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.
IFU-10808[A]