TROUBLESHOOTING GUIDE FOR DISK DIFFUSION SUSCEPTIBILITY TESTING

ABERRANT RESULT CORRECTIVE ACTION EFFECT ON CURRENT DAY'S REPORTS Tetracycline zone too large pH of medium too low.(A,B) Adjust pH to 7.2 to 7.4 Do not report test results until and clindimycin zone too before pouring media. corrective action has been small with Commercial media should taken and a new batch of E. coli or S. aureuscontrol not have pH problems. medium demonstrates strains. Report to manufacturer. acceptable results. Tetracycline zone too pH of medium too high. Get a new lot. (Incubation in small and clindamycin CO2may alter agar surface (A,B) zone too large with pH.) S. aureus or E. colicontrol strain. Aminoglycoside zone too Ca2 and/or Mg2 too high in Acquire a new lot of agar Do not report amino- glycoside small with P. aeruginosa medium.(C,D) medium that will meet QC results on P. aeruginosa or spp. Acinetobactercontrol criteria. until zone sizes meet QC strain. standard. Aminoglycoside zone too Ca2 and/or Mg2 too low in large with P. medium.(C,D) aeruginosa control strain. Zones universially too Adjust inoculum to a Hold results until repeat QC is Inoculum too light. large on control plates. McFarland 0.5 turbidity within limits (6 hour reading). (B,D) standard. Nutritionally poor medium. Use only Mueller Hinton Do not report until Mueller Agar medium. Hinton Agar is used. Slow-growing organism. Use minimum inhibitory Do not report any slow-grower (not seen with controls) concentration (MIC) disk results. procedure only. ABERRANT RESULT PROBABLE CAUSE CORRECTIVE ACTION EFFECT ON CURRENT DAY'S REPORTS Use 4-5mm depth. Do not use this batch or lot of Improper medium depth medium for testing. (too thin)(E,F) Zones universially too Adjust inoculum to a Hold results until repeat QC is Inoculum too heavy. small on control plates. McFarland 0.5 turbidity within limits (6 hour reading). (C,F) standard. Use 4-5mm depth. Do not use this batch or lot of Agar depth too thick. medium for testing. (minor)(D,F) Methicillin zone Change methicillin disks or Report methicillin only if Methicillin degrading decreasing over days or during refrigerator storage. use oxacillin or nafcillin as within QC range. weeks with control the routine disk. (G) organisms. Methicillin zone Methicillin being degraded Do not use coxacillin disks, PROBABLE CAUSE

indeterminate in disk test. by strong beta-lactamase producing staphylococci.

since this drug may not detect in vitromethicillin resistance. Carbenicillin zone Resistant mutant has been Change Pseudomonascontro Repeat carbenicillin disk only. disappears selected for testing. l strain every two weeks and Read at 6 hours. Report other withPseudomonas control. whenever resistant mutants disks as usual. appear within the carbenicillin zone. Cephalothin zone with Unknown. Record actual reading. Report actual test result. E. coli control is too small. S. aureus from a patient Check testing temperature. Report result obtained at 35 May be two different was resistant to methicillin organisms. Temper- ature Test must be performed at degrees C. or 30 degrees C. one day and sensitive the shift from 37 to 35 degrees 35 or 30 degrees C. for next. C. can dramatically alter methicillin (oxacillin or the zone size in this case. nafcillin) and S. aureus.
(H)

A single disk result above Error in reading. Fuzzy or below the control limit. zone edge. Transcription error. Bad disk.

Report other disks following standard protocol. Repeat the test for the out-of-control disk before reporting actual test result. Read repeat QC at 6 hours. ABERRANT RESULT PROBABLE CAUSE CORRECTIVE ACTION EFFECT ON CURRENT DAY'S REPORTS Disk may not be pressed Statistically, one may expect firmly onto the agar an occasional out-of-range surface. (Bad disks usually result. Values usually fall demonstrate a trend toward within range on retesting. being out of control.) Colonies within zone of Mixed culture. Isolate, identify and retest Do not report results of this inhibition. pure cultures only. plate. Resistant mutants within Gram stain or do another test Report as resistant. zone. (See methicillin to rule out contamination. above.) Very large zones with Do not use disk agar anaerobes. diffusion procedure to test anaerobes! With colistin, growth seen "Prozone-like" Confirm with MIC. Report disk test as resistant. immediately adjacent to phenomenon. disk, then larger zone at endpoint (Occurs with colistin when testing Serratia spp. and someEnterobacter spp.). The methicillin disk test Mueller Hinton Broth is No action necessary with Report disk result when tested shows "resistant" but an inadequate in this case. A disk test. To be expected if at 30 degrees C. to 35 degrees

Note error. Recheck error and ask for a second opinion.

MIC shows "sensitive" for S. aureus.

Zones overlap.

modified broth used in some commercial MIC systems frequently eliminates this problem. Low methicillin content in disk.(I) Disks too close together.
(E,J)

Mueller Hinton Broth is C. used in MIC test. Use broth with 2% NaCl if MIC testing is necessary. Use new disks. Use no more than 12 disks Repeat test. on a 150mm plate and 4 to 5 disks on a 100mm plate. CORRECTIVE ACTION EFFECT ON CURRENT DAY'S REPORTS Place disks no closer than 15mm from the edge of the plate. Use properly adjusted Repeat test before reporting. inoculum and repeat test. Report as usual if confident in results.

ABERRANT RESULT

PROBABLE CAUSE

Zones indistinct with single colonies noted on the plate. Indistinct zones with sulfamethoxazole with or without trimethoprim or with trimethoprimalone. Enterococcus appears sensitive to aminoglycoside disks. "Zone within a zone"

Poorly streaked plate. Inadequate inoculum.

Use commercial thymidineThymidine in medium inhibits the action of these free plates. Disregard small amount of growth within the antimicrobics.(J) zone as with sulfonamides. Test only ampicillin, Assessment of erythromycin, aminoglycosides is inaccurate in disk test.(F,G) nitrofurantoin, tetracycline and vancomycin. A swarming Proteusspp. Read the wide distinct zone and disregard the growth that swarmed over. Feather edges of zones Take half the distance from around penicillin or the inner zone to outermost ampicillin disks usually zone as measure mark. occur with beta-lactamasenegative strains of S. aureus. Sulfonamides Disregard growth from disk margin to the major inner zone. Beta-lactamase-positive H Use inside zone. aemophilus influenzae with penicillin or ampicillin.

Report only ampicillin, erythromycin, nitrofurantoin, tetracycline and vancomycin forEnterococcus. Report Proteus spp. phenomenon from outer distinct zone. Report zone as described.

Report outer zone.

Call physician if meningitis.

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