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Millipore Technical Publications

Poster - PDA Annual trade show in Washington and Basel, October 2001

The Effect of Residual VHP on Microorganism Recovery when Performing Air Monitoring in Isolators

Lit Number:PDA Annual trade show in Washington and Basel, October 2001
Year:2001
AuthorSerge Ohresser, Ph.D, Millipore Corporation


Patent Pending on Pyruvate Application using MairT Agar Cassette

Presentation Outline
Introduction
  • Current Situation
  • Conclusion & New Study Objective
Evaluation of Additives
  • Catalase
  • Issues & Conclusions
  • Pyruvate
  • Historical Review
  • Preliminary & Performance Tests
  • Experimental procedure
  • Results
  • Conclusion
  • Evaluation of Gamma Sterilization
Conclusion


Current Situation:
Residual VHP Negatively Effects Microbial Recovery when Performing Air Monitoring

  • The use of vaporous hydrogen peroxide (e.g. VHP) as a sanitant in barrier systems such as isolators is well documented.
  • Microbiological Air monitoring is performed after the sanitization and aeration cycles to confirm that the isolator maintains a germ free environment between decontamination cycles.
  • M Air T ™ Millipore Air Tester, is an agar impaction air tester for microbial analysis in critical areas.
  • Residual levels of vapor hydrogen peroxide can remain in the barrier and inhibit the growth of micro-organisms after concentration on agar media, rendering microbiological air monitoring difficult.

MAirT Isolator
The M Air T Isolator Pump and Sampling Head have been Designed for Air Monitoring in Isolators and Barrier Environments


Current Situation:
Residual VHP Negatively Effects Media used for Microbiological Air Monitoring

  • The use of vaporous hydrogen peroxide (e.g. VHP) as a sanitant in barrier systems such as isolators is well documented.
  • Microbiological Air monitoring is performed after the sanitization and aeration cycles to confirm that the isolator maintains a germ free environment between decontamination cycles.
  • M Air T ™ Millipore Air Tester, is an agar impaction air tester for microbial analysis in critical areas.
  • Residual levels of vapor hydrogen peroxide can remain in the barrier and inhibit the growth of micro-organisms after concentration on agar media, rendering microbiological air monitoring difficult.

Current Situation:
Protocol used to Determine the Effect of Residual VHP on Bacterial Recovery

  • 1000 liters of air were sampled using TSA media in an isolator post sanitization and aeration.
  • Residual VHP was determined to be 0.3ppm.
  • The control was done in a Laminar flow hood, providing microbial-free air as well as VHP-free air.
  • Inoculum of 10-100 cfu of Staphylococcus aureus, Micrococcus luteus, Candida albicans, Pseudomonas aeruginosa, Bacillus subtilis, Aspergillus niger or Staphylococcus hominis were applied to the media and incubated 18h at appropriate temperature.

Current Situation:
As Low as 0.3 ppm Residual VHP can Impact Recovery




Conclusion & New Study Objective

  • Residual: 0.3 ppm residual VHP inhibited growth of 6 USP & EP pharmacopoeia microorganisms as well as 1 environmental isolate.
  • New Study Objective: To optimize a media, for use in isolators, that can mediate the effects of residual VHP and prevent false negative test results.

Evaluation of Additives: Catalase

  • Catalase is known to degrade peroxide through an enzymatic reaction, leading to H2O and O2 as end products.
  • Catalase was incorporated to TSA media at different concentrations.
  • Air sampling of 1000 L was performed on TSA media supplemented with catalase and as a control on standard TSA media, in isolator after sanitization and aeration, with 3 ppm residual VHP.
  • Control of same media was tested in a laminar flow hood.
  • An inoculum of 10-100 cfu of Staphylococcus aureus was applied to the media and incubated 18h at 37°C.

Evaluation of Additives: Catalase
Issue: Air Bubbles in Media due to Enzymatic Reaction




Evaluation of Additives: Catalase
Issue: Low Microbial Recovery due to Catalase



Evaluation of Additives: Catalase
Conclusions

  • Residual VHP inhibits growth of micro-organisms after air sampling: the objective is to optimize a media that can mediate the effects of residual vapor hydrogen peroxide.
  • Catalase, as an additive, displays two major drawbacks:
  • Air bubbles due to the enzymatic reaction
  • Low microbial recovery.
  • Further investigation required to find another solution.

Evaluation of Additives: Pyruvate
Historical Review

  • Baird-Parker & Davenport, 1965 (J. Appl. Bacteriol.): effect of pyruvate supplemented in recovery medium (1%) on the isolation of Sta. Aureus after heat treatment and after storage of frozen or dried cells.
  • Lee & Hartman, 1989 (Journal of Food Protection). Optimal pyruvate concentration (0.02%) for the recovery of coliforms from food and water. Pyruvate degrades peroxide, which is produced by most aerobically growing cells.
  • Mizunoe et al., 1999 (Arch Microbiol). Restoration of culturability of starvation-stressed and low-temperature-stressed E. coli cells by using H2O2-degrading compounds.

Evaluation of Additives: Pyruvate
Protocol used to Select the Optimal Pyruvate Concentration

  • Pyruvate was incorporated into TSA media at different concentrations.
  • Air sampling of 1000 L was performed on TSA media supplemented with pyruvate in an isolator, post sanitization and aeration. Residual VHP was 3 ppm.
  • Control of same media was tested in a laminar flow hood.
  • An inoculum of 10-100 cfu of Staphylococcus aureus was applied to the media and incubated 18h at 37°C.

Evaluation of Additives: Pyruvate
Selection of the Optimal Pyruvate Concentration




Evaluation of Additives: Pyruvate
No Air Bubbles !




Evaluation of Additives: Pyruvate
Protocol Used to Evaluate the Performance of Pyruvate on Selected Strains

  • Pyruvate was incorporated to TSA media at a concentration of 1%. Media devoid of pyruvate was tested in the same time as a negative control.
  • Air sampling of 1000 L was performed on TSA media supplemented or not with pyruvate in an isolator, post sanitization and aeration. Residual VHP was 8 ppm.
  • Control of same media was tested in a laminar flow hood.
  • Inoculum of 10-100 cfu of Staphylococcus aureus, Micrococcus luteus, Candida albicans, Pseudomonas aeruginosa, Bacillus subtilis, Aspergillus niger or Staphylococcus hominis were applied to the media and incubated 18h at appropriate temperature.

Evaluation of Additives: Pyruvate
Pyruvate Protects all Strains Tested Against 8 ppm Residual VHP



Evaluation of Additives: Pyruvate
Protocol used to Determine the Effect of Gamma (g)-Sterilization on Pyruvate Activity

  • Pyruvate was incorporated to TSA media at a concentration of 1%.
  • The media was g-sterilized. Non g-sterilized media was tested in the same time as a control.
  • Air sampling of 1000 L was performed on TSA media supplemented with pyruvate, g-sterilized or non g-sterilized, in an isolator, post sanitization and aeration. Residual VHP was 8 ppm.
  • Control of same media was tested in a laminar flow hood.
  • An Inoculum of 10-100 cfu of Staphylococcus aureus, Micrococcus luteus, Candida albicans, Pseudomonas aeruginosa, Bacillus subtilis, Aspergillus niger or Staphylococcus hominis was applied on the media and incubated 18h at appropriate temperature.

Evaluation of Additives: Pyruvate
Gamma (g)- sterilization does not Effect Pyruvate Activity



Evaluation of Additives: Pyruvate
Conclusions

  • The optimal Pyruvate concentration in TSA media was 1%.
  • Pyruvate at 1% protects all strains tested against up to 8 ppm VHP.
  • Pyruvate activity is not affected by gamma-sterilization.

Presentation Conclusion
  • Millipore has demonstrated that residual VHP as low as 0.3 ppm can inhibit the growth of microorganisms after concentration on the agar, rendering microbiological air monitoring difficult.
  • Catalase was the initial logical solution to neutralize residual VHP. This solution demonstrated poor recovery and air bubbles that formed in the agar, thus rendering the colonies difficult to count.
  • Millipore optimized the MAirT, TSA agar cassette by supplementing it with 1% pyruvate. Data presented proves that this formulation will tolerate as much as 8 ppm residual VHP ensuring growth promotion and avoiding any false negative results when air sampling.