Use of Ozone for Inactivation of Bacteria and Viruses in Cryostats

The biocidal efficacy of ozone has been investigated in cryostats at -20°C and +30°C and 30-120 min exposure time. Stainless steel coupons were inoculated with 50 μl bacterial or viral test suspension. The tests were performed using Staphylococcus aureus and Mycobacterium terrae as test bacteria and Polyoma virus (Simian virus 40, SV 40). The inoculated coupons were exposed to a combination of ozone and UV generated by UV lamps in the cryostats. To measure the level of disinfection achieved, the number of surviving bacteria recovered from the coupons and, respectively, the virus infectivity titer was determined. Reduction factors were calculated by dividing the initial titer by the titer of recovered viable bacteria or virus. Bactericidal efficacy at -20°C was demonstrated by inactivation of S. aureus by at least 5 log10 units in an automatic defrost/60 min ozone cycle in all test positions, which allowed to remove the +30°C/120 min test from the testing panel. In a 30 min treatment, a disinfection rate of 5 log10 units was achieved in some, but not all test positions. Mycobacterium terrae, the substitute for the pathogenic M. tuberculosis, was inactivated by >5.3 log10 units in all tested locations using a 120-minute ozone cycle at +30°C. The infective virus titer was reduced by at least 5 log10 units in the same ozone cycle on the pressure plate and chuck positions. Apart from this, significant reductions in viable cell and virus numbers have been shown for all disinfection protocols and test positions, thus demonstrating the principle efficacy of a combination of gaseous ozone and UV as a disinfectant in cryostats.


Introduction
In cryotomy, the risk of transmittance of health-care associated pathogens from tissue samples and surfaces is of particular concern. For optimal staff protection, it is often desirable to reduce the risk of contamination by a simple disinfection routine that can be conveniently applied between sectioning sessions. For this reason, there has been interest in treating cryostat chambers with a fumigant that in a rapid and safe way will inactivate pathogens on all surfaces and even in spaces.
Ozone, as a highly potent oxidant, is able to inactivate a broad range of microorganisms and viruses. The susceptibility of micro-organisms and viruses to ozone has been tested in a large number of investigations but ozone´s mode of action against microorganisms is only incompletely understood. Some studies suggest that molecular ozone oxidizes proteins and unsaturated fatty acids in biological membranes, leading to cell lysis or damage of viral envelopes and capsids. Ozone may also disrupt cellular enzyme activity and react with nucleic acids. Hydroxyl radicals from ozone decomposition are also highly reactive, but unspecific oxidants [1][2][3][4][5][6][7].
High-level antimicrobial activity of ozone on surfaces or in air has been demonstrated in only few studies. The biocidal efficacy of ozone is determined by a variety of factors, besides concentration and exposure time, including temperature, relative humidity, presence or absence of organic matter, micro-organism type, surface composition and structure, and aggregation. Pathogens may be adsorbed to cryostat surfaces or embedded within tissue debris. It is evident that the complexes that result may be much more resistant to ozone than pathogens in the free state [8].
The aim of the present study was to examine the disinfection efficacy of ozone combined with UV treatment in cryostats, making use of the ability of gaseous ozone to diffuse throughout the space and penetrate into crevices. Four parameters are the major contributors to pathogen inactivation in a cryostat: temperature, ozone concentration, UV irradiance and exposure time. The Gram-positive bacterium Staphylococcus aureus, Mycobacterium terrae and Polyoma virus (Simian virus 40, SV40) have been employed as standard surrogate organisms in carrier tests [9][10][11]. The experiments were performed at a working temperature of -20°C and, in addition, at +30°C with variable exposure time, using two cryostat models.

Test instruments
Two cryostats were used in the experiments. A TISSUE TEK ® CRYO3 ® PLUS cryostat was used for ozone treatment at -20°C, and a TISSUE TEK ® CRYO3 ® FLEX (Sakura Finetek USA, Inc., Torrance, CA) were equipped with new low pressure mercury vapor lamps with quartz glass lamp sleeves as ozone generating systems.

Ozone measurements
The ozone generating lamps show major emission lines at 254 nm ("UV-C") and 185 nm ("VUV"). Absorption of VUV by oxygen molecules in the ambient air leads to photolysis of oxygen molecules and formation of ozone by reaction of oxygen atoms with other oxygen molecules. Simultaneously, photolysis of ozone occurs by UV-C absorption, in addition to spontaneous decomposition to oxygen. Thus, a steady state ozone concentration gradient is generated in the cryostat tub during lamp operation [12].
The time course of ozone formation in the cryostats at various positions and with different disinfection schemes has been measured using a 2B Technologies (Boulder, CO, USA) 106-M ozone monitor. The total ozone doses were determined as the sum of single concentration measurements made in 10 sec intervals over complete ozonation cycles. The equilibrium concentration is reached within 20-30 min lamp operation. At the end of the cycle, ozone is rapidly purged from the cryostat tub. The ozone levels were uniform throughout the cryostat tub (± 2%). In the TISSUE TEK ® CRYO3 ® PLUS, the average total doses were 32,100, 75,360 and 180,440 ppm*min in the 30, 60, and 120 min treatments at -20°C, respectively. In the TISSUE TEK ® CRYO3 ® FLEX chamber, the ozone concentration was slightly higher than in the TISSUE TEK ® CRYO3 ® PLUS after 30 and 60 min at -20°C and thus a slightly higher disinfection rate can be expected. When the chamber temperature in the TISSUE TEK ® CRYO3 ® FLEX was raised to +30°C, an ozone dose of 78.460 ppm*min was reached in a 120 min disinfection cycle.

Test strains
As a test strain for bactericidal activity, the gram positive bacterium Staphylococcus aureus ATCC 6538 was used. The frozen bacterial stock was suspended in tryptic soy broth (TSB) and incubated at 35 ± 1°C for 18-24 h. 0.1 mL aliquots of the liquid culture were plated on tryptic soy agar (TSA) and incubated at 35 ± 1°C for 18-24 h. For the test suspension, bacteria were rinsed off a plate with 10 mL sterile deionized water (SDW), homogenized by vortexing with sterile glass beads and washed twice with SDW by centrifugation (2.000 g, 20 min, 5°C). After the third centrifugation step, the pellet was re-suspended in 3 mL SDW. The suspension was homogenized by vortexing with sterile glass beads at maximum speed for 3 min and diluted to 108-109 colony forming units per mL (cfu/mL) with SDW. The test suspensions were stored in a fridge and used within 4 h. The tests were performed without adding interfering substances.
Mycobacterium terrae ATCC 15755 was employed for demonstrating tuberculocidal activity [13,14]. The bacterial stock was suspended in Middlebrook 7H9 broth with OADC (MBB) (Merck Chemicals, Darmstadt, Germany) and incubated at 36 ± 1°C for 48 h. 0.1 mL aliquots of the liquid culture were spread on Middlebrook 7H10 agar with OADC (MBA) (Merck Chemicals, Darmstadt, Germany), sealed in polyethylene bags and incubated at 36 ± 1°C for about 21 days. The bacteria were rinsed off a plate with 10 mL SDW, homogenized by vortexing with sterile glass beads and washed twice with SDW by centrifugation (2.000 g, 20 min, 5°C). Afterward, the pellet was re-suspended in 3 mL SDW. The suspension was homogenized by vortexing (3 min) with sterile glass beads at maximum speed and diluted to 108-109 colony forming units per mL (cfu/mL) with SDW. The test suspensions were stored in a fridge and used for two experiments on consecutive days. The tests were performed without adding interfering substances.
Polyoma virus (Simian Virus 40, SV40), strain 777 (MikroLab GmbH, Bremen, Germany) was multiplied in African green monkey kidney cells (CV 1 cells) to produce high titers of infectious viruses. The cell culture originated from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK). CV 1 cells were cultivated at 37 ± 2°C with 5% CO 2 . Virus suspensions with cell detritus were frozen and thawed two times. The cell detritus was then separated by low speed centrifugation and the supernatant was stored at ≤-60°C. Virus suspension were prepared in virus test medium without serum (Dulbecco´s modified Eagle´s medium, supplemented with L-glutamine and 100 units/mL penicillin, 130 µg/mL streptomycin). During inactivation experiments, the virus test suspension was stored in a refrigerator (+4 to +7°C).

Inactivation experiments
Sterilized stainless steel coupons (20 x 1 mm) of the same material as the cryostat tub (SAE grade 304, bead blast with round glass beads mesh 140/170, passivated per AMS2700E type 2 class 2) were each inoculated with 50 µL test suspension (Figure 1). The suspensions were evenly spread using fine glass loops. After drying to optical dryness in a laminar flow cabinet, the inoculated coupons were transferred into the cryostat, equilibrated to -20°C, and then subjected to the ozone treatment schemes described in Table 1. The coupons were variably placed in up to five test positions ( Figure 2) in triplicate. Ozone treatment for 60 and 120 min was preceded by a 60 min defrost period. Ozone was generated throughout the treatment periods. Afterward, it was rapidly purged from the cryostat chamber.
In control experiments, the coupons were removed from the instrument before start of the ozone treatment routine. In Polyoma virus, positive virus inactivation controls with formaldehyde were performed. Interference of possible ozone reaction products with susceptibility of the CV 1 cell line was excluded in additional control experiments.  For recovery of virus particles after treatment, the coupons were immediately transferred into jars containing 0.5 g glass beads (0.25-0.5 mm) and 1 mL virus test medium. The jars were vortexed for one minute to recover the virus particles. A defined volume of the eluate and dilutions thereof were inoculated on cultures of receptive target cells. After 17-19 days, the cell cultures were checked for cytopathic effects and the 50% tissue culture infective dose (TCID50/ml) was calculated using the Spearman-Kärber method [15]. Reduction factors were calculated from the initial and recovered viable bacterial numbers and virus titers.

Results and Discussion
The results of inactivation tests with ozone are presented in Table 1.
Significant reductions in viable cell and virus numbers have been demonstrated in all ozone treatment protocols and positions tested. Despite measurements of a more or less uniform ozone steady state concentration throughout the cryostat chamber, significant differences in disinfection efficacy between test positions have been observed and require further investigation about the effect of the combination of ozone, UV, temperature and time.  Table 1: Results of different ozone treatment regimes on reduction in microbial numbers.
Based upon the provisions of the German DGHM guideline [9] and the European norm EN 13697 [10], bactericidal efficacy for a disinfectant is shown by demonstrating a reduction in cell counts by at least 5 log10 units. The present investigation confirms bactericidal efficacy of the 60 min ozone treatment at -20°C for S. aureus in all test positions. In the 30 min period at -20°C, a disinfection rate of 5 log10 units was achieved in some, but not all test positions. M. terrae required a 120 min treatment at +30°C for a reduction rate of at least 5 log10 units.
Based upon the provisions of the German Association for the Control of Virus Diseases and the Robert Koch Institute [11], virucidal efficacy for a virucidal agent is shown by demonstrating a virus titer reduction of at least 4 log10 units. The present investigation does confirm virucidal efficacy of the 120 minute ozone treatment at -20°C on the pressure plate and at +30°C on the pressure plate and chuck positions.
Ozone has shown to be very effective against most vegetative bacteria and viruses. In general, bacterial spores, mycobacteria and non-enveloped viruses are more resistant than vegetative bacteria and lipid-enveloped viruses [3,[16][17][18][19]. The spore coat in bacterial endospores is a protective barrier against ozone [20], therefore, bacterial spores are more resistant than their vegetative forms. Compared to non-enveloped viruses, enveloped forms are more sensitive to chemical disinfectants because they require an intact lipid envelope to infect host cells, and this envelope can be damaged by chemical and physical agents. Prions might be more susceptible to ozone than some bacteria [21][22][23].
Reliable prediction of microbial disinfection efficiency is difficult because of the wide range of pathogens that may be present. This can be resolved by employing suitable surrogate organisms in disinfection testing. In the present study, S. aureus, M. terrae and Polyoma virus have been used. S. aureus is a standard Gram-positive test bacterium representing vegetative bacterial forms (except mycobacteria), whereas M. terrae and the relatively small, non-enveloped Polyoma virus belong to the most resistant bacteria and viruses, respectively. M. terrae is a surrogate for the cause of tuberculosis, M. tuberculosis, in disinfectant testing. Polyoma virus is a non-enveloped standard test virus for virucidal activity with high resistance against various disinfectants [9][10][11]13,14].
Bactericidal acitivity against S. aureus and M. terrae, together with virucidal activity against Polyoma virus thus allow the conclusion that the +30°C/120 min ozone disinfection routine in the present study achieved intermediate-level to high-level disinfection according to the CDC definition [24] in the pressure plate and chuck test positions. At -20°C, low-level disinfection is achieved in all test positions by ozone treatment for 60 minutes. However, a 5 log10 reduction was achieved with S. aureus within 60 min at -20°C.