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close this bookBasic Laboratory Procedures in Clinical Bacteriology (WHO; 1991; 128 pages)
View the documentPreface
View the documentIntroduction
open this folder and view contentsQuality assurance in microbiology
close this folderPart I. Bacteriological investigations
open this folder and view contentsBlood
open this folder and view contentsCerebrospinal fluid
open this folder and view contentsUrine
open this folder and view contentsStool
open this folder and view contentsLower respiratory tract infections
open this folder and view contentsUpper respiratory tract infections
open this folder and view contentsSexually transmitted diseases
open this folder and view contentsPurulent exudates, wounds, and abscesses
open this folder and view contentsAnaerobic bacteriology
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
open this folder and view contentsPart II. Essential media and reagents for isolation and identification of clinical pathogens
View the documentSelected further reading
View the documentSelected WHO publications of related interest
View the documentBack Cover
 

Technical factors influencing the size of the zone in the disc diffusion method

Inoculum density

If the inoculum is too light, the inhibition zones will be larger although the sensitivity of the organism is unchanged. Relatively resistant strains may then be reported as susceptible. Conversely, if the inoculum is too heavy, the zone size will be reduced and susceptible strains may be reported as resistant. Usually optimal results are obtained with an inoculum size that produces near confluent growth.


Fig. 9. Zone diameters for the determination of susceptibility with the standard disc diffusion method

Table 14. Interpretative chart of zone sizesa

 

Diameter of zone of inhibition (mm)

Antibiotic or chemotherapeutic agent

Disc potency

Resistant

Intermediate/
moderately susceptible

Susceptible

amikacin

30 μg

≤ 14

15-16

≥ 17

ampicillin when testing:

       

- Enterobacteriaceae

10 μg

≤ 13

14-16

≥ 17

- Enterococcus faecalis

10 μg

≤ 16

-

≥ 17

benzylpenicillin when testing staphylococci

10 IU

≤ 28

-

≥ 29

ceftriaxone

30 μg

≤ 13

14-20

≥ 21

cefuroxime sodium

30 μg

≤ 14

15-17

≥ 18

cefalotin

30 μg

≤ 14

15-17

≥ 18

chloramphenicol

30 μg

≤ 12

13-17

≥ 18

clindamycin

2 μg

≤ 14

15-20

≥ 21

co-trimoxazole

25 μg

≤ 10

11-15

≥ 16

erythromycin

15 μg

≤ 13

14-22

≥ 23

gentamicin

10 μg

≤ 12

13-14

≥ 15

nalidixic acid

30 μg

≤ 13

14-18

≥ 19

nitrofurantoin

300 μg

≤ 14

15-16

≥ 17

oxacillin when testing:

       

- staphylococci

1 μg

≤ 10

11-12

≥ 13

- pneumococci

1 μg

≤ 19

-

≥ 20

piperacillin when testing:

       

- Enterobacteriaceae

100 μg

≤ 17

18-20

≥ 21

- Pseudomonas

100 μg

≤ 14

15-17

≥ 18

sulfonamides

300 μg

≤ 12

13-16

≥ 17

tetracycline

30 μg

≤ 14

15-18

≥ 19

tobramycin

10 μg

≤ 12

13-14

≥ 15

trimethoprim

5 μg

≤ 10

11-15

≥ 16

 

a National Committee for Clinical Laboratory Standards. Voluntary consensus standards for clinical laboratory testing. Villanova, PA, NCCLS, 1990.

Timing of disc application

If the plates, after being seeded with the test strain, are left at room temperature for periods longer than the standard time, multiplication of the inoculum may take place before the discs are applied. This causes a reduction in the zone diameter and may result in a susceptible strain being reported as resistant.

Temperature of incubation

Susceptibility tests are normally incubated at 35 °C for optimal growth. If the temperature is lowered, the time required for effective growth is extended and larger zones result. When a heterogeneous resistant strain of Staphylococcus aureus is being tested against meticillin (oxacillin), the resistant portion of the population can be detected at 35 °C. At higher temperatures the entire culture appears to be susceptible. At 35 °C or lower temperatures, resistant colonies develop within the zone of inhibition. These resistant colonies can be seen more easily if the plate is left for several hours at room temperature before the result is read. Such colonies should always be identified to check whether they are contaminants.


Fig. 10. Template for uniform placement of susceptibility discs on plates of 90 mm diameter

Incubation time

Most techniques adopt an incubation period of between 16 and 18 hours. In emergencies, however, a provisional report may be made after 6 hours. This is not recommended as a routine and the result should always be confirmed after the conventional incubation time.

Size of plate, depth of agar medium, and spacing of the antibiotic discs

Susceptibility tests are usually carried out with 9 - 10 cm plates and no more than 6 or 7 antibiotic discs on each plate. If larger numbers of antibiotics have to be tested, two plates, or one 14-cm diameter plate, is to be preferred. Excessively large inhibition zones may be formed on very thin media; the converse is true for thick media. Minor changes in the depth of the agar layer have negligible effect. Proper spacing of the discs is essential to avoid overlapping of the inhibition zones or deformation near the edge of the plates (see Fig. 10).

Potency of the antibiotic discs

The diameter of the inhibition zone is related to the amount of drug in the disc. If the potency of the drug is reduced owing to deterioration during storage, the inhibition zone will show a corresponding reduction in size.

Composition of the medium

The medium influences the size of the zone by its effect on the rate of growth of the organism, the rate of diffusion of the antibiotic, and the activity of the agent. It is essential to use the medium appropriate to the particular method.

The many factors influencing the zone diameters that may be obtained for the same test organism clearly demonstrate the need for standardization of disc diffusion methods. Only if the conditions laid down in a particular method are closely followed can valid results be obtained. Alteration of any of the factors affecting the test can result in grossly misleading reports for the clinician.

The precision and accuracy of the method should be monitored by establishing the quality control programme described below. Variations can then be immediately investigated and corrective action taken to eliminate them.

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