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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 .

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