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ANTIMICROBIAL SUSCEPTIBILITY TESTING (AST)
The disc diffusion method, which although developed in 1947¹ remains as the most widely used test. Performed with care, using adequate controls, it is as accurate as more costly and complicated tests.

In common with all other methods it cannot mimic the in vivo environment but it does uniquely show the effect of a changing antimicrobial concentration (first rising and then falling) on an increasing microbial population. The minimum inhibitory concentration (MIC) of the antimicrobial appears at the edge of the zone of inhibition which represents the interaction of a critical concentration of antimicrobial at a critical time on a critical microbial population². The methods are simple but the interactions are very complex and the accuracy and precision of the test is based on the following critical components:

CULTURE MEDIUM
The medium chosen for the test must have been manufactured and tested specifically for AST. Oxoid manufactures and recommends the following agar media:

i Diagnostic Sensitivity Test Agar (DST Agar) CM0261 which was the first medium specifically designed for this test and is still very popular.
ii HR Medium CM0845 - a chemically defined medium for susceptibility testing with antifungal agents.
iii HTM (Haemophilus Test Medium) CM0898 was specifically formulated for the susceptibility testing of Haemophilus influenzae. The medium is based on Mueller-Hinton Agar to which a supplement (SR0158) containing NAD and Haematin is added.
iv Iso-Sensitest Agar CM0471 was developed as a semi-synthetic AST medium in which undefined protein hydrolysates were reduced to the minimum level which would allow optimum growth of a wide range of organisms and the `free’ cation strength adjusted to give correct MIC results. Its popularity rests on its reproducible performance.
v Mueller-Hinton Agar CM0337 was originally formulated to grow pathogenic neisseria and it was adopted for use in the Bauer-Kirby test⁴. Its variable performance was strongly criticised but following agreement between culture media manufacturers and representatives of the Clinical Laboratory Standards Institute and the FDA a uniform medium is now produced which must meet the CLSI specifications⁵. Therefore Mueller-Hinton medium suitable for AST will carry a statement on the label that it meets CLSI standards (M6-A2).
vi Sensitest Agar CM0409 the first semi-defined AST medium which remains popular with laboratories which have designed methods around this specific formulation³.
vii Wilkins-Chalgren Anaerobic Agar CM0619 was developed as a medium for the growth of anaerobic bacteria. Its relative lack of inhibitory action towards anaerobes made it the medium of choice for anaerobic agar dilution tests⁶. Anaerobic disc diffusion tests can be carried out but it is essential to include adequate controls which can monitor the effect of anaerobiosis on antimicrobials.

All the above media have broth versions of their formulations (except DST and HTM.). This allows dilution or pre- enrichment to be carried out in the same medium. It is also helpful when carrying out MIC tests in broth to standardise disc diffusion zones to establish the breakpoint categories of S,I,R.

Many of the constituents used in culture media affect the precision and accuracy of AST results. The agar must allow free diffusion of the antimicrobial from the disc. Variations in pH and ionic strength will cause differences in zone sizes. Blood can reduce the zone size of highly protein-bound antimicrobials e.g. fusidic acid. Changes in `free’ electrolyte content will affect aminoglycosides, tetracyclines and polymyxins. Glucose will enlarge the zones of antimicrobials against organisms which are adversely affected by a fall in pH following fermentation of the sugar. Thymidine and thymine levels have to be monitored and reduced, if necessary, to prevent antagonism of trimethoprim and sulphonamides. All AST media must be specifically tested with critical `drug-bug’ combinations to measure their performance and to ensure that they meet the quality specifications. The latter tests are carried out by the manufacturer, to ensure that the medium conforms to regulatory standards, they should also be carried out in an abbreviated form in the user laboratory to monitor the performance of all of the components of AST.

ANTIMICROBIAL DISCS
The paper discs used in the diffusion method are made from paper which conforms to the WHO⁷and FDA⁸ standards.
Impregnation of the discs ensures that prepared solutions of antimicrobials are accurately applied across the paper. The drying procedures used do not affect this uniform distribution or cause deterioration in activity of the antimicrobial.

When each cartridge of 50 discs is sealed together with a molecular sieve in a foil covered see through blister, the discs contain less than 2% moisture and are stored at low temperatures. Real-time shelf life studies of the discs in their packaging demonstrate that they meet the stated storage life printed on the labels.

STORAGE
Discs must be stored at -20°C if kept for long periods. Storage at 2-8°C is suitable for discs currently being used or to be used very soon. Discs should be returned to the refrigerator as quickly as possible after use. The most common cause of moisture reaching the discs and causing destruction of labile antimicrobials is condensation of warm laboratory air on cold discs removed from the refrigerator.

It is important to allow the cartridge blister pack to reach room temperature before exposing the discs, a period of one hour is generally sufficient. Use discs in order of expiry date, which is valid only for unopened blisters stored under proper conditions. Once a cartridge has been opened it needs to be stored in a dessicated environment at 2-8°C for no more than one week.

INOCULUM
One of the very critical factors for accuracy and precision in disc diffusion tests is the inoculum preparation. It is therefore important to use a technique which will always yield a uniform suspension of the correct number of organisms.

Various techniques are described in which suspensions of pre-grown organisms are prepared or small inocula are incubated for fixed periods of time. It is important that more than one colony is sampled (4-10 cols) to ensure a representative sample of the organism has been taken.
Some form of standardisation of the final suspension is necessary and it should be noted that different organisms will display different opacities of solution to yield a dense but not confluent growth.

The Oxoid Turbidometer provides the inoculum density standardisation for 0.5 McFarland necessary to ensure accurate reproducible results.

OTHER FACTORS INFLUENCING THE RESULTS
Temperature of incubation - the incubators should be checked for satisfactory performance and their recording thermometers should show air temperatures of 35-37°C with fluctuations of not more than 2°C. Agar plates should not be placed in high stacks because the middle plates will take longer to reach the incubator temperature and this delay could cause overlarge zones.

A routine procedure should be established so that inoculated plates have discs applied not later than 15 minutes after inoculation. This prevents a pre-incubation of organisms before the antimicrobial discs are applied. Similarly once the discs have been applied, plates should be placed in the incubator within a 15 minute interval, to prevent pre-diffusion of the antimicrobial at room temperature.

Uniformity of agar depth - plates should be poured on leveled surfaces, using dishes with flat bottoms, to ensure a uniform depth of agar (~4mm in depth).
Application of discs - it is essential that discs are in intimate contact with a moist agar surface. Therefore either use dispensers which have a tamping action on the discs or press them separately after application. Do not over dry the agar surface before applying the discs.
Incubation period - ensure uniform times of incubation for the plates, 16-18 hours at 35-37°C is usually satisfactory.
Interpretation of zone sizes - after incubation the plates are removed and the zones of inhibition noted and measured. The diameter of the zone (including the diameter of the disc) is measured to the nearest millimetre, using calipers and compared to the appropriate standard method.
Where other systems are used the zone size breakpoints should have been determined using MIC/zone comparative tests, following the particular methodology chosen.
Control cultures - it is essential that each laboratory maintains adequate control over AST methods by testing reference cultures at regular intervals. Daily tests may be required if new media and discs are constantly being used.

The following reference strains are used in the CLSI test method.
Staphylococcus aureus ATCC® 25923
Escherichia coli ATCC® 25922
Pseudomonas aeruginosa ATCC® 27853
Haemophilus influenza ATCC® 49247
Neisseria gonorrhoeae ATCC® 49226
Streptococcus pneumoniaeATCC® 49619
Escherichia coli ATCC® 35218 for testing compounds containing beta lactamase inhibitors.

Records of the results obtained with these reference strains should be maintained in log books and any deviation of zone sizes from the range accepted should be investigated.

INTERPRETATION OF RESULTS
For details of methodology and interpretational criteria, please refer to the current published standards:
British Society for Antimicrobial Chemotherapy (BSAC)
CDS
Clinical Laboratory Standards Institute (CLSI) formerly NCCLS
Updated CLSI and FDA tables (PDF)
Comite de l’Antibiogramme
EUCAST

References
1 Bondi A., Spauling E. H., Smith E. D. & Dietz C. C. (1947) Amer. J. Med. Sci. 214. 221-225.
2 Cooper H. E. (1964) The theory of antibiotic zones. In Kavanagh F. (Ed) Analytical Microbiology. Academic Press. New York. 1-86.
3 Bell S. M. (1975) Pathology. 7. l-48 (Suppl).
4 Bauer A. W., Kirby W. M. M., Sherris J. C. & Turck M. (1966) Amer. J. Clin. Path. 45. 493-496.
5 Pollock H. M. et al. (1986) J. Clin. Microbiol. 24. 1-6.
6 Nat. Comm. Clin. Lab. Standards (1979) Proposed reference dilution procedure for AST of anaerobic bacteria. NCCLS PSM-11. Villanova. Pa. USA.
7 WHO (1977) Tech. Rep. Ser. No. 610.
8 FDA (1978) Codes of Fed. Rebs. 21. Part 460.

Note
The list of antimicrobial discs manufactured, the generic names, the symbols and range of levels available are supplied in the Oxoid Product List and the list is updated annually.

THE OXOID DISC DISPENSER
An enhanced system for antimicrobial susceptibility testing incorporating the ergonomically designed Disc Dispenser. It has a simple one-handed operation and is fully height-adjustable to cater for various depths of media. The cover and base of the dispenser are fully interlocked to prevent the ingress of moisture. Cartridges `click’ positively into their correct locations and a plastic skirt ensures that each agar plate is precisely centred every time the dispenser is used.

ANTIMICROBIAL SUSEPTIBILITY TESTING MEDIA
Media for Antimicrobial Susceptibility Testing (coded CM) should be stored in their closed containers at an even temperature in a cool, dry place, away from direct light.