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Introduction
The quality of air surround us is important to our health1. It is not only pollutants in the air such as volatile organic compounds (VOCs) but also microorganisms distributed in it that should be concerned. Several pathogens that cause infectious diseases such as influenza, tuberculosis, melioidosis, bacterial meningitis, and anthrax can infect people through inhalation of contaminated droplets or particles in the air into human body 2, 3. The incidence of emerging infectious diseases was also increased due to the climate change4. Moreover, indoor airborne microflora has been shown to be implicated as the etiology of hypersensitivity pneumonitis, allergic rhinitis and some type of asthma5, 6, 7. Therefore, the indoor air quality due to the present of several microorganisms is recently become highly concerned8, 9. Many methods were used for indoor air purification to prevent the spreading or to control these microorganisms such as HEPA air filtration10, short ultraviolet wavelength (UV-C) light11, photocatalytic oxidation (PCO) 12 plus UV air purifiers with positively and negatively charges cluster ion generator13, 14. Each method has some limitations such as HEPA air filtration that could not trap virus particles or mycoplasma due to their small size. UV-C does not efficiently kill the microorganisms if they are not directly exposed to the UV light. Moreover, the light could cause some health hazard to human in that it could not be operated during work. Ion generator (IG) is a new technology that splits the airborne molecules of water into positively charged hydrogen (H+) and negatively charged oxygen (O2-). The generated ions have a property of forming clusters around microscopic particles and microorganisms and create groups of highly reactive OH- radicals that can kill the germs in the air. In developing countries, with limited research budget for set up the high quality of Biosafety level 2 or 3 as required for microbiology work, therefore, the efficacy of the IG was evaluated whether it would be able to control microbial contaminants in laboratories or rooms that need clean air.
Materials and Methods
Microorganisms
Bacteria and fungi strains used in this study were obtained from the stock culture in Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand. The bacteria used were Burkholderia pseudomallei, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Streptococcus pneumoniae and fungi were Aspergillus fumigatus, Rhizopus sp, Pennicillium sp, Microsporum sp. and Candida albicans. All bacterial strains were cultured in Brain Heart Infusion (BHI) broth and nutrient agar (NA) except S. pneumoniae that used blood agar whereas the fungi were cultured in potato dextrose agar (PDA).
Ion Generator
Two ion generators (PCI model IG-A10TA), provided by Sharp Thai Co. Ltd, were used throughout this study. The model was evaluated for their capacity for the area of 10 m2 according to the manufacturer instruction.
Research Design
The studies were done in 3 separated conditions (Fig. 1).The number of bacterial or fungi colony (CFU) was determined before and after being exposed to the ion generated from Plasmacluster Ion® generator machine at the duration of 30, 60 or 120 min.
Condition I: The efficacy of ion generator machine to reduce the microorganisms in culture plates. The experiment was performed in a plexi glass chamber (size of 100 x 50 x 50 cm). Nine NA plates were freshly spread with either 102 CFU of each bacteria or each fungal strain. The plates were left opened in the chamber and then the IG was operated. After 30 min of exposure, 3 plates were removed from the chamber and did so after 60 and 120 min of exposure. The plates were incubated for an appropriated period of time and temperature depending on the microorganisms before being evaluated for the number of colonies in comparison with those exposured in the chamber without ion generator.
Condition II: The efficacy of ion generator machine to kill the microorganisms in the air. The evaluation was performed in the same chamber. The broth containing 104-108 CFU/ml (5 ml) of bacteria or 103-104 spores/ml (5 ml) of fungi, one at a time, were sprayed into the chamber that contained 9 sterile NA plates that just left opened. After operated for 30 min, 3 plates were removed and did so after 60 and 120 min of exposure. The plates were incubated and the number of colony was counted in comparison with that in the chamber without ion generator.
Condition 3: The efficacy of ion generator machine to reduce the microorganisms present in the atmosphere. The experiment was done in 2 microbiology laboratory rooms (size of 6 x 4 m2 and 7.35 x 3.13 m2). The number of microorganisms in each room was detected before and after the ion generator machine was operated. Four NA plates were placed at one meter from each corner of the room for 1 hour to detect the number of microorganisms present in the room and then removed. After that, another 4 plates were placed at the same positions and the machine was operated for 1 hour. All plates were incubated and the number of colonies was counted.
All experimental designs were approved by human ethical committee of Khon Kaen University, Khon Kaen, Thailand.
Results and discussion
The number of bacterial count on solid media (Fig. 2) is displayed in Figures 3. The results demonstrated that the IG did not have any significant effect on the tested organisms when they were grown on solid media. This same observation was obtained when fungi were used to evaluate (data not shown). The generated ions may not be able to kill the microorganisms on the surface of the agar plates due to the instability of the ions on moisture surface. However, some reports indicated the negative ion is more effective than the positive one in reducing the gram-negative and gram-positive bacteria by detecting the microorganisms growing on agar plates15, 16. From our design experiment, the ions were designed to generate very close to the plates surface. The possible mechanisms responsible for bactericidal process proven by other experiments was ozone that plays a major role and electroporation as a secondary role and not directly by the negative ions itself16. However, the ozone was known to have toxic effect to human health17. As IG works by emitting positive and negative ions into the air which change into OH- radicals when adhered or surround to the surface of airborne pathogens and destroy them14. Moreover, the IG was reported to generate less than 10 ppb (£0.01 ppm) of ozone.
When IG efficacy was tested by using microorganisms in a spray condition, it could kill all of the tested bacteria and most of fungi within 30 min (Fig. 4). The killing efficacy at 30 min to tested bacteria was ranged from 73.1-99.1%. However, when the operation time was extended to 60 or 120 min, the killing capacity was not much different except for P. aeruginosa that was increased from 73.1% to 91.30%. E.coli was found to be the most susceptible whereas P. aeruginosa was the least susceptible. The maximum killing efficacy of IG on E. coli, P. aeruginosa, B. pseudomallei, B. subtilis and S. pneumoniae were 99.3%, 91.3%, 90.1%, 87.96%, and 83.1%, respectively. B. pseudomallei, which was classified in category B of possibly being used as a bio-weapon by CDC, USA, was very sensitive to IG killing. In fungal experiment, the percentage of killing tested fungi ranged from 42.6 to 93.9%. Microsporum sp. and Rhizopus sp. were found to be the most susceptible in all time course (92.0 to 95.3% and 91.9 to 93.9%, respectively) whereas A. fumigatus was quite resistant (42.6 to 53.9%). At 30 min of IG exposure, the percentage of killing of A. fumigatus, C. albican, Rhizopus sp and Microsporum sp were 44.4%, 76.1%, 91.9% and 95.3% respectively. When the exposure time was extended for 60 or 120 min, the killing efficacy was not significantly different. This result suggested that fungus spores are more resist to IG. This finding is not unexpected as fungus spores have a rigid structure.
The operation of IG in the two microbiology laboratories could reduce microorganisms in the air at the mean of 10-14% in 1 hour. This result was less efficient than expected. This might be due to the size of the room (22-24 m2) that were larger than that (10 m2) given in the specification of the machine model IG-A10TA (10 m2). The low number of microorganisms (10-14 colonies/m2) in the control group (no IG operated) found in the environment of the room tested might be another reason.
Figure 1 Illustration of the chamber and design for condition I and II. The size of the chamber is 100 x 50 x 50 cm. In condition I, 9 NA plates (3 sets) containing approximately 102 CFU of each bacteria or fungal strains were left opened in the chamber. After IG operation, 3 plates at a time were removed simultaneously after 30, 60 and 120 min (A). In condition II, liquid culture containing 104-108 CFU/ml (5 ml) of bacteria or 103-104 spores/ml (5 ml) of fungal strains were sprayed into the chamber. Nine sterile NA plates were put into the chamber and the IG was operated. Three plates were removed after 30, 60 or 120 min of the operation time (B).
Figure 2 The appearance of colonies on solid agar plate as a result after each microorganism in sprayed form was exposed to IG in comparison with without exposure. The appearance of colony without IG operation (1, 2 and 3) and with IG operation (4, 5 and 6) for 30 (1 and 4), 60 (2 and 5) and 120 (3 and 6) min. of Escherichia coli (A), Bacillus subtilis (B), Burkholderia pseudomallei (C), Pseudomonas aeruginosa (D), Streptococcus pneumoniae (E), Candida albicans (F), Aspergillus fumigatus (G) and Rhizopus sp (H).
Figure 3 The efficacy of IG against bacteria and yeast on agar plates. The average colony counts of E. coli, P. aeruginosa, B. subtilis, B. pseudomallei, S. pneumoniae and C. albicans after 30, 60 and 120 min with or without exposure to IG were shown. Each bar represents mean ± SD.
Conclusion
The IG could efficiently kill bacteria in the air if being used as specified but not on the agar plates. This condition is suitable for using in routine microbiology laboratory where bacteria are generally stored on solid media. As fungal spores are more resist to the ions, the IG model with HEPA filter should be more suitable and the suitable size of the room with a correct IG model should be concerned to obtain an efficient mechanism for prevention of the airborne pathogens.
Acknowledgements
We would like to thank Sharp Thai Co. Ltd, for providing the Ion generator machine model IG-A10TA to be tested and Melioidosis Research Center, Department of Microbiology, Faculty of Medicine, Khon Kaen University for financial and laboratory facility supports. The authors have no conflict of interest to this report.
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