Laboratory Diagnosis of Gonorrhoea

Antibiotic Susceptibility Testing of N. Gonorrhoeae

Two main techniques are available for antimicrobial susceptibility testing:

     disc diffusion tests

     agar plate dilution techniques, as either          

     full minimum inhibitory concentration (MIC) determination, or

     breakpoint tests

The disc diffusion technique is widely employed as the initial susceptibility test method in SEAR countries. The other techniques of agar incorporation will be progressively introduced as expertise develops in the region.

Disc diffusion technique

The Australian Gonococcal Surveillance Programme (AGSP) method is used. This method is based on the Calibrated Dichotomous Sensitivity (CDS) test (Bell, 1975). 

CDS derived antibiotic sensitivity test

Materials

1.      Inoculating wire: a straight nichrome wire (Nichrome SWG24, 0.56 mm diameter). The wire should be 10 cm in length and cut with sharp scissors so that the end is square.

2.      Loop holder for wire.

3.      2.5 mL sterile normal saline in test tubes.

4.      Rubber teat, Pasteur pipettes.

5.      Clear plastic ruler (mm) or vernier callipers.

6.      Incubator.

7.      CO2 source.

Procedure

1.      Sample colony with a straight wire.

2.      Prepare a suspension in 2.5 ml normal saline.

3.      Inoculate a pre-dried chocolate agar plate.

4.      Distribute the inoculum by rocking.

5.      Remove excess inoculum.

6.      Dry the plate at room temperature.

7.      Load the plate with antibiotic discs.

8.      Incubate for 18 hours.

9.      Measure the annular radii.

10.   Interpret zone sizes.

Medium

Chocolate agar comprising Columbia agar base (Oxoid, BBL or LABM) are suitable; other commercial brands should be evaluated for their quality) with 8% horse blood ‘chocolated’ at 70 ºC for 30 minutes (see page 10). 20 mL agar is dispensed into a 90 mm dia Petri dish so that the depth in each plate is approximately 4 mm.

The plates are stored in an inverted position in the refrigerator before use and are used within seven days of preparing.

On the day of the test, the plates are dried by inverting them without the lid in an incubator at 35 ºC for one hour. (Longer drying may be required in a humid climate.)

Inoculum

1.      The inoculum is prepared from a typical colony of at least 2 mm diameter grown overnight on chocolate agar or LBA.

2.      After flaming and cooling the wire, the inoculum (107 cfu/ml) is prepared as follows.

     Pass the straight wire through a colony until it touches the surface of the agar. Move the wire across so that most of the colony is picked up. Bacterial material must be visible on the tip of the straight wire after removal. 

     A heavy inoculum will cause a slight decrease in zone sizes. A light inoculum will cause a marked increase in zone sizes.

     Where possible, the inoculum should be obtained by stabbing to get a single isolated colony. However, if only small colonies are available, it may be necessary to collect 3–5 colonies before the material is visible on the tip of the straight wire.

     Inoculate 2.5 mL saline in a test tube by rotating the straight wire at least 10 times with the tip touching the bottom of the test tube.

Make sure that the material has come off from the tip into the saline. Some gonococci produce ‘sticky’ colonies and the material may have to be teased apart.

Inoculation of plates

1.      Mix the inoculum using a Pasteur pipette at least 10 times and check that there are no lumps left in the suspension.

2.      Flood the dried chocolate agar plate and remove excess inoculum.

3.      Allow the plate to dry at room temperature. This may take 10–15 minutes. Plates must not be left longer than 15 minutes after the inoculum has dried. (If plates are not dry by this time, pre-drying has not been sufficient; see the section on medium, page 28.)

4.      Apply antibiotic discs using sterile forceps or a disc dispenser. Ensure that discs are applied evenly but do not reapply discs after initial contact with the agar.

5.      Low potency discs are recommended in this method. Up to 6 discs can be applied on a single 90 mm dia plate.

6.      Incubate the plates at 35 ºC in air containing 5%–7% CO2 for 18 hours.

Discs and disc strengths