The importance of the contribution of biogenic particles to organic ambient aerosol has become increasingly apparent in recent years. Bioparticles might be allergenic, harmful themselves or carriers of harmful substances. Moreover, because of their size, they might contribute significantly to the mass concentration of atmospheric particulate matter (PM). In this context, methodological advances in the determination of this PM component are of growing importance to complement air quality issues. This study was aimed at bioaerosol identification and bioparticle size characterization by using a cascade impactor as sampling medium and by combining epifluorescence microscopy and flow cytometry as detection techniques. Following procedures described in Amalfitano and Fazi (2008), the extraction of particles from collection filters was performed using a detaching solution composed by chelating agents and detergents (i.e., sodium pyrophosphate and Tween® 80). The liquid suspension was double stained with the nucleic acid specific dyes Sybr Green I and Propidium Iodide. After staining, a subaliquot of each liquid suspension (50 µl) was air-dried on a well glass slide to double-check the integrity of biogenic particles by microscopy. The efficiency of the detaching procedure was evaluated by performing three subsequent extraction steps. After each extraction the filters were microscopically inspected to rule out the presence of undetached bioparticles. Particle detection and enumeration were performed by flow cytometry. This technology allows the rapid (up to 1000 events per second) and multiparametric detection of single particles passing through a laser interrogation point (Figure 1). The light scatter signals at forward and side angles give information on the size and inner complexity of each particle. Moreover, various fluorescent dyes can be used to selectively identify those particles containing components of biological origin (e.g., DNA, RNA) (Chen & Li, 2005). Figure 2 shows the particle distribution within 10 size classes in the range 0.18 - 18 um. The total abundance of bioparticles was 3.5x104 particles/m3. Most of the fluorescent particles were found within the Aerodynamical Diameter (AD) size classes 1.8 - 3.2 (9.0x103 particles/m3) and 3.2 - 5.6 µm (1.1x104 particles/m3). Only 6.3% of the total particles were retrieved in the size range below 1 µm. After three consecutive extractions, the particle recovery efficiency was, on average, 92%, with lower efficiency (78%) for particles with AD<5.6 µm. Recognizing to bioaerosol an important role for the determination of PM composition and mass concentration, novel data on biogenic particles represent a valid complement to allow a more complete evaluation of the air quality. For such purposes, flow cytometry seems an appropriate analytical tool to provide rapid and multi-parametric data for a better understanding of the bioaerosol size distribution and concentration in the atmospheric environment.
Flow Cytometry as a rapid and multi-parametric tool to characterize bioaerosol in atmospheric particulate matter.
Amalfitano S;Marcovecchio F;Perrino C
2015
Abstract
The importance of the contribution of biogenic particles to organic ambient aerosol has become increasingly apparent in recent years. Bioparticles might be allergenic, harmful themselves or carriers of harmful substances. Moreover, because of their size, they might contribute significantly to the mass concentration of atmospheric particulate matter (PM). In this context, methodological advances in the determination of this PM component are of growing importance to complement air quality issues. This study was aimed at bioaerosol identification and bioparticle size characterization by using a cascade impactor as sampling medium and by combining epifluorescence microscopy and flow cytometry as detection techniques. Following procedures described in Amalfitano and Fazi (2008), the extraction of particles from collection filters was performed using a detaching solution composed by chelating agents and detergents (i.e., sodium pyrophosphate and Tween® 80). The liquid suspension was double stained with the nucleic acid specific dyes Sybr Green I and Propidium Iodide. After staining, a subaliquot of each liquid suspension (50 µl) was air-dried on a well glass slide to double-check the integrity of biogenic particles by microscopy. The efficiency of the detaching procedure was evaluated by performing three subsequent extraction steps. After each extraction the filters were microscopically inspected to rule out the presence of undetached bioparticles. Particle detection and enumeration were performed by flow cytometry. This technology allows the rapid (up to 1000 events per second) and multiparametric detection of single particles passing through a laser interrogation point (Figure 1). The light scatter signals at forward and side angles give information on the size and inner complexity of each particle. Moreover, various fluorescent dyes can be used to selectively identify those particles containing components of biological origin (e.g., DNA, RNA) (Chen & Li, 2005). Figure 2 shows the particle distribution within 10 size classes in the range 0.18 - 18 um. The total abundance of bioparticles was 3.5x104 particles/m3. Most of the fluorescent particles were found within the Aerodynamical Diameter (AD) size classes 1.8 - 3.2 (9.0x103 particles/m3) and 3.2 - 5.6 µm (1.1x104 particles/m3). Only 6.3% of the total particles were retrieved in the size range below 1 µm. After three consecutive extractions, the particle recovery efficiency was, on average, 92%, with lower efficiency (78%) for particles with AD<5.6 µm. Recognizing to bioaerosol an important role for the determination of PM composition and mass concentration, novel data on biogenic particles represent a valid complement to allow a more complete evaluation of the air quality. For such purposes, flow cytometry seems an appropriate analytical tool to provide rapid and multi-parametric data for a better understanding of the bioaerosol size distribution and concentration in the atmospheric environment.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
