TROUBLESHOOTING GUIDE FOR DISK DIFFUSION SUSCEPTIBILITY TESTING

ABERRANT RESULT PROBABLE CAUSE CORRECTIVE ACTION EFFECT ON CURRENT DAY'S REPORTS
Tetracycline zone too large and clindimycin zone too small with
E. coli or S. aureus control strains.
pH of medium too low. (A,B) Adjust pH to 7.2 to 7.4 before pouring media. Commercial media should not have pH problems. Report to manufacturer. Do not report test results until corrective action has been taken and a new batch of medium demonstrates acceptable results.
Tetracycline zone too small and clindamycin zone too large with
S. aureus or E. coli control strain.
pH of medium too high. (A,B) Get a new lot. (Incubation in CO 2 may alter agar surface pH.)
Aminoglycoside zone too small with P. aeruginosa Acinetobacter control strain. Ca 2 and/or Mg 2 too high in medium. (C,D) Acquire a new lot of agar medium that will meet QC criteria. Do not report amino- glycoside results on P. aeruginosa or spp. until zone sizes meet QC standard.
Aminoglycoside zone too large with P. aeruginosa control strain. Ca 2 and/or Mg 2 too low in medium. (C,D)
Zones universially too large on control plates. Inoculum too light.
(B,D)
Adjust inoculum to a McFarland 0.5 turbidity standard. Hold results until repeat QC is within limits (6 hour reading).
Nutritionally poor medium. Use only Mueller Hinton Agar medium. Do not report until Mueller Hinton Agar is used.
Slow-growing organism. (not seen with controls) Use minimum inhibtory concentration (MIC) procedure only. Do not report any slow-grower disk results.
ABERRANT RESULT PROBABLE CAUSE CORRECTIVE ACTION EFFECT ON CURRENT DAY'S REPORTS
Improper medium depth (too thin) (E,F) Use 4-5mm depth. Do not use this batch or lot of medium for testing.
Zones universially too small on control plates. Inoculum too heavy.
(C,F)
Adjust inoculum to a McFarland 0.5 turbidity standard. Hold results until repeat QC is within limits (6 hour reading).
Agar depth too thick. (minor) (D,F) Use 4-5mm depth. Do not use this batch or lot of medium for testing.
Methicillin zone decreasing over days or weeks with control organisms. Methicillin degrading during refrigerator storage. (G) Change methicillin disks or use oxacillin or nafcillin as the routine disk. Report methicillin only if within QC range.
Methicillin zone indeterminate in disk test. Methicillin being degraded by strong beta-lactamase producing staphylococci. Do not use coxacillin disks, since this drug may not detect in vitro methicillin resistance.
Carbenicillin zone disappears with Pseudomonas control. Resistant mutant has been selected for testing. Change Pseudomonas control strain every two weeks and whenever resistant mutants appear within the carbenicillin zone. Repeat carbenicillin disk only. Read at 6 hours. Report other disks as usual.
Cephalothin zone with
E. coli control is too small.
Unknown. Record actual reading. Report actual test result.
S. aureus from a patient was resistant to methicillin one day and sensitive the next. May be two different organisms. Temper- ature shift from 37 to 35 degrees C. can dramatically alter the zone size in this case. (H) Check testing temperature. Test must be performed at 35 or 30 degrees C. for methicillin (oxacillin or nafcillin) and S. aureus . Report result obtained at 35 degrees C. or 30 degrees C.
A single disk result above or below the control limit. Error in reading. Fuzzy zone edge. Transcription error. Bad disk. Note error. Recheck error and ask for a second opinion. 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 firmly onto the agar surface. (Bad disks usually demonstrate a trend toward being out of control.) Statistically, one may expect an occasional out-of-range result. Values usually fall within range on retesting.
Colonies within zone of inhibition. Mixed culture. Isolate, identify and retest pure cultures only. Do not report results of this plate.
Resistant mutants within zone. (See methicillin above.) Gram stain or do another test to rule out contamination. Report as resistant.
Very large zones with anaerobes. Do not use disk agar diffusion procedure to test anaerobes!
With colistin, growth seen immediately adjacent to disk, then larger zone at endpoint (Occurs with colistin when testing
Serratia spp. and some Enterobacter spp.).
"Prozone-like" phenomenon. Confirm with MIC. Report disk test as resistant.
The methicillin disk test shows "resistant" but an MIC shows "sensitive" for S. aureus . Mueller Hinton Broth is inadequate in this case. A modified broth used in some commercial MIC systems frequently eliminates this problem. No action necessary with disk test. To be expected if Mueller Hinton Broth is used in MIC test. Use broth with 2% NaCl if MIC testing is necessary. Report disk result when tested at 30 degrees C. to 35 degrees C.
Low methicillin content in disk. (I) Use new disks.
Zones overlap. Disks too close together. (E,J) Use no more than 12 disks on a 150mm plate and 4 to 5 disks on a 100mm plate. Repeat test.
ABERRANT RESULT PROBABLE CAUSE CORRECTIVE ACTION EFFECT ON CURRENT DAY'S REPORTS
Place disks no closer than 15mm from the edge of the plate.
Zones indistinct with single colonies noted on the plate. Poorly streaked plate. Inadequate inoculum. Use properly adjusted inoculum and repeat test. Repeat test before reporting.
Indistinct zones with sulfamethoxazole with or without trimethoprim or with trimethoprimalone. Thymidine in medium inhibits the action of these antimicrobics. (J) Use commercial thymidine-free plates. Disregard small amount of growth within the zone as with sulfonamides. Report as usual if confident in results.
Enterococcus appears sensitive to aminoglycoside disks. Assessment of aminoglycosides is inaccurate in disk test. (F,G) Test only ampicillin, erythromycin, nitrofurantoin, tetracycline and vancomycin. Report only ampicillin, erythromycin, nitrofurantoin, tetracycline and vancomycin for Enterococcus .
"Zone within a zone" A swarming Proteus spp. Read the wide distinct zone and disregard the growth that swarmed over. Report Proteus spp. phenomenon from outer distinct zone.
Feather edges of zones around penicillin or ampicillin disks usually occur with beta-lactamase-negative strains of S. aureus . Take half the distance from the inner zone to outermost zone as measure mark. Report zone as described.
Sulfonamides Disregard growth from disk margin to the major inner zone. Report outer zone.
Beta-lactamase-positive Haemophilus influenzae with penicillin or ampicillin. Use inside zone. Call physician if meningitis.

For more information on Disk Diffusion Testing, see the HUGO™ keywords:

"Disk Diffusion Testing"
"QC Zone Diameters"
"Inoculation Procedures"

REFERENCES

A. Garrod, L.P., Lambert, H.P. and O'Grady, F., et al. 1973. "Laboratory control", in Garrod, L.P., Lambert , H.P., and O'Grady F., (eds): Antibiotics and Chemotherapy , 4th ed. Edinburgh, Churchill-Livingston; pp. 451-485.

B. Thornsberry, C. 1974. "The agar diffusion antimicrobial susceptibility test", in Balows, A., (ed): Current Techniques for Antibiotic Susceptibility Testing . Springfield, IL, Charles C. Thomas; pp. 6-16.

C. Reller, L.B., Schoenknecht, F.D., and Kenny M.A., et al. 1974. Antibiotic susceptibility testing of Pseudomonas aeruginosa : selection of a control strain and criteria for magnesium and calcium content in media. J Infect. Dis. ; 130:454-463.

D. Balos, A., and Gavan, T.L. 1980. "Quality control methods for in vitro antibiotic susceptibility testing", in Lorian V (ed) Antibiotics in Laboratory Medicine . Baltimore, Williams & Wilkins; pp. 409-417.

E. Acar, J.F. 1980. "The disc susceptibility test", in Lorian V., (ed): Antibiotics in Laboratory Medicine . Baltimore, Williams & Wilkins; pp. 24-54.

F. Performance Standards for Antimicrobic Disc Susceptibility Test , M2-A2S2 (suppl 2). 1982. Clinical Laboratory Standards Institute (CLSI - formerly NCCLS), Villanova, PA.

G. Thornsberry, C. Gavan, T.L. and Gerlach, E.H. 1977. "New developments in antimicrobial agent susceptibility testing", in Sherris J.C., (ed): Cumitech 6 . Washington D.C., American Society for Microbiology; pp. 1-13.

H. Thornsberry, C., Caruthers, J.Q. and Baker, C.N. 1973. Effect of temperature on the in vitro susceptibility of Staphylococcus aureus to penicillinase-resistant penicillins. Antimicrob Agents Chemother. ; 4:263-269.

I. Thornsberry, C. and McDougal, L.K. 1983. Susceptibiltiy tests for methicillin-resistant staphylococci: successful use of broth microdilution. J. Clin. Microbial. ; 18:1084-1091.

J. Performance Standards for Antimicrobic Disc Susceptibility Test : M-2, 2nd ed. 1972. Clinical Laboratory Standards Institute (CLSI - formerly NCCLS), Villanova, PA.


The chart above was prepared by J. Michael Miller, Ph.D., Clyde Thornsberry, Ph.D., and Carolyn Baker, BS; from the Bacteriology Training Section, Centers for Disease Control, Atlanta, GA 30333. 1984. Reprinted with permission of Carolyn Baker, CDC.

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