Validation of the Direct Plate Method Described in the NCCLS Document M40: Quality Control of Microb

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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

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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 Stuarts transport media
(without charcoal) from two manufacturers to
support the growth of organisms over the length
of their shelf life.
Objective
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Swabs Tested

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

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Testing Schedule

• Amies transport media was tested after 3, 6, 9,
  12, 18 and 21 months of storage at room
  temperature.
• Liquid Stuarts transport media was tested after
  3, 6, 9, 12 and 18 months of storage at room
  temperature.

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Testing Schedule

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

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Strains Tested

• S. pyogenes 19615
• S. pneumoniae 49619
• H. influenzae 10211
• P. aeruginosa 27853
• N. gonorrhoeae 43069
• S. pneumoniae 6305 added at 18 months

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Strains Tested contd

• P. anaerobius 27337
• F. nucleatum 25586
• B. fragilis 25285
• P. acnes 6919

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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

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Methods contd

• 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.

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Methods contd

• The results presented here are the 21 month Amies
  which represent the end of the shelf life for the
  Amies transport swab.

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Results Amies 21 months
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Results Amies 21 months
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Results Amies 21 months
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Results Amies 21 months
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Results Amies 21 months
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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.

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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.

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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

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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.

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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.

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Conclusions

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

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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.

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Ongoing Investigation

• A new study to be undertaken to determine the
  reproducibility of the M40 document protocol.