1. MATERIALS AND METHODS
Arrangements of tests
This evaluation has been performed in the laboratory of Jyväskylä Professional University, Dep. of Technology and Logistics, Rajakatu, Jyväskylä.
The first 35 tests (in the beginning of July) were intended in the optimization of testing equipment (Figure 1).
Following values of main parameters were then chosen :
1. Air pressure for spraying of the microbial suspension from ”Spira” (est. 10% of the constant output pressure in the laboratory)
2. ATP concentrations (related to cell counts) of bacterial suspensions (200 +/-50 RLU)
3. Length of spraying period (3 min)
4. Length of sampling period (1 min)
5. Length of chamber cleaning by continuous flow of air between trials (min. 10 min)
The densities of bacteria in suspensions for aerosol generation with ”Spira” were controlled with luminometric ATP Assay (BIOORBIT 1253 Luminometer by BIO-ORBIT Oy, Finland, and ATP Biomass Kit HS by BIOTHEMA AB, Sweden).
Densities of fungal spores in suspensions were analyzed with microscopical countings, respectively. Certain variation of densities were needed for statistical purposes but ATP values of bacteria were limited inside a range of tens to hundreds RLU units and count of spores inside a range of tens per field (with 100* objective and 10* ocular) in every case.
Figure 1. Schematic presentation of test equipment for the air cleaners.
All microbes (Table 1) were aerosolized into air cleaner during periods of 3 minutes from suspensions in sterile 0.9% NaCl buffer solutions. Average speed of air inflow into air cleaner during aerolization was 5 km/h. Air flow was stopped after aerolization and microbes were immediately sampled on nutrient agar plates with 6-stage Andersen sampler from the sampling chamber. Sampling periods were either 5 minutes (M. luteus) or 1 minute (other microorganisms). Plates were then incubated for 1 day to 5 days, depending on the microbial species involved, in +30oC.
Colonies from both total 6-stage set (bacteria and yeast) and from two last stages (fungal spores) were calculated, and results were transformed into units ”CFU/m3”.
A special care has been taken to avoid any bacterial rests of previous tests in sampling chamber. The efficacy of cleaning measures was confirmed by frequent control samplings (Andersen sampling and contact plate analyses of the sampling after trials).
Test organisms were chosen with certain criteria. All bacteria have environmental origin and are not real pathogens. Genus Bacillus includes common airborne bacteria which are capable to produce very tolerant cell forms called spores. Micrococcus is frequently found in air sampling because it produces pigments which cover it against radiation of sunshine. Escherichia is also a common bacterium, both in aquatic environments and human colon, acting therefore as an indicator organism of fecal pollution. Among fungi were Penicillium and Saccharomyces chosen – first of them because its universal distribution as an actively sporulating mould, the second one as an internationally accepted yeast species for different types of testing.
Table 1. The test organisms used in the study.
|Test organism||Type of organism||Strain code1||Nutrient agar2||Incubation (d)|
|Bacillus subtilis||Gram-positive, aerobic, sporulating, rod-shaped bacterium||E-97009T||TYGA||2|
|Escherichia coli||Gram-negative, facultatively anaerobic, rod-shaped bacterium||ATCC 25922||TYGA||1|
|Micrococcus luteus||Gram-positive, aerobic, pigmented, cocci-shaped bacterium||E-93442T||TYGA||4|
|Penicillium chrysogenum||A common mould||D-88381||MEA||5|
|Saccharomyces cerevisiae||A common yeast||B-62001||MEA||2|
1 Strain codes of culture collection by ATCC (E.coli) or by VTT (other organisms)
2 TYGA = Tryptone Yeast Extract Glucose Agar, MEA = Malt Extract Agar
Referring the prerequisites set for Poisson-distributed colony count data, statistically confirmed results were derived from samples with min. 30 colonies.
Reductions of microbial densities in air were calculated as follows:
Reduction (%) = 100 * (Cc – Ct)/Cc
Where Cc = cumulative colony count in ANDERSEN plate set when treatment was OFF and Ct = cumulative colony count on ANDERSEN plate set when treatment was ON.
Statistical significances of reductions were estimated by taking into account one basic feature of Poisson distribution: average of repeats (C) = variance of repeats. Confidence ranges were therefore estimated with following equations:
C +/- √C gives confidence range of 66%
C +/- 2√C gives confidence range of 95%
Whenever colony counts reach the value of 30 in all tests, estimates of reductions were also statistically more reliable because ”overlapping” of confidence ranges of control and test results do not exist. Several repeats of tests also helps to confirm reduction values by minimizing the effect of random errors of the tests.
Reductions of microbial densities in air, treated with “Aero 600BM”, are collected into following figures. Figure 2 shows percentual reductions of every test organism, Figure 3 describes the microbial densities in air, with and without UV and ionizator treatments.
Two clusters of reduction values are visible: one between 0% and 90%, another near or even over 99%.
Reductions of test organisms after treatment with Aero 600BM
Number of tests per organism = 3
Figure 2. Percentual reductions of microbial densities with Aero 600BM.
EVALUATION OF Aero 600BM:
REDUCTIONS OF MICROBIAL DENSITIES IN AIR
B = Bacillus subtilis,
E = Escherichia coli,
M = Micrococcus luteus,
P = Penicillium chrysogenum,
S = Saccharomyces cerevisiae
diagonal lines = reductions 0%, 90% and 99%
Figure 3. Microbial densities of test organisms in air with/without UV&ionizator treatments.
BAILEY,N.T.J. 1981. Statistical Methods in Biology. 2nd Ed. Edward Arnold. London, England.
BROCK Biology of Microorganisms 2003. Ed: Madigan, M.T., Martinko, J.M. & Parker,J. 10th Ed. Pearson Education, Inc. Upper Saddle River, U.S.A.
LYNCH,J.M. & HOBBIE,J.E. 1988. Micro-organisms in Action: Concepts and Applications in Microbial Ecology. 2nd Ed. Blackwell Scientific Publications. Oxford, England
MÄKINEN,Y. & RANTIO-LEHTIMÄKI,A. 1979. Diurnal Variation of Airborne Fungal Spores in Turku, Finland, in 1974. Reports from the Aerobiology Laboratory, University of Turku. Turku, Finland.
NIEMELÄ, S. 1979. Mikrobiologisen havaintoaineiston tilastollisen käsittelyn alkeet. Helsingin yliopiston Mikrobiologian laitoksen julkaisuja 17. Helsinki, Finland.
NIEMELÄ,S.I., VÄÄTÄNEN,P., MENTU,J., JOKINEN,A., JÄPPINEN,P. & SILLANPÄÄ,P. 1985. Microbial Incidence in Upper Respiratory Tracts of Workers in the Paper Industry. Appl.Environ.Microbiology, July 1985, p. 163-168.
RANTIO-LEHTIMÄKI,A. 1977. Research on Airborne Fungus Spores in Finland. Grana 16: 163-165.
RANTIO-LEHTIMÄKI,A., POHJOLA,a. & MÄKINEN,Y. (manuscript). Spore Composition in a Garbage Disposal Plant in Turku.
The Shorter Bergeys’s Manual of Determinative Bacteriology 1977. Ed: HOLT,J.G. 8th Edition. The Williams & Wilkins Company, Baltimore, U.S.A.
TOMMILA,E. 1965. Fysikaalinen kemia. 3. painos. Kustannusosakeyhtiö Otava, Helsinki.