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close this bookHealth and Environment in Sustainable Development - Five Years after the Earth Summit (WHO, 1991, 128 p.)
close this folderPart I. Bacteriological investigations
close this folderAntimicrobial susceptibility testing
View the documentIntroduction
View the documentGeneral principles of antimicrobial susceptibility testing
View the documentClinical definition of terms “resistant” and “susceptible”: the three-category system
View the documentIndications for routine susceptibility tests
View the documentChoice of drugs for routine susceptibility tests in the clinical laboratory
View the documentThe modified Kirby-Bauer method
View the documentDirect versus indirect susceptibility tests
View the documentTechnical factors influencing the size of the zone in the disc diffusion method
View the documentQuality control

Quality control

The need for quality control in the susceptibility test

The final result of a disc diffusion test is influenced by a large number of variables. Some of the factors, such as the inoculum density and the incubation temperature, are easy to control, but a laboratory rarely knows the exact composition of a commercial medium or the batch-to-batch variations in its quality, and it cannot take for granted the antimicrobial content of the discs. The results of the test must therefore be monitored constantly by a quality control programme, which should be considered part of the procedure itself.

The precision and accuracy of the test are controlled by the parallel use of a set of control strains, with known susceptibility to the antimicrobial agents. These quality control strains are tested using exactly the same procedure as for the test organisms. The zone sizes shown by the control organisms should fall within the range of diameters given in Table 13. When results regularly fall outside this range, they should be regarded as evidence that a technical error has been introduced into the test, or that the reagents are at fault. Each reagent and each step in the test should then be investigated until the cause of the error has been found and eliminated.

Standard procedure for quality control

The quality control programme should use standard reference strains of bacteria that are tested in parallel with the clinical cultures. They should preferably be run every week, or with every fifth batch of tests, and, in addition, every time that a new batch of Mueller-Hinton agar or a new batch of discs is used.

Standard strains for quality control

Staphylococcus aureus (ATCC 25923)
Escherichia coli (ATCC 25922)
Pseudomonas aeruginosa (ATCC 27853)

These cultures can be obtained from national culture collections. They are commercially available in the form of pellets of desiccated pure cultures.

Cultures for day-to-day use should be grown on slants of nutrient agar (tryptic soy agar is convenient) and stored in the refrigerator. They should be subcultured on to fresh slants every 2 weeks.

Preparing the inoculum

The cultures may be inoculated into any type of broth, and incubated until the broth is turbid. Each broth should be streaked on to an agar plate and incubated overnight. Single colonies should then be picked off and submitted to susceptibility tests as described on “Procedure”.

Placing antimicrobial discs

After the inoculum has been streaked on to the plates, as described on “Procedure”, the appropriate discs should be applied. The discs to be selected for each control strain are listed in Table 13.

Reading the plates

After 16 to 18 hours’ incubation, the diameters of the inhibition zones should be measured with a ruler and recorded, together with the date of the test, on a special quality control chart. This chart should display data for each disc - strain combination. The chart is labelled in millimetres, with an indication of the range of acceptable zone sizes. An example of such a chart is shown in Fig. 11. When the results consistently fall outside the acceptable limits, action should be taken to improve the quality of the test.

Grossly aberrant results, which cannot be explained by technical errors in the procedure, may indicate contamination or sudden changes in the susceptibility or growth characteristics of the control strain. If this occurs, a fresh stock-strain should be obtained from a reliable source.

Fig. 11. Quality control chart for antimicrobial susceptibility testing

Control disc: Ampicillin disc 10 mg. Control strain: E. coli ATCC 25922