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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

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.

Objective

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

105, 106 and 107 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

Starplex Copan

CFU CFU

P. aeruginosa 27853 (105)

Baseline 455 420

4° C 130 130

S. pyogenes 19615 (106) (107)

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 (106) (107)

Baseline 185 68

RT 51 3

4° C 230 5

28° C 0 0

S. pneumoniae 49619 (106) (107)

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 (106) (106)

Baseline 357 417

RT 0 27

4° C 35 53

28° C 0 0

N. gonorrhoeae 43069 (106) (107)

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 (106)

Baseline 378 358

RT 357 65

4° C TNTC 27

28° C 221 83

P. acnes 6919 (106) (107)

Baseline 365 304

RT 0 0

4° C 9 17

28° C 0 0

Results – Amies 21 months

Starplex Copan

CFU CFU

P. anaerobius 27337 (106)

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 (107).

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.