APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1987, p. 683-686
Vol. 53, No. 4
0099-2240/87/040683-04$02.00/0 Copyright © 1987, American Society for Microbiology
An Evaluation of the Sporicidal Activity of Ozone JAMES R. RICKLOFF
Research Department, American Sterilizer Company, Erie, Pennsylvania 16514 Received 7 April 1986/Accepted 26 December 1986
This study was undertaken to determine the feasibility of sterilizing surfaces with ozone-saturated water by the methods of the Association of Official Analytical Chemists (AOAC). Initially, it was determined that there was no apparent difference in ozone resistance between spores of Bacillus subtilis and Clostridium sporogenes when they are suspended in water. Both species were inactivated by a 10-min exposure at ambient temperature. Resistance was increased when the spores were dried on AOAC carriers. Viable organisms were recovered after an exposure of 40 min at ambient temperature. An increase in the reactor water temperature to 600C did not improve the effectiveness of the ozone in sterilizing AOAC carriers. Dried spores of C. sporogenes were more resistant than B. subtilis spores because of a greater accumulation of organic matter on the carriers. No significant sporicidal activity was demonstrated after 40 min for spores of either species when they were inoculated on silk suture loops. The data suggest that organic loading and poor ozone penetrability are key factors in effecting the ability of ozone to sterilize surfaces rapidly.
Ozone is known to possess broad-spectrum antimicrobial properties (1, 2, 4). Because of a variety of problems associated with its use in the past, the only practical application of ozone was limited to potable water disinfection, primarily in Europe. With the introduction of highly efficient and reliable ozone-generation equipment, however, a number of other applications have been developed and applied on a worldwide basis (10). The health care industry could derive benefits if high concentrations of ozone dissolved in water demonstrated rapid sporicidal activity. A potential application for ozone in hospitals is in the sterilization of heat-sensitive medical equipment or instruments. Currently, such items are exposed to a high-level disinfectant, i.e., 2% glutaraldehyde, between each patient procedure. The increase in the number of applications in which thermolabile equipment is used has generated considerable controversy regarding such disinfection practices (7, 8). Under usual conditions, high-level disinfectants cannot guarantee the sterility of the treated object but can only reduce the risk of infection. Sterilization, on the other hand, provides a degree of assurance that all microbial life, including highly resistant bacterial spores, has been destroyed (16). The major drawback to sterilization is that it is usually limited to lengthy exposure to ethylene oxide, which, if it were mandatory, would require hospitals to purchase multiple pieces of equipment at a high initial cost. Therefore, practical considerations still favor disinfection, but a rapid, i.e., less than 30 min, method of sterilization would satisfy all of the diverse requirements associated with these items. This study was undertaken to determine the feasibility of rapidly sterilizing surfaces with ozonated water. Because the sporicidal activity of ozone has not been studied in detail, experiments were performed to determine if high concentrations of ozone dissolved in water could destroy bacterial spores in suspension. On successful completion of these studies, surface sterilization tests were undertaken by the methods of the Association of Official Analytical Chemists (AOAC). The AOAC sporicidal test is the standard method used by the Environmental Protection Agency to designate a chemical germicide as a sterilant (7). The use of large numbers of spores in the presence of organic soil makes the test highly stringent. This provides adequate assurance that the germicides passing the test are most likely effective
sterilants. Experiments were also performed with spores inoculated on AOAC penicylinders in the absence of organic soil to determine the ability of ozone to sterilize relatively clean surfaces. MATERIALS AND METHODS Test orgailisms, The spore-forming organisms selected for this study included Bacillus subtilis ATCC 19659 and Clostridium sporogenes AtCC 3584. Spores of B. subtilis were prepared with soil extract medium, while soil extract-eggmeat medium was used for the propagation of C. sporogenes spores, as described by AOAC sporicidal test methods (12). Preparation of spores for testing. Before testing of the effectiveness of ozone against spores in suspension was begun, 72-h cultures of B. subtilis and C. sporogenes (10 ml each) were initially filtered through sterile cotton to remove cellular debris. The filtered cultures were centrifuged for 20 min at 18,800 x g, and then the pellets were suspended in an equal volume of sterile distilled water. This washing procedure was repeated 3 times. Both suspensions were then heat shocked at 80°C for 20 min to ensure the absence of any vegetative cells. The spore suspensions were refrigerated until use. A 1-ml fraction of each suspension was diluted 4,000-fold in distilled water and stirred before the ozone was added. The initial spore population in the reactor was determined by plating out dilutions of a 1-ml sample in tryptic soy agar (Difco Laboratories, Detroit, Mich.). For C. sporogenes, the plates were initially placed in a GasPak Anaerobic System (BBL Microbiology Systems, Cockeysville, Md.) prior to incubation of both test organisms at 370C for 48 h. Spore-inoculated porcelain penicylinders and silk suture loops were prepared for testing by AOAC sporicidal test methods (12). Porcelain penicylinders were also inoculated with washed spores of each test organism that were prepared as described above. Ozone. The ozone was generated from oxygen at an operating pressure of 16 lb/in2 with a high-frequency ozone generator (model GL-1; PCI Ozone Corp., West Caldwell, N.J.). The flow rate was set at 5 ft3 of oxygen per h, which permitted ozone gas phase concentrations to reach 8% (by weight; 107 mg of applied dosage per liter). Initially, the determination of the aqueous ozone concentration was per683
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RICKLOFF
formed by a modification of the method described by Shechter (15). A 1-ml sample was added to 5 ml of 2% neutral potassium iodide and 4 ml of distilled water. The standard curve was linear from 0.3 to 2.0 mg of ozone per liter. Dissolved ozone concentrations averaged approximately 10 mg/liter, but values could not be accurately reproduced at these saturated levels. Therefore, instead of monitoring the aqueous concentration, the incoming ozoneoxygen mixture was monitored with an ozone monitor (model HC; PCI Ozone Corp.) to maintain a constant input of ozone to the batch reactor. Exposure of suspended spores. Ozone was added continuously to spore suspensions through a porous glass diffuser placed at the bottom of a Lexan (General Electric Plastics) cylinder. The batch reactor (height, 620 mm; diameter, 100 mm) held a total volume of 4,000 ml of distilled water. A stainless steel sample port (Conax Corp., Buffalo, N.Y.) was threaded through the side of the reactor near the center of the water column for sampling purposes. This fitting permits the monitoring of a sealed chamber without altering the conditions under study. Spore survival determinations at ambient temperature (approx. 20°C) were made, after specified contact times, with the use of a 10 cm3 sterile, disposable syringe and an 18-gauge needle (American Pharmaseal Laboratories, Glendale, Calif.). The needle was passed through the silicone septum of the sample port and was flushed several times prior to sample removal. Each 10-ml sample was immediately transferred to a sterile test tube containing 100 ,u of 1% sodium thiosulfate and mixed to destroy any residual ozone. A control experiment was performed to show that this quantity of sodium thiosulfate would not inhibit spore germination. A 5-ml fraction of the treated sample was then aseptically transferred to test tubes containing 5 ml of double-strength Trypticase soy broth (BBL) for recoVery of B. subtilis or double-strength fluid thioglycolate medium USP (B3BL) for C. sporogenes. Each sample was incubated at 37°C for 7 days before the presence or absence of growth was determined. Exposure of spore-inoculated carriers. A different reactor was required to facilitate the use of carriers. The apparatus consisted of a 2,000-ml beaker (height, 175 mm; diameter, 125 mm; Pyrex; Coming Glass Works, Corning, N.Y.) containing 1,600 ml of distilled water and a glass diffuser for continuous ozonation. A glass funnel over the top of the reactor was used to direct excess ozone to a vent. Each lot of cal-riers was verified for sufficient resistance to 2.5 N hydrochloric acid, as specified by AOAC test methods (12). Penicylinders were placed in and suture loops were attached to a removable polyethylene basket that was designed to permit ozonated water and bubbles to freely circulate around the carriers. The carrier basket was added to the reactor prior to the addition of ozone. Experiments were performed at ambient temperature (approx. 20°C) and 60°C. A hot plate
(Tek Stir; American Hospital Supply Corp., Evanston, Ill.) water temperature at 60°C during specified contact times, the carrier the reactor and placed in a laminar air flow hood. The penicylinders and suture loops were aseptically transferred to tubes of fluid thioglycolate medium. The tubes were incubated at 37°C for 7 days before the presence or absence of growth was determined. Experiments were performed to determine whether the small amounts of ozonated water carried over during transfer (determined to be less than 10 ,ul) was inhibitory to recovered test spores. Testing was performed by adding was used to maintain the the ozone bubbling. After basket was removed from
APPL. ENVIRON. MICROBIOL.
TABLE 1. Sporicidal activity of ozone-saturated water against spores in suspensiona Contact time
(min) 1 2 4 8 16
No. of samples (of 5) producing growth from spore suspensions of: C. sporogenes B. subtilis ATCC 19659b ATCC 3584C
3 1 0 0 0
4 1 1 0 0
a The concentration of the ozone gas phase was 8% (by weight); temperature, approx. 20°C. b Initial population, approx. 1.6 x 107 CFU/5-ml sample. c Initial population, approx. 2.8 x 107 CFU/5-ml sample.
approximately 100 spores of each test organism to tubes containing 10 ml of their respective recovery media. Ozonated water (10-mg/liter aqueous concentration) was added to a series of the inoculated tubes in fractions of 100, 200, or 500 ,ul. Standard plate counts were performed on half of the samples, while those remaining were incubated at 37°C for 12 h before examination for growth. RESULTS AND DISCUSSION It is difficult to compare the results of the few studies in which the sporicidal activity of ozone has been discussed (4, 9, 11, 13) because of the differences in test conditions and the citing of ozone dosages by some and residuals by others. In spite of this, greater ozone dosages or longer contact times would be required for the effective destruction of bacterial spores when compared with vegetative forms. Because it was my intention in this study to determine if spores could be rapidly inactivated, i.e., in less than 30 min, the ozone dosage was increased to permit higher than normal (compared with water treatment levels) dissolved ozone concentrations. The first series of experiments was designed to determine the rate of destruction for spores in suspension at ambient temperature with dissolved ozone concentrations exceeding 10 mg/liter. The response of the two spore suspensions to the ozonation is shown in Table 1. The washed spores of both test organisms exhibited a similar rapid reduction in numbers during their exposure to ozone dissolved in distilled water and ozonated bubbles in the batch reactor. Viable organisms were not recovered after an 8-min exposure for either B. subtilis or C. sporogenes. Farooq et al. (6) have emphasized the importance of ozone residual and ozone-containing bubbles to the inactivation of microorganisms. Operation of the ozone generator at 8% (by weight) ozone and injection of the gas stream into a batch reactor is perhaps the best method for accomplishing this simultaneous exposure of the test organisms to ozone. The effect of ozone-containing bubbles on bacterial spores, however, was not confirmed in this study. Ozone resistance was increased when either spore type was dried on AOAC carriers. Viable organisms were recovered from a small percentage (10 to 20%) of the penicylinders after a 40-min exposure to ozone at ambient temperature (Table 2). Dahi (3) has stated that microorganisms encapsulated in organic material survive longer, not because of the slow penetration of ozone or its radicals but because free radicals react with the material before reaching the vital sites of the microbes. This may help to explain the decreased
SPORICIDAL ACTIVITY OF OZONE
VOL. 53, 1987
effectiveness of ozone at elevated temperatures and the difference in resistance between the test organisms when they were inoculated and dried on carriers. Farooq et al. (5) observed that the degree of microorganism inactivation by ozone improved remarkably at higher temperatures, despite the decreased solubility of ozone and the corresponding lower residual concentrations. It should be noted that the microorganisms were suspended in water and not in the presence of organic matter, which permitted their rapid oxidation by ozone, free radical species, or both. The results in Table 2 show that the effectiveness of ozone was decreased when the reactor water temperature was increased to 60°C. The heavy organic load present on the AOAC penicylinders would, as Dahi (3) suggested, consume the free radicals before their contact with all the spores. Therefore, dissolved ozone residuals, which would be greater at 20°C, may be more effective in destroying microorganisms than free radicals when in the presence of organic
matter.
A longer ozone contact time is required to inactivate spores as the level of organics is increased. This may explain the increased ozone resistance of the C. sporogenes spores relative to that of the B. subtilis spores when each was inoculated on porcelain penicylinders (Table 2). It was visually apparent during carrier preparation that the soil extract-egg-meat medium, which was used to propagate C. sporogenes spores, contributed much more debris to the carriers than the soil extract medium used for B. subtilis. Experiments were performed to verify this by inoculating porcelain penicylinders with washed spores of each test organism. The washing procedure effectively removed the spores from their respective growth media and cellular debris. The results in Table 3 show that organic matter is responsible for the difference in ozone resistance between the organisms studied. The rate of kill was significantly faster when the spores were removed from their protective organic cover. The difference in the number of positive carriers for each species after a 5-min exposure could be explained by the initial difference in spore populations. Spores that were inoculated on silk suture loops (data not shown) were more resistant to ozone than were those inoculated on penicylinders. Every carrier produced growth in the recovery media following a 40-min exposure to ozone to ambient temperature and 60°C for both test organisms. Longer contact times were not examined during this study. These results cannot be interpreted solely on the basis of the organic matter present. It has been shown that AOAC suture loops form many tiny bubbles on their surfaces when forced TABLE 2. Sporicidal activity of ozone-saturated water at 20 and 60°C, as measured by AOAC test methods'
approx.
No. of carriers producing growth/no. inoculated with spores of: Contact time
(min)
B. subtilis ATCC
200C
5 10 20 30 40
1/10 1/10 1/10 0/10 2/10
19659b
C. sporogenes ATCC 3584C
600C
200C
600C
6/8 8/8 0/8 1/8 0/8
8/8 8/8 5/8 3/8 1/8
8/8 8/8 6/8 6/8
gas
TABLE 3. Sporicidal activity of ozone-saturated water against spores washed free from their growth media prior to being dried on porcelain penicylindersa Contact time (min)
5 10 20 30 40
No. of carriers producing growth/no. inoculated with spores of: C. sporogenes B. subtilis ATCC 3584c ATCC 19659b
1/8 0/8 0/8 0/8 0/8
8/8 0/8 0/8 0/8 0/8
a The ozone gas phase concentration was 8% (by weight); temperature, approx. 20'C. b Initial population, approx. 1.0 x 104 CFU per carrier. c Initial population, approx. 5.1 x 105 CFU per carrier.
under a chlorine solution (H. W. Anderson, S. R. Anderson, C. Zaner, and C. H. Harrison, U.S. Patent 4,418,055, November 1983). The addition of a chlorine-compatible surfactant was required to improve carrier wetability, which, in turn, increased the efficacy of the solution. Bubble formation may also have prevented the ozone from contacting all portions of the suture loops, and subsequently, a significant number of spores was able to survive. The addition of a surfactant may also increase the effectiveness of ozone penetration; however, this practice could lead to the sudden foaming of the water (14). Another disadvantage of adding a surfactant would be the requirement for a rinsing cycle following ozonation if the articles to be sterilized were, for example, medical instruments. In conclusion, spore suspensions were rapidly inactivated by exposure to ozone-saturated water at ambient temperature. No survivors were recovered after an 8-min exposure when dissolved ozone concentrations exceeded 10 mg/liter. No apparent difference in ozone resistance between the two bacterial species was observed. The results of the AOAC sporicidal tests show that spores dried on carriers are more resistant to ozone than those suspended in water. Viable spores were being recovered from some porcelain penicylinders after a 40-min contact time at ambient temperature. An increase in the reactor water temperature to 600C did not improve the effectiveness of ozone in sterilizing AOAC carriers. Spores of C. sporogenes that were inoculated on porcelain penicylinders were more resistant to ozone than were B. subtilis spores because of the greater accumulation of organic matter. Sporicidal activity was not demonstrated for spores of either species when they were inoculated on silk suture loops. The presence of many small bubbles on the surface of these carriers may have prevented adequate exposure to ozone. Therefore, the results of this study suggest that surface sterilization with ozone-saturated water is not feasible within the exposure times indicated. Organic loading, poor ozone penetrability, or both were key factors in these findings. The addition of surfactants may enhance the efficacy of ozone; however, this would negate the attractiveness of having a residuefree sterilant, which would require no aseptic rinsing. Sonication may also prove beneficial, although this and the use of surfactants were not investigated during this study.
3/8
Spores were dried on porcelain penicylinders. The concentration of the phase was 8% (by weight). b Initial population, approx. 1.5 x 104 CFU per carrier. c Initial population, approx. 6.5 x 105 CFU per carrier.
a
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ACKNOWLEDGMENTS I thank Raymond C. Kralovic and William C. Schmidt for technical assistance and Millie Schilling for typing the manuscript.
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RICKLOFF
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