The disc diffusion method, originally described in 1966,¹ is well standardized and has been widely evaluated. Official agencies have recommended it, with minor modifications, as a reference method which could be used as a routine technique in the clinical laboratory.

 

¹ BAUER, A. W. et al. Antibiotic susceptibility testing by a standardized single disc method. American journal of clinical pathology, 44: 493-496 (1966).

Reagents

Mueller-Hinton agar

1. Mueller-Hinton agar should be prepared from a dehydrated base according to the manufacturer’s recommendations. The medium should be such that control zone sizes within the published limits are produced (see Table 13). It is important not to overheat the medium.

2. Cool the medium to 45-50 °C and pour into the plates. Allow to set on a level surface, to a depth of approximately 4 mm. A 9-cm plate requires approximately 25 ml of medium.

3. When the agar has solidified, dry the plates for immediate use for 10 - 30 minutes at 35 °C, by placing them in the upright position in the incubator with the lids tilted.

4. Any unused plates may be stored in a plastic bag, which should be sealed and placed in the refrigerator. Plates stored in this way will keep for 2 weeks.

To ensure that the zone diameters are sufficiently reliable for testing susceptibility to sulfonamides and co-trimoxazole, the Mueller-Hinton agar must have low concentrations of the inhibitors thymidine and thymine. Each new lot of Mueller-Hinton agar should therefore be tested with a control strain of Enterococcus faecalis (ATCC 29212 or 33186) and a disc of co-trimoxazole. A satisfactory lot of medium will give a distinct inhibition zone of 20 mm or more that is essentially free of hazy growth or fine colonies.

Antibiotic discs

Any commercially available discs with the proper diameter and potency can be used. Stocks of antibiotic discs should preferably be kept at - 20°C; the freezer compartment of a home refrigerator is convenient. A small working supply of discs can be kept in the refrigerator for up to 1 month. On removal from the refrigerator, the containers should be left at room temperature for about 1 hour to allow the temperature to equilibrate. This procedure reduces the amount of condensation that occurs when warm air reaches the cold container. If a disc-dispensing apparatus is used, it should have a tight-fitting cover and be stored in the refrigerator. It should also be allowed to warm to room temperature before being opened.

Table 13. Zone diameter limits for control strains^a

 

^a National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disc susceptibility tests. Tentative standard. 4th ed. Villanova, PA, USA, NCCLS, 1988.

^b Sulfafurazole.

Turbidity standard

Prepare the turbidity standard by pouring 0.6 ml of a 1% (10 g/litre) solution of barium chloride dihydrate into a 100-ml graduated cylinder, and filling to 100 ml with 1% (10 ml/litre) sulfuric acid. The turbidity standard solution should be placed in a tube identical to the one used for the broth sample. It can be stored in the dark at room temperature for 6 months, provided it is sealed to prevent evaporation.

Swabs

A supply of cotton wool swabs on wooden applicator sticks should be prepared. They can be sterilized in tins, culture tubes, or on paper, either in the autoclave or by dry heat.

Procedure

To prepare the inoculum from the primary culture plate, touch with a loop the tops of each of 3 - 5 colonies, of similar appearance, of the organism to be tested.

Transfer this growth to a tube of saline.

When the inoculum has to be made from a pure culture, a loopful of the confluent growth is similarly suspended in saline.

Compare the tube with the turbidity standard and adjust the density of the test suspension to that of the standard by adding more bacteria or more sterile saline.

Proper adjustment of the turbidity or the inoculum is essential to ensure that the resulting lawn of growth is confluent or almost confluent.

Inoculate the plates by dipping a sterile swab into the inoculum. Remove excess inoculum by pressing and rotating the swab firmly against the side of the tube above the level of the liquid.

Streak the swab all over the surface of the medium three times, rotating the plate through an angle of 60 ° after each application. Finally, pass the swab round the edge of the agar surface. Leave the inoculum to dry for a few minutes at room temperature with the lid closed.

The antibiotic discs may be placed on the inoculated plates using a pair of sterile forceps. It is convenient to use a template (see Fig. 10) to place the discs uniformly.

A sterile needle tip may also be used to place the antibiotic discs on the plate.

Alternatively, an antibiotic disc dispenser can be used to apply the discs to the inoculated plate.

A maximum of seven discs can be placed on a 9-10 cm plate. Six discs may be spaced evenly, approximately 15 mm from the edge of the plate, and 1 disc placed in the centre of the plate. Each disc should be gently pressed down to ensure even contact with the medium.

The plates should be placed in an incubator at 35 °C within 30 minutes of preparation. Temperatures above 35 °C invalidate results for oxacillin/meticillin.

Do not incubate in an atmosphere of carbon dioxide.

After overnight incubation, the diameter of each zone (including the diameter of the disc) should be measured and recorded in mm. The results should then be interpreted according to the critical diameters shown in Table 14.

The measurements can be made with a ruler on the under-surface of the plate without opening the lid.

If the medium is opaque, the zone can be measured by means of a pair of calipers.

A template (see Fig. 9) may be used to assess the final result of the susceptibility tests.

The endpoint of inhibition is judged by the naked eye at the edge where the growth starts, but there are three exceptions:

 

• With sulfonamides and co-trimoxazole, slight growth occurs within the inhibition zone; such growth should be ignored.

• When β-lactamase-producing staphylococci are tested against penicillin, zones of inhibition are produced with a heaped-up, clearly defined edge; these are readily recognizable when compared with the sensitive control, and regardless of size of zone of inhibition, they should be reported as resistant.

• Certain Proteus species may swarm into the area of inhibition around some antibiotics, but the zone of inhibition is usually clearly outlined and the thin layer of swarming growth should be ignored.

Interpretation of the zone sizes

 

• Using a template. When the zone sizes are compared with the template (see Fig. 9) the result - susceptible, resistant, or intermediate - can be read at once: “susceptible”, when the zone edge is outside the black circle; “resistant”, when there is no zone, or when it lies within the white circle; and “intermediate”, when the edge of the zone of inhibition lies on the black circle.

• Using a ruler. When the zone sizes are measured in mm, the results should be interpreted according to the critical diameters given in Table 14.