Part of Thermo Fisher Scientific
Disc diffusion is one of the most simple and widely used methods for sensitivity
testing. Disc testing is an extremely flexible technique, with a wide variety
of antibiotics and concentrations available, which can easily be interchanged
as required. This guide is designed to illustrate where potential sources of
error may enter the test, and how to minimise these as much as possible.
The disc diffusion test not only categorises resistant, intermediate and susceptible
organisms through quantitative results, but also provides a visual indication
of:
• Inoculum level
• Presence of contamination
• Antagonism and synergy between adjacent antibiotics
• ß-lactamase activity
Although disc diffusion is a simple test to perform, to obtain accurate and
reproducible results attention to detail and care must be taken, since relatively
small changes in operating procedure can affect the results obtained.
The initial consideration must be investment in quality products with which
to perform AST.
If Mueller-Hinton agar is the medium of choice then this should be produced
to conform with the NCCLS M6-A which tightly controls many of the variable factors
in the medium e.g. cation concentration, pH, antibiotic inhibitor levels. If
these are uncontrolled, the results obtained could be greatly affected. Although
no international standards currently exist for Iso-sensitest agar or Diagnostic
sensitivity test agar, users should ensure that their supplier adheres to strict
quality systems to ensure a reliable product e.g. ISO 9001 and ISO 9002.
Once the AST products are in the laboratory, they must be stored as per the
manufacturer’s instructions, as temperature and moisture variance will
cause the products to degrade. Media preparation protocols must be strictly
adhered to, since a number of factors in this process can influence the zone
sizes achieved:
Depth of agar
For sensitivity testing the depth of the agar is usually recommended to be
4mm in the centre of the plate (approximately 25ml in a 90mm plate). Variation
in depth will affect the zone sizes – if the agar is too thin, larger
zones will appear since the volume is decreased, and the effective antibiotic
concentration increased. If the agar is too thick, smaller zones will appear
since the effective antibiotic concentration has been decreased. If the agar
is intentionally thin, then small modifications to other factors will have
a disproportionate effect.
pH
The pH of the prepared medium needs to be within the range stated by the
manufacturer – this is normally around neutral. If the pH is outside
the stated parameters the zone sizes will be altered, with the degree of
alteration determined by the antibiotic group. For example, an increase in
pH will cause a decrease in penicillin and quinolone zones; conversely, the
same increase will increase zone diameters for macrolide and aminoglycosides.
pH should form part of the routine quality control tests for each batch of
medium prepared. Media which falls outside the manufacturer’s pH specifications
should not be used for sensitivity testing.
Storage of prepared media
Once prepared, plates must be stored appropriately so that they do not become
contaminated, or too dry (for example, through prolonged storage, or stored
near a fan). The moisture in the medium plays an important role in disc diffusion:
when the antibiotic discs are applied to the agar, moisture from the plate
is absorbed, dissolving the antibiotic, and allowing it to diffuse into the
agar. Thus, a reduced moisture content will impede the flow of antibiotic
and result in smaller zones. Suppliers of ready prepared media will state
an expiry date on their products, plates should not be used beyond this date.
Laboratories preparing their own plates should validate their storage conditions
with respect to expected zone sizes.
Inoculum level
The inoculum level is one of the most important, and variable factors in susceptibility
testing, and will have a profound effect on the zone size obtained. The zone
edge is determined when the critical mass is achieved. If the inoculum is
heavy, then the critical mass will be achieved in a shorter time and the
zone will be smaller. If the inoculum is light, the critical mass takes longer
to develop, the antibiotic diffuses further and a larger zone is achieved.
The recommended level of inoculum to use in sensitivity testing varies between
the standard methods, and can be up to 100 fold difference; each inoculum
will work perfectly well with the standard method and breakpoints given for
that method.
The impact of the inoculum level on zone size is highlighted by comparing quality control parameters for the various standard methods. In the following example the only variable is the inoculum level, both methods use Mueller-Hinton agar with the Staphylococcus aureus ATCC 25923 .
Zone size criteria (mm) |
||
Antibiotic |
NCCLS 0.5 McFarland |
DIN 1:100 dilution of 0.5 McFarland |
Penicillin G 10 IU |
26 - 37 |
35 - 45 |
It is therefore vital to adhere as closely as possible to the recommended inoculum
level since the zone size interpretational criteria are specific for a particular
inoculum level and medium. Turbidometers, and density control references are
available to aid in this area since small variations here can significantly
alter the final result.
Once the inoculum has been applied to the agar plate, the discs should be applied
as soon as possible. Delays here will allow the organism a chance to begin replication
before the antibiotic is applied, potentially resulting in smaller zone sizes.
Disc Storage
The antibiotic discs must be stored according to manufacturer’s instructions
i.e. between -20°C and + 8°C in a sealed, desiccated environment. Cartridges
not in use should be stored unopened in their original packaging in order to
prevent moisture ingress. This is extremely important since it is well known
that moisture is a major cause of antibiotic degradation. Opened cartridges
must be refrigerated and sealed in the disc dispenser or other suitable container
when not in use. The desiccant in the base of the dispensers must be regularly
recharged to help maintain the moisture free environment. After regeneration,
the desiccant should be checked to ensure that the indicator dye has returned
to its original colour. If a strong colour is not achieved, the desiccant should
be replaced. After refrigeration, the dispensers need to be allowed to reach
room temperature before the lid is removed to prevent condensation and moisture
ingress. Opened cartridges should not be stored for more than one week; cartridges
containing ß-lactamase inhibitors (e.g. clavulanic acid, tazobactam and
sulbactam) are particularly susceptible to degradation by moisture. If cartridges
are
kept open for longer than one week, then QC testing should be performed on
a daily basis to validate the stability of the antibiotics. Antibiotic cartridges
and packaging will have an expiry date, discs must not be used once this date
has been exceeded.
Dispenser adjustments
Before the antibiotics are inserted into the dispenser, the height of the “skirt” at
the base needs to be adjusted to match the depth of the agar. If the dispenser
is incorrectly adjusted the discs can be imbedded into the agar. As a consequence,
the prongs that press the discs into place on the agar surface can become contaminated
which can then contaminate successive plates. If the dispenser becomes contaminated,
then the prongs can be cleaned using a swab soaked in alcohol. This procedure
should be performed on a weekly basis, as a preventative measure unless gross
contamination takes place when immediate cleaning is required.
If the dispenser
is set too high, the discs may not be pressed onto the agar surface. This can
lead to uneven spacing of the discs, and consequently overlapping
or unreadable zones (see below Figure 1 where one disc has been dropped at
the edge of the plate). If insufficient contact between the discs and the agar
takes place moisture may not be drawn into the disc and the antibiotic would
not diffuse into the plate. This would be especially problematic with large
molecular weight antibiotics such as vancomycin and teicoplanin, which routinely
have difficulty diffusing into agar (see below Figure 2 – each of these
discs should have produced a zone).
Incubation
Once the discs have been applied to the agar, the plates need to be incubated
as soon as possible. Delays before incubation will allow the antibiotic to
diffuse out before the organism starts to grow and will result in larger
zone sizes. Plates incubated in large stacks will have a poor transfer of
heat to the middle of the stack, thus the antibiotics will diffuse before
the test gets to temperature potentially resulting in larger zone sizes.
It has been shown that a single plate on a metal shelf will take 1 hour to
warm to within 1°C of the incubator temperature, however plates stacked
five deep will take up to four hours for the centre plate to reach the same
temperature.
Atmospheric conditions
The atmospheric conditions under which an organism is incubated will also affect
the zone sizes. For example a Streptococcus pneumoniae or Haemophilus influenzae incubated aerobically will not thrive, and so will appear more sensitive
to the antibiotics under test. However, incubating these organisms in CO2 will
cause a drop in the pH which will affect the zones sizes achieved to different
degrees (as already discussed). For these fastidious organisms,
the factors of pH and growth rate will be taken into account when the breakpoints
are set.
Incubation temperature
Incubation temperatures are normally set at 35 – 37°C for the standard
methods, and this is optimal for most human pathogens. If the temperature is
lower then the rate of growth will be reduced resulting in an increased time
to reach the critical mass. An increased time to reach the critical mass will
allow a greater diffusion of the antibiotics and a larger zone size. An increased
incubation temperature will also result in larger zones, since the organisms
may be compromised at elevated temperatures, and the antibiotics will diffuse
easier due to viscosity changes within the agar.
Measurement of zones
All the standard methods advocate the accurate measurement of zone sizes, especially
when zones are close to the breakpoint. Unaided visual interpretation is
very subjective and can vary between users from day to day. Callipers and
automated reading systems are available. Some laboratories use templates
of the break points which can be used for comparison. These should be prepared
for use with different organism groups.
Quality control
Quality control checks should ideally be performed on a daily basis to monitor
all aspects of susceptibility testing: media (depth, pH etc), inoculum level,
antibiotic disc integrity, incubation temperature and atmospheric conditions.
Quality control testing is performed with reference cultures (e.g. ATCC®,
NCTC) where known results are expected. If the results fall outside the expected
parameters, it will indicate potential problems with the day’s samples
since all tests should all be exposed to the same conditions. Reasons for
the failure can be investigated and resolved quickly. Standard control organisms
are commercially available to enable laboratories to use fresh cultures for
QC. Repeated sub-culturing of control strains can often result in contamination
and loss of the control strain.
Standard Methods
There are numerous standard methods available in different geographical regions,
users should regularly check for method updates, since methodology changes,
breakpoint changes and the introduction of new antibiotics can have a significant
impact on the interpretation the results obtained.
Summary
Although the disc diffusion test is simple to perform, there are key areas which
need to be controlled:
Disc storage conditions
Media manufacture
Inoculum level
Incubation conditions
Accurate and reproducible results rely on quality materials and attention to
detail. Clinicians must have confidence in laboratory test results; these can
only be achieved by adhering to proper procedures.
Useful Sources of Reference
Antibiotics in Laboratory Medicine Lorian
Antibiotic and Chemotherapy O’Grady, Lambert, Finch and Greenwood