Validation of the Direct Plate Method Described in the NCCLS Document M40: Quality Control of Microbiological Transport Devices

Transcription

1 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

2 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.

3 Swabs Tested The swabs tested were the Amies Starplex Starswab II (Starplex Scientific Inc.) and the Copan Venturi Transystem (Copan Diagnostics Inc).

4 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.

5 Testing Schedule Swabs from freshly prepared lot numbers were obtained from both manufacturers. The same lot numbers were used for the entire study.

6 Strains Tested S. pyogenes S. pneumoniae H. influenzae P. aeruginosa N. gonorrhoeae S. pneumoniae 6305 added at 18 months

7 Strains Tested cont d P. anaerobius F. nucleatum B. fragilis P. acnes 6919

8 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

9 Methods cont d Baseline counts were established by immediately plating out the 0 hour swab onto appropriate solid media and incubating. After hr of incubation, colonies were counted. Test swabs were held at room temperature, 4º C and 28º C for 24 (GC) and 48 hours.

10 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.

11 Results Amies 21 months P. aeruginosa (10 5 ) Starplex CFU Copan CFU Baseline C S. pyogenes (10 6 ) (10 7 ) Baseline RT C C 77 31

12 Results Amies 21 months Starplex Copan CFU CFU S. pneumoniae 6305 (10 6 ) (10 7 ) Baseline RT C C 0 0 S. pneumoniae (10 6 ) (10 7 ) Baseline RT C C 0 0

13 Results Amies 21 months Starplex Copan CFU CFU H. influenzae (10 6 ) (10 6 ) Baseline RT C C 0 0 N. gonorrhoeae (10 6 ) (10 7 ) Baseline RT C C 0 0

14 Results Amies 21 months Starplex Copan CFU CFU B. fragilis (10 6 ) Baseline RT C TNTC C P. acnes 6919 (10 6 ) (10 7 ) Baseline RT C C 0 0

15 Results Amies 21 months P. anaerobius (10 6 ) Starplex CFU Copan CFU Baseline RT C C 0 0

16 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.

17 Results Amies 21 months S. pneumoniae 6305 and 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 was recovered from 4º C holding only from the Starplex transport system.

18 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

19 Conclusions The dilutions chosen were designed to yield baseline colony counts of CFUs. With some organisms, the dilution yielding the appropriate baseline colony counts differed between the two swab systems and between testing times.

20 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.

21 Conclusions Recovery of anaerobes continues to be better at 4º C.

22 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.

23 Ongoing Investigation A new study to be undertaken to determine the reproducibility of the M40 document protocol.