Validation of the Direct Plate Method Described in the NCCLS Document M40: Quality Control of Microbiological Transport Devices C. Brosnikoff and R. Rennie Medical Microbiology Laboratory University of Alberta Hospitals Edmonton, AB, Canada
Objective The objective of this study was to use NCCLS M40-P (Quality Control of Microbiological Transport Systems) to determine the ability of Amies and liquid Stuart s transport media (without charcoal) from two manufacturers to support the growth of organisms over the length of their shelf life.
Swabs Tested The swabs tested were the Amies Starplex Starswab II (Starplex Scientific Inc.) and the Copan Venturi Transystem (Copan Diagnostics Inc).
Testing Schedule Amies transport media was tested after 3, 6, 9, 12, 18 and 21 months of storage at room temperature. Liquid Stuart s transport media was tested after 3, 6, 9, 12 and 18 months of storage at room temperature.
Testing Schedule Swabs from freshly prepared lot numbers were obtained from both manufacturers. The same lot numbers were used for the entire study.
Strains Tested S. pyogenes 19615 S. pneumoniae 49619 H. influenzae 10211 P. aeruginosa 27853 N. gonorrhoeae 43069 S. pneumoniae 6305 added at 18 months
Strains Tested cont d P. anaerobius 27337 F. nucleatum 25586 B. fragilis 25285 P. acnes 6919
Methods Make 0.5 McFarland standard of each organism Serially dilute each organism to the required dilution Inoculate each transport medium with 100 µl of organism suspension 10 5, 10 6 and 10 7 were the dilutions tested
Methods cont d Baseline counts were established by immediately plating out the 0 hour swab onto appropriate solid media and incubating. After 24 48 hr of incubation, colonies were counted. Test swabs were held at room temperature, 4º C and 28º C for 24 (GC) and 48 hours.
Methods cont d The results presented here are the 21 month Amies which represent the end of the shelf life for the Amies transport swab.
Results Amies 21 months P. aeruginosa 27853 (10 5 ) Starplex CFU Copan CFU Baseline 455 420 4 C 130 130 S. pyogenes 19615 (10 6 ) (10 7 ) Baseline 364 255 RT 224 38 4 C 274 118 28 C 77 31
Results Amies 21 months Starplex Copan CFU CFU S. pneumoniae 6305 (10 6 ) (10 7 ) Baseline 185 68 RT 51 3 4 C 230 5 28 C 0 0 S. pneumoniae 49619 (10 6 ) (10 7 ) Baseline 264 249 RT 25 3 4 C 187 26 28 C 0 0
Results Amies 21 months Starplex Copan CFU CFU H. influenzae 10211 (10 6 ) (10 6 ) Baseline 357 417 RT 0 27 4 C 35 53 28 C 0 0 N. gonorrhoeae 43069 (10 6 ) (10 7 ) Baseline 330 319 RT 1 0 4 C 18 1 28 C 0 0
Results Amies 21 months Starplex Copan CFU CFU B. fragilis 25285 (10 6 ) Baseline 378 358 RT 357 65 4 C TNTC 27 28 C 221 83 P. acnes 6919 (10 6 ) (10 7 ) Baseline 365 304 RT 0 0 4 C 9 17 28 C 0 0
Results Amies 21 months P. anaerobius 27337 (10 6 ) Starplex CFU Copan CFU Baseline 193 223 RT 0 0 4 C 23 9 28 C 0 0
Results Amies 21 months P. aeruginosa, S. pyogenes, and B. fragilis were recovered from both systems at RT and 4º C. H. influenzae was recovered from 4º C from both systems; RT holding from Copan only.
Results Amies 21 months S. pneumoniae 6305 and 49619 were recovered from RT holding from Starplex only; both systems demonstrated recovery at 4º C but at higher dilutions for Copan (10 7 ). N. gonorrhoeae 43069 was recovered from 4º C holding only from the Starplex transport system.
Results Amies 21 months P. anaerobius and P. acnes were recovered only at 4º C from both systems F. nucleatum was not recovered from either system
Conclusions The dilutions chosen were designed to yield baseline colony counts of 30 300 CFUs. With some organisms, the dilution yielding the appropriate baseline colony counts differed between the two swab systems and between testing times.
Conclusions The Amies transport media continues to support the recovery of most organisms, especially at 4º C, right to the end of their shelf life and beyond, although higher dilutions are needed for recovery of some organisms.
Conclusions Recovery of anaerobes continues to be better at 4º C.
Conclusions Differences in colony counts at different dilutions and testing times for the commercial swab transport systems indicates that the NCCLS M40 document protocol is not precise. Resolution of these issues may require further developments to the document protocol.
Ongoing Investigation A new study to be undertaken to determine the reproducibility of the M40 document protocol.