatec
Mecillinam 2–8 10 14–23 14–18

55
16 1 13 24 13 19
Nalidixic acid 32 – 16 30 17 – 18 28–36
Nitrofurantoin 64 – 32 200 19 – 20 25–30 23–27
Norfloxacin 8 – 4 2 15 – 16 34–37 32–36
Trimethoprim 4 – 2 2.5 16 – 17 28–34 20–26

The information shown in bold is tentative. Tentative breakpoints are based on information that is currently available to the Working Party, and for some antimicrobial/organism combinations
few data are available or breakpoints are based on information from a single laboratory. Breakpoints will remain tentative for 1 year from when first published.
BSAC disc susceptibility testing method

If an organism is isolated from multiple sites, for example from blood and urine, interpretation of susceptibility should be made with regard to the systemic site, that is, if the blood isolate is
resistant and the urine isolate susceptible, both should be reported similarly (resistant), irrespective of the results obtained using interpretative criteria for the urine isolates.
For agents not listed, criteria given for systemic isolates may be used for urinary tract isolates (see Tables V and VI).
Direct sensitivities on urine samples may be performed as long as the inoculum obtained is equivalent to semi-confluent growth.
In the absence of a definitive organism identification, use the recommendations most appropriate for the presumptive identification, accepting that on some occasions the interpretation may be
incorrect. A more cautious approach is to use two systemic recommendations.
a
β-Lactamase-producing strain.
b
Cefadroxil, report as for cephalexin.
c
The susceptibility of Proteus spp. which swarm up to the disc, can be difficult to interpret.
 Table XVI. MIC and zone breakpoints for urinary tract isolates (Gram-positive cocci)

Interpretation of zone diameters (mm)

Staphylococcus Acceptable zone range

MIC breakpoint (mg/L) Enterococci saprophyticus Group B streptococci
Disc NCTC ATCC
Antibiotic R I S content (g) R I S R I S R I S 6571 25923

Ampicillin 64 – 32 25 19 – 20 25 – 26 25 – 26

Cephalexina 64 – 32 30 – – – – – – 23 – 24
Ciprofloxacin 8 – 4 1 – – – 17 – 18 – – – 25–32 17–22
Co-amoxiclav 64 – 32 20/10 20 – 21 27 – 28 27 – 28
Fosfomycin/ 256 – 128 200/50 19 – 20 19 – 20 – – –
glucose-6-phosphate

56
Mecillinam 128 – 64 50 – – – 9 – 10 – – –
Nalidixic acid 32 – 16 30 17 – 18 – – – – – –
Nitrofurantoin 64 – 32 200 14 – 15 19 – 20 19 – 20 21–25 20–26
Norfloxacin 8 – 4 2 15 – 16 – – – – – –
Trimethoprimb 4 – 2 2.5 – – – 14 – 15 – – – 25–30 20–28

The information shown in bold is tentative. Tentative breakpoints are based on information that is currently available to the Working Party, and for some antimicrobial/organism combinations
few data are available or breakpoints are based on information from a single laboratory. Breakpoints will remain tentative for 1 year from when first published.
If an organism is isolated from multiple sites, for example from blood and urine, interpretation of susceptibility should be made with regard to the systemic site, that is, if the blood isolate is
resistant and the urine isolate susceptible, both should be reported similarly (resistant), irrespective of the results obtained using interpretative criteria for the urine isolates.
Direct sensitivities on urine samples may be performed as long as the inoculum obtained is equivalent to semi-confluent growth.
The Working Party suggest that isolates of Staphylococcus epidermidis and Staphylococcus aureus should be treated as systemic infections because they are usually associated with more serious
infections.
a
J. M. Andrews for the BSAC Working Party on Susceptibility Testing

Cefadroxil, report as for cephalexin.

b
Trimethoprim not active in vivo against enterococci.
 BSAC disc susceptibility testing method
aware that this is common practice in many laboratories
and therefore suggest a method that gives the correct
inoculum size for a reasonable proportion of positive blood
cultures. The method varies according to the Gram reac-
tion of the infecting organism.
8.2.1 Gram-negative bacilli
Using a venting needle, place one drop in 5 mL of sterile
water and use this to inoculate IsoSensitest or equivalent
agar.
8.1.2 Gram-positive organisms
It is not always possible to accurately assess the genera of
Gram-positive organisms from the Gram’s stain. However,
careful observation of the morphology, coupled with some
clinical information should make an ‘educated guess’ cor-
rect most of the time.
8.1.2.1 Staphylococci and enterococci
Using a venting needle, place three drops in 5 mL of sterile
water and use this to inoculate IsoSensitest or equivalent
agar.
8.1.2.2 Pneumococci, ‘viridans’ streptococci and diph-
theroids
Using a venting needle, place one drop in the centre of an
IsoSensitest or equivalent agar supplemented with 5%
57
horse blood, and spread evenly over the entire surface of
the plate. If the inoculum is not correct and growth is not
semi-confluent or the culture is mixed the test must be
repeated.
References
1. Report of the Working Party on Antibiotic Sensitivity Testing of
the British Society for Antimicrobial Chemotherapy. (1991). A guide
to sensitivity testing. Journal of Antimicrobial Chemotherapy 27,
Suppl. D., 1–50.
2. Andrews, J. M., Brown, D. F. J. & Wise, R. (1996). A survey of
antimicrobial susceptibility testing in the United Kingdom. Journal of
Antimicrobial Chemotherapy 37, 187–8.
3. Andrews, J. M. (2001). The development of the BSAC standard-
ized method of disc diffusion testing. Journal of Antimicrobial
Chemotherapy 48, Suppl. 1., 29–42.
4. Inderlied, C. B. & Nash, K. A. (1996). Antimycobacterial agents:
In vitro susceptibility testing, spectra of activity, mechanisms of
action and resistance, and assays for activity in biologic fluids. In
Antibiotics in Laboratory Medicine, (Lorian, V., Ed.), pp. 127–75.
Williams and Wilkins, Baltimore, MD.
5. Brown, D. F. J. (2001). Detection of methicillin/oxacillin resist-
ance in staphylococci. Journal of Antimicrobial Chemotherapy 48,
Suppl. 1., 65–70.