By Russ Nyberg
Editor's note: See the November print issue of ICT for tables and graphs referred to in this article.
Spurred by the recently published methods for substituting the use of chlorine dioxide gas as a replacement for those using formaldehyde gas, an increasing number of individuals are moving in that direction. The question now arises as to how long the exposure time needs to be at various ClO2 concentrations to accomplish an equally effective decontamination. The document published was NSF/ANSI Standard 49, Biosafety Cabinetry; Design, Construction, Performance and Field Certification published by NSF International. This document requires that the process produces a log-6 population (1 million) reduction of spores of resistant bacteria. The resistant spore-forming organism is Bacillus atrophaeus.(1) Usually these spores are applied to a small piece of filter paper, enclosed in a primary package and used as Spore Strips to evaluate the decontamination process. The strips are placed in the most difficult areas for the gas to reach within a BSC or room and then the gassing process begins. Following gassing, the spore strips are collected and aseptically transferred to a tube of growth media and incubated. The strips are then checked after incubation for signs of growth. If the tubes of media show signs of growth, the process was not successful at killing all the bacterial spores and the decontamination cycle is a failed cycle. If the media remains free of growth the cycle would be considered successful and a 6-log reduction was achieved.
In order to accomplish a successful decontamination, one needs to deliver a known concentration of chlorine dioxide gas for the correct amount of exposure time needed to kill a spore strip providing confidence in a successful cycle. In order to find out what the minimum exposure time would be with various concentrations of chlorine dioxide gas, a test chamber was constructed where one could accurately control ClO2 concentration, relative humidity and exposure time.
The test chamber constructed was approximately 10 cubic feet in volume. The chamber had an air sampling port, access door, steam entry port, a means of stirring chamber liquids and a retrieval tube for extracting spore strips after various exposure times to the ClO2 gas. The chamber sat on two large magna stirrers so that large glass petri dishes containing water and a stir bar could be stirred when placed directly above the magna stirrer.
As a means of generating a known quantity of chlorine dioxide gas in the test chamber, we decided to use some ClO2 generating tablets called MB-10 provide by Quip Labs. These tablets can be placed into the chamber petri dishes each containing approximately 20 ml of tap water. The magna stirrers helped to stir the tablet/tap water mixture so that the ClO2 gas generated would be distributed throughout the test chamber. Knowing our chamber volume, we could use four MB-10 ClO2 tablets (two tablets in each petri dish) to generate a chamber ppm volume of 600 ppm ClO2 gas.
Once we were ready to test the spore strips, we placed 20ml tap water in each petri dish, placed a rack of approx 100 log-6 atrophaeus spore strips in a sealed container into the test chamber, set two MB-10 tablets next to each petri dish and introduced steam into the chamber for 10 seconds to generate a chamber RH of 70+ percent. Once the ClO2 tablets were placed into the tap water of each petri dish and stirring began, the chamber air volume was sampled for ClO2 ppm concentration. Once the chamber gas concentration was at and maintained at 600 ppm, the spore strip container lid was removed so that the spore strips exposure to the generated gas would begin. The chamber air was tested for concentration using a Hach Test Kit, ClO2 Colorimeter II. Throughout the testing exposure phase the ClO2 gas concentration remained within a plus or minus 10 percent of the targeted 600ppm. During exposure, spore strips were removed from the chamber at 5-minute intervals over a total exposure period of 40 minutes. Once removed from the chamber, the spore strips were assayed for viable remaining population as per USP 31, Total Viable Spore Count.(2) The viable spores still present on the spore strips after each exposure interval are shown in Table 1.
Table 1 shows that at a ClO2 concentration of 600 ppm, after an exposure of 40 minutes, two CFUs or spores survived the process. To achieve a full all kill of the Log-6 spore strip it would have taken an additional 5-plus minutes or a total time of 45 minutes. A new trial was run where the ClO2 concentration was increased to 1,000ppm. The CFUs after exposure are shown in Table 2.
Based upon the data from Table 2, if one was generating a ClO2 volume concentration of approx. 1,000 ppm, a full decontamination could be accomplished as quickly as 30-plus minutes.
A third test run was performed with a lower ClO2 concentration to see what affect upon time a low concentration would produce. A ClO2 concentration of 250ppm was selected. Table 3 shows the surviving CFU results after the strips were assayed for population.
Table 3 shows that after 40 minutes of exposure at 250ppm of ClO2 gas, 23 viable spores were still present. Extrapolating out in time would show that at approximately 45 minutes we would find an all-kill.
With all three concentrations illustrated above, one must insure that the minimum concentration target has been achieved and once it has, start your exposure time.
Log-6 biological indicator (BI) spore strips can be used to verify a successful cycle. Now that three different ClO2 concentrations were used and the kill times for each is known, one can determine the amount of time necessary to reduce the population of the spore strip by one log. This can be shown by plotting the population reductions on a semi-log graph. The resulting amount of time necessary to reduce the BI population by one log (at specific test parameters) is known as the D-Value for that particular spore strip. Thus if a D-Value for a spore strip were listed to be 5 minutes at 600ppm ClO2 gas concentration, it would take 5 minutes to eliminate one log in population or 30 minutes to reduce all six logs of a six log BI. If one were to graph all three concentrations to show rate of population reduction, it would look like Graph 1.
Graph 1: Population reduction over exposure time using different ClO2 concentrations and log-6 BI spore strips.
Using the regression data from Graph 1, the D-Value for each ClO2 concentration can be determined and they are listed in Table 4.
One can now use the above data to determine how long at various ClO2 concentrations an exposure time would need to be to achieve a 6-log reduction in a BI population and comply with NSF Standard 49. At a concentration for example of 1,000 ppm of ClO2 gas, the exposure time needed to reduce the population one Log (D-Value) would be 4.6 minutes. Thus a 6-log BI would require a minimum exposure time of 27.6 minutes to reduce 6 logs at that concentration.
As shown in Tables 1-3, when the testing exposure period ended, there were still a few surviving spores left on the BI. In Table 1 we had two survivors and as many as 23 in Table 3. With a known resistance D-Value for the spore strips and the certified population of the strips, one can calculate the approximate kill exposure time needed. This is done by multiplying the log of the population times the D-Value.
Using the spore strips used with the 1,000 ppm concentration as an example, the population is 2.7log-6 and the D-Value is 4.6 minutes. Multiplying the log of the population (6.43) times the D-Value of 4.6 minutes we find the calculated kill time to be 29.5 minutes. In actual testing, we found that the actual kill time would be a little longer than 30 minutes. Thus we need to add a cushion or safety factor into the calculation. To do this and increase our confidence that by exposure end all organisms will be dead, we can use a calculation found in United States Pharmacopeia (USP). USP has already established such a cushion for sterilization cycles such as Steam, Ethylene Oxide, Dry Heat, etc. It could also be applied to ClO2. It is the USP calculation for survive/kill times.(3) Using this calculation one will be adding approximately a 4-minute safety margin onto a calculated exposure time. The USP calculation is: (Log of the BI population + 4) (D-Value). Thus if our BI had a population 2.0 X 106 and the D-Value was 7.0 minutes at 600ppm, we would calculate the log of the population (6.3) + 4 (10.3) and then times the D-Value of 7.0 minutes would equal a kill time of 72 minutes at an exposure concentration of 600ppm. If one would not use the USP +4 added to the calculation, the calculated kill time would be 44 minutes. The 44 minutes would be very close to where the actual testing results with 600 ppm were shown to be. However, allowing for unexplained variables that can occur, the USP time of 72 minutes gives us a very high confidence level of successful, repeatable cycles.
In order to accomplish the above, one must be using a calibrated BI spore strip that has a certified D-Value or resistance and a listed population. All B. atrophaeus spore strips do not necessarily have an acceptable resistance to ClO2 or even provide a certified resistance to ClO2. In a study done in 1989,(4) testing results with ClO2 gas and various Bacillus species showed that some atrophaeus (subtilis) BIs and spore discs showed very little to no resistance to the ClO2 gas exposures.
In conclusion, if one is using a certified BI for ClO2 gas and has the means to pre-determine the gas concentration to be delivered, using the USP survive/kill calculation a very accurate and reliably successful decontamination/sterilization cycle exposure time can be used that is not excessively long in exposure duration. Rather than use an alternative to ClO2 gas and leave the cycle run overnight, one can decontaminate from start to finish in a matter of a few hours and get on with the day's work.
Russ Nyberg is the director of tech support/biological indicators for Raven Labs (a Division of Mesa Labs) in Omaha, Neb. He may be reached at (800) 728-5702 or e-mail at: Russ@RavenLabs.com
1. Bacillus atrophaeus, non-pathogenic gram positive spore forming bacteria of demonstrated resistance to ClO2 gas.
2. USP 31, <55> Biological Indicators-Resistance Performance Tests, Total Viable Spore Count, page 69.
3. USP 31, <61> Microbial Tests, Survive and Kill Time, page 71.
4. Jeng D and Woodworth A. Chlorine dioxide gas sterilization under square-wave conditions. Applied and Environmental Microbiology. February 1990, pages 514-519.