Rapid counting of liquid-borne microorganisms by light scattering spectrometry.

Fast and sensitive techniques are needed to determine microorganism presence in liquid samples. In this research, the feasibility of using light scattering spectrometry for enumerating the biological particles in liquid samples was investigated. A particle size spectrometer was used to count six commonly found microbial species suspended in liquid with and without microbiological stains applied: Pseudomonas fluorescens, Micrococcus spp. vegetative cells and Bacillus subtilis var. niger endospores were stained with Acridine Orange and Crystal Violet, while Cladosporium cladosporioides, Penicillium melinii and Aspergillus versicolor fungi were stained with Acridine Orange and Lactophenol Cotton Blue. The counts obtained with the spectrometer were compared with those obtained with a phase-contrast microscope. It was found that the spectrometer counted about 32 % of non-stained B. subtilis endospores and this percentage increased to almost 90 % for stained endospores. Among the investigated species of fungi, the counting efficiency of P. melinii was the only one significantly affected by the application of the stain Lactophenol Cotton Blue: the fraction of counted fungal spores increased from 64 % (non-stained spores) to about 100 % (stained spores). The observed difference in counting efficiency may serve as a basis for differentiating biological from non-biological particles in liquid samples.

Assessment of electrical charge on airborne microorganisms by a new bioaerosol sampling method.

Bioaerosol sampling is necessary to monitor and control human exposure to harmful airborne microorganisms. An important parameter affecting the collection of airborne microorganisms is the electrical charge on the microorganisms. Using a new design of an electrostatic precipitator (ESP) for bioaerosol sampling, the polarity and relative strength of the electrical charges on airborne microorganisms were determined in several laboratory and field environments by measuring the overall physical collection efficiency and the biological collection efficiency at specific precipitation voltages and polarities. First, bacteria, fungal spores, and dust dispersed from soiled carpets were sampled in a walk-in test chamber. Second, a simulant of anthrax-causing Bacillus anthracis spores was dispersed and sampled in the same chamber. Third, bacteria were sampled in a small office while four adults were engaged in lively discussions. Fourth, bacteria and fungal spores released from hay and horse manure were sampled in a horse barn during cleanup operations. Fifth, bacteria in metalworking fluid droplets were sampled in a metalworking simulator. It was found that the new ESP differentiates between positively and negatively charged microorganisms, and that in most of the tested environments the airborne microorganisms had a net negative charge. This adds a signature to the sampled microorganisms that may assist in their identification or differentiation, for example, in an anti-bioterrorism network.

Release of Streptomyces albus propagules from contaminated surfaces.

The release of Streptomyces albus propagules from contaminated agar and ceiling tile surfaces was studied under controlled environmental conditions in a newly developed aerosolization chamber. The experiments revealed that both spores and cell fragments can be simultaneously released from the colonized surface by relatively gentle air currents of 0.3 ms(-1). A 100 x increase of the air velocity can result in a 50-fold increase in the number of released propagules. The aerosolization rate depends strongly on the type and roughness of the contaminated surface. Up to 90% of available actinomycete propagules can become airborne during the first 10 min of the release process. Application of vibration to the surface did not reveal any influence on the aerosolization process of S. albus propagules under the tested conditions. This study has shown that propagules in the fine particle size range can be released in large amounts from contaminated surfaces. Measurement of the number of S. albus fragments in the vicinity of a contaminated area, as an alternative to conventional air or surface sampling, appears to be a promising approach for quantitative exposure assessment.

Effect of electrical charges and fields on injury and viability of airborne bacteria.

In this study, the effects of the electric charges and fields on the viability of airborne microorganisms were investigated. The electric charges of different magnitude and polarity were imparted on airborne microbial cells by a means of induction charging. The airborne microorganisms carrying different electric charge levels were then extracted by an electric mobility analyzer and collected using a microbial sampler. It was found that the viability of Pseudomonas fluorescens bacteria, used as a model for sensitive bacteria, carrying a net charge from 4100 negative to 30 positive elementary charges ranged between 40% and 60%; the viability of the cells carrying >2700 positive charges was below 1.5%. In contrast, the viability of the stress-resistant spores of Bacillus subtilis var. niger (used as simulant of anthrax-causing Bacillus anthracis spores when testing bioaerosol sensors in various studies), was not affected by the amount of electric charges on the spores. Because bacterial cells depend on their membrane potential for basic metabolic activities, drastic changes occurring in the membrane potential during aerosolization and the local electric fields induced by the imposed charges appeared to affect the sensitive cells' viability. These findings facilitate applications of electric charging for environmental control purposes involving sterilization of bacterial cells by imposing high electric charges on them. The findings from this study can also be used in the development of new bioaerosol sampling methods based on electrostatic principles.

Fungal fragments as indoor air biocontaminants.

The aerosolization process of fungal propagules of three species (Aspergillus versicolor, Penicillium melinii, and Cladosporium cladosporioides) was studied by using a newly designed and constructed aerosolization chamber. We discovered that fungal fragments are aerosolized simultaneously with spores from contaminated agar and ceiling tile surfaces. Concentration measurements with an optical particle counter showed that the fragments are released in higher numbers (up to 320 times) than the spores. The release of fungal propagules varied depending on the fungal species, the air velocity above the contaminated surface, and the texture and vibration of the contaminated material. In contrast to spores, the release of fragments from smooth surfaces was not affected by air velocity, indicating a different release mechanism. Correlation analysis showed that the number of released fragments cannot be predicted on the basis of the number of spores. Enzyme-linked immunosorbent assays with monoclonal antibodies produced against Aspergillus and Penicillium fungal species showed that fragments and spores share common antigens, which not only confirmed the fungal origin of the fragments but also established their potential biological relevance. The considerable immunological reactivity, the high number, and the small particle size of the fungal fragments may contribute to human health effects that have been detected in buildings with mold problems but had no scientific explanation until now. This study suggests that future fungal spore investigations in buildings with mold problems should include the quantitation of fungal fragments.

Efficiency of final cleaning for lead-based paint abatement in indoor environments.

The effectiveness of procedures used for the final indoor cleaning after active lead-based paint abatement were evaluated in a 830 ft3 test chamber. Dry and wet scraping and dry machine sanding were applied to wooden doors obtained from lead-hazard control sites. The airborne particle concentration and size distribution were monitored using a real-time particle size spectrometer. Particulates were also collected on filters and analyzed for total dust and lead. The resulting airborne lead mass was determined for each cleaning procedure, and the potential floor lead loading resulting from the dust settling was calculated. Wipe samples were collected to measure the actual floor lead loading. The effectiveness of final cleaning was evaluated first for dry abatement methods. Various cleaning work practices were tested by applying wet and dry debris sweeping as well as no sweeping in combinations with wet and dry removal of plastic sheeting. Considerable resuspension of leaded particles was detected during dry sweeping: the airborne lead mass increase ranged between 65 and 220 percent. However, this increase did not exceed 22 percent when wet sweeping was applied. Minimal or no resuspension was found when the plastic was folded with leaded debris inside (no sweeping was performed prior to the sheeting removal). During folding activity, the "clean" (uncovered) floor surface may be significantly contaminated with leaded dust from workers' shoes and cleaning tools. The first HEPA vacuuming resulted in a 15- to 20-fold decrease of the airborne lead mass; however, it was not sufficient to reduce the floor lead loading to the U.S. Department of Housing and Urban Development (HUD) clearance level of 40 microg/ft2, as determined by wipe sampling. Wet mopping following the first HEPA vacuuming was proven to be effective to reduce the lead loading significantly below 40 microg/ft2. The second HEPA vacuuming resulted in further reduction of the airborne lead mass concentration. The floor lead loading remained much lower than 40 microg/ft2. These results were confirmed in the tests when using wet scraping. Overall, the HUD-recommended cleaning protocol was found to be sufficient in reducing the floor lead loading below 40 microg/ft2. At the same time, several modifications are proposed in this study to further improve the cleaning effectiveness.

Dynamic monitoring of the dust pickup efficiency of vacuum cleaners.

This study evaluated a new method that uses an optical aerosol photometer for dynamically monitoring dust pickup efficiency during vacuuming. In the first stage of this study the new method was compared with built-in dirt sensors installed by vacuum cleaner manufacturers. Through parallel testing it has been shown that the widely available built-in dirt sensors are not sensitive enough to register small (< 53 microm) dust particles. Therefore, only the optical photometer was used in the rest of the experiments of this study to monitor the dust pickup efficiency while the vacuum cleaner was operated with different nozzles on clean and soiled carpet and vinyl sheet flooring. This method also was used to monitor dust pickup efficiency when vacuuming carpets originating from lead-contaminated homes. The dust pickup efficiencies obtained with the optical aerosol photometer have been compared with the surface lead concentrations found during different stages of cleaning. Results indicate that the dust mass concentration registered with the optical aerosol photometer at the nozzle outlet correlates well with the dust mass collected in the vacuum cleaner filter bag and with the surface lead level. Therefore, dynamic dust pickup monitoring can provide valuable information about the efficiency of cleaning when a vacuum cleaner is used. This suggests that a small aerosol photometer similar to a light-scattering smoke detector would be beneficial in vacuum cleaners used for cleaning surfaces contaminated with leaded dust and biological particles (including allergens).

Method for Evaluating Germicidal Ultraviolet Inactivation of Biocontaminated Surfaces.

Safety issues related to work-site conditions often deal with potential worker exposure to infectious airborne microorganisms due to their dissemination in indoor air and contamination of surfaces. Germicidal ultraviolet (GUV) radiation is used in health-care settings and other occupational environments for microbial inactivation. In this study, a new methodology for determining the efficiency of GUV microbial inactivation of surfaces was developed and evaluated. The method utilizes identical chambers in which test microorganisms are irradiated on agar surfaces at different humidity and irradiation intensity levels. The effects of GUV intensity and exposure time on microbial inactivation were examined for Micrococcus luteus and Serratia marcescens. It was found that at low humidity levels (20-25%) both organisms can be inactivated with at least 95% efficiency if the GUV intensity exceeds 50 µW/cm2 for at least 3-5 min (corresponding to a dose of ~ 10 mJ/cm2). The radiation dose needed for effective inactivation of S. marcescens, as measured by a UV meter near the microbial sample, was found not to be affected by the humidity level, whereas that of M. luteus increased at higher humidities. The findings of this study can be used to determine sufficient GUV inactivation doses for occupational environments with various microbial contaminations.