Chlorine Dioxide Gas Decontamination of Bacillus Atrophaeus and
Chlorine Dioxide Gas Decontamination of Bacillus Atrophaeus and Geobacillus Stearothermophilus Biological Indicators #14 Henry S. Luftman and Michael A. Regits DRS Laboratories Allentown, Pennsylvania Abstract In anticipation of a validation program for the use of chlorine dioxide (CD) gas in the decontamination of laminar flow biological safety cabinets, the use of Bacillus atrophaeus (BA) and Geobacillus stearothermophilus (GS) endospores for biological indicators (BIs) of CD gas decontaminations were studied. This work includes studies of BIs having either paper or steel substrates. BI analyses were performed with both enumeration and fractionation methods. Among the conclusions it was found that in using paper GS BIs, results from enumeration analysis proved to be too variable to monitor CD decontaminations. Targeted CD exposure levels provided significantly less than a 6 -log spore reduction of GS spores on steel substrates, but were very effective for GS BIs with paper substrates. BA spores on paper proved to be suitable and repeatable for validation work with either enumeration or fractionation analysis. Formaldehyde decontamination conditions used in standard practices were largely ineffective at decontaminating the GS indicators. Materials and Methods • Chlorine Dioxide generation • Fixed mass of CD via Reaction (2) for all data except that in Table 1 (Reaction (1)) • CD concentration monitored by spectrometer in real time, used to determine dosages • Containment device • Most experiments in biological safety cabinet • Data in Tables 1 and 2 from other enclosures • Humidification • Most with steam • Data in Tables 1 and 2, cold water nebulizer Biological Safety Cabinet for most of studies Test BI Locations Chlorine Dioxide Gas (Cl. O 2) Formaldehyde (F), Chlorine Dioxide (CD) and Hydrogen Peroxide (HP) • Potent to bacteria – F, HP, CD • Non-carcinogen – HP, CD • “True” gas, penetrating – F, CD • Non-reactive with cellulose – F, CD • No residue – HP, CD Biological Indicator (BI) Characteristics • Endospore Species • B. atrophaeus (BA, formerly subtilus)– formaldehyde, ethylene oxide, dry heat • G. stearothermophilus (GS) – hydrogen peroxide, steam • Both have been used for CD • Population – typically ~106 – can buy lower or higher • Various substrates (paper, stainless steel) and envelopes BI Analysis • Fractionation (Go / No Go) • Drop strip into media and incubate 3 -7 days • BA: 30 -35 o. C, GS: 55 -60 o. C incubation • Look for turbidity and/or color change in media • Result All spores inactive or at least one spore active • Enumeration (Log Reduction) • Macerate strip (glass beads) • Heat shock • Plate into molten media agar and incubate (2 -5 days) • Count colony forming units (CFU’s) • Compare to positive control for log-kill • Much more info than one G/NG, but requires better control and more expensive Results (cont. ) Table 4. GS BI cellulose strips in CD BSC experiment Table 5. GS BIs in formaldehyde BSC experiment Table 1. BI strip enumeration (CFU) following CD dose of 0. 4 mg-hr/L at 70% RH Each ratio represents the number of positively testing BIs over the total number used within that trial. Humidity 70%-85% RH. Geobacillus stearothermophilus Fractionation (cont. ) Introduction • Decontamination mechanism: Selective oxidation (no chlorination, more like ozone) • Generated on site via reaction: • (1) Cl 2(g) + 2 Na. Cl. O 2 2 Cl. O 2(g) + 2 Na. Cl • (2) 5 Na. Cl. O 2 + 4 H+(aq) 4 Cl. O 2(g) + 2 H 2 O + Na. Cl + 4 Na+(aq) • Visible green gas • Humidification required, 65 -80% RH Results (cont. ) • Biological Indicators • Spores: B. atrophaeus and G. stearothermophilus • Suppliers: Apex Laboratories, SGM Biotech, and STERIS • Substrates: paper (strip) and stainless steel • Envelope materials: Tyvek, Mylar, and bare • Indicator Placement • See Figure CD dose intended to be sub-lethal. SGM in Tyvek/Mylar pouches, STERIS strips bare. • BA Results • Lab 1 : No surviving spores from STERIS, >1000 from SGM • Lab 2: Similar • Spore strip effect • GS Results • Lab 1: Similar high (~105) survival among BIs • Lab 2: Lower (100 – 101) survival Lab effect • Similar results for BI sets stored for 1 week prior to analysis Table 2. BI results from 2 -ft 3 test chamber experiment • CD Neutralization • For data in Tables 6 and 7 • Exposed BIs submerged in 1 wt% sodium thiosulfate for 1 min Values for GS BI disks correspond to number positive out of two disks used. BA results indicate the observed logreduction from the count of control strips, based upon enumeration analysis. • Formaldehyde experiments (Table 5) • 0. 3 g/ft 3 depolymerized paraformaldehyde Humidity supplied by ultra-sonicator. • BI Analysis • Enumeration: Three separate laboratories • Go/No-go: In-House • Media: Trypticase soy broth Table 3. GS BI disks in CD BSC experiment Dose > 4. 3 mg-hr/L yields < 0. 5 CFU/BI All BIs were impregnated with ~2 x 106 endospores of Geobacillus stearothermophilus. Each data point corresponds to accumulated results form 3 to 5 BIs from a single experimental run. Tables 6 and 7. Survival (CFU) BA spores from paper indicators (~10 6 spores initially) following treatment with CD at 0. 1 mg-hr/L, either with or without immersion in 1. 0% sodium thiosulfate (ST) General increase (~ x 2) recovery of BA spores if paper BIs treated with sodium thiosulfate prior to incubation. Table 8. BA BIs in CD BSC experiment B. Atrophaeus Fractionation • < 6 -log reduction of stainless steel BIs at 7. 3 mg-hr/L • > 6 -log reduction of paper BIs at 1. 5 mg-hr/L Each ratio represents the number of positively testing BIs over the total number used within that trial. Humidity 65 -75% RH. Results Surviving CFUs from BIs following Chlorine Dioxide Gas Exposure • Paper BIs in BSC with chlorine dioxide gas: 6 -log reduction at dose ≥ 1. 1 mg-hr/L • Paper and stainless steel BIs in BSC with formaldehyde gas (nom. 8000 ppm): spore reduction with 19 hr exposure < 4 -log with steel BIs, < 6 -log with paper BIs Significantly different effect of CD between paper and stainless steel BIs Formaldehyde has limited effect of G. stearothermophilus spores, paper or steel substrates Geobacillus Stearothermophilus Fractionation • Stainless steel BIs in test chamber • Generally 6 -log reduction at > 1 mg-hr/L dose • Stainless steel BIs in BSC • < 6 -log reduction at 6. 3 mg-ht/L • < 5 -log reduction at 1. 7 mg-hr/L • Difference attributed to methods of humidification – cold water vapor possibly dampens BI, increasing local CD absorption. Discussion and Conclusion • Greater resistance of stainless steel vs. paper Bis for CD • 2 -dimensional islanding at higher spore doses on steel • Greater gas access to spores distributed on paper fibers • Potential enhancement of CD if paper becomes moistened • Variability of enumeration for GS Bis • Analysis • Biological Indicators • Method of maceration • Spore clustering • Growth media • BI substrates • Other manufacturer variables • Method of counting colonies • Storage • Time lapse until spore preparation • Formaldehyde gas vs. CD decontamination • Standard formaldehyde methods yield less than a 6 -log reduction for G. stearothermophilus spores • CD gas is more successful (6 -log reduction) with GS if the BI has a paper substrate • While both are effective against B. atrophaeus spores on paper, CD is not as effective with steel substrates • Analysis of CD decontamination was most reproducibly monitored using B. atrophaeus spores on paper substrates using fractionation analysis. • Process variables included BI spore species, substrate and vendor, as well as the method and laboratory used for microbiological analysis.
- Slides: 1