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 are 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 will be tested after 3, 6,
  9, 12, 18 and 20 months of storage at room
  temperature
• Liquid Stuarts transport media will be tested
  after 3, 6, 9, 12 and 18 months of storage at
  room temperature
• Swabs from freshly prepared lot numbers were
  obtained from both manufacturers. The same lot
  numbers were/are 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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Results Amies 12 months

• P. aeruginosa, S. pyogenes and B. fragilis were
  recovered from both systems at all holding
  temperatures
• S. pneumoniae was recovered from 4º C from both
  systems RT holding from Starplex only
• H. influenzae was recovered from 4º C from both
  systems RT holding from Copan only
• GC was recovered after 4º C holding from Starplex
  only

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Results Amies 12 months contd

• P. acnes was recovered from Starplex only
• P. anaerobius was not recovered from either
  system
• F. nucleatum failed to grow even in the baseline
  counts

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Results Stuarts 12 months

• P. aeruginosa and S. pyogenes were recovered from
  both systems
• S. pneumoniae and GC were recovered from both
  systems but only at 4º C
• H. influenzae was recovered from both systems at
  4º C, but only Copan demonstrated recovery from
  RT

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Results Amies 18 months
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Results Amies 18 months
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Results Amies 18 months
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Results Amies 18 months

• P. aeruginosa, S. pyogenes, S. pneumoniae 49619
  and B. fragilis were recovered from both systems
• S. pneumoniae 6305 was recovered from 4º C from
  both systems RT holding from Starplex only
• H. influenzae was recovered from 4º C from both
  systems RT holding from Copan only

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Results Amies 18 months

• P. acnes was recovered from Starplex only
• P. anaerobius was recovered from both systems at
  4º C
• F. nucleatum failed to grow even in the baseline
  counts

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Results Stuarts 18 months
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Results Stuarts 18 months
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Results Stuarts 18 months

• P. aeruginosa and S. pyogenes were recovered from
  both systems
• S. pneumoniae 6305 showed recovery from both
  systems at 4º C, but only Starplex had recovery
  from RT
• S. pneumoniae 49169 only showed recovery at 4º C
• H. influenzae and GC were recovered from both
  systems at 4º C, but only Copan demonstrated
  recovery from RT

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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
• Recovery of anaerobes is better at
• 4º C

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Conclusions

• Liquid Stuarts media demonstrated better recovery
  from 4º C, especially the more fastidious
  organisms
• It has demonstrated good recovery to the end of
  the stated shelf life (12 month Copan and 18
  month Starplex)

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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

• Testing at 21 months will be performed on Amies
  transport
• media to complete this study.
• A new study to be undertaken to determine the
  reproducibility of the M40 document protocol.