Collection of airborne micro-organisms on Nuclepore filters, estimation and analysis—CAMNEA method

The total number of airborne micro-organisms collected on Nuclepore filters was determined by acridine orange staining and epifluorescence microscopy. The viable fraction of the total numbers varied significantly when actinomycete and fungal spores from different environments were stored on the filter surface for 1 week, although the microflora composition was not altered. A high correlation between viable and total counts was noted in environments where the airborne flora was dominated by fungal spores, while a low correlation was found for airborne bacteria. Peak values of the total counts registered in some work environments varied between 10⁷ and 10¹¹ micro-organisms/m³. Size analysis showed a dominating fraction of respirable micro-organisms (aerodynamical diameter < 5 μm). The investigation shows that it is of the utmost importance to combine viable counts with total count enumeration in the study of exposure to micro-organisms in work-related situations.     

Study of airborne fungal spores in Madrid, Spain

The concentration of fungal spores in the atmosphere of Madrid was recorded and analyzed for the year 2003. Airborne spores were sampled continuously with a Hirst-type spore trap located on the roof of a building of the School of Pharmacy, at about 8 m above ground level. Correlation between the mean daily spore concentrations and meteorological variables were explored by means of Spearman’s correlation analyses. Seventy spore types were identified, of which the most numerous were Cladosporium, Aspergillaceae (conidia), Coprinus, Agaricales (basidiospores), Ustilago (teliospores) and Pleospora (ascospores). These six types of spores represented more than 70% of the total. Cladosporium represented 41% of the total fungal spores, while Ustilago spores, the concentrations of which in May and June exceeded 47% of the monthly total spore count, constituted the second most important group. Spores reached their highest concentrations in the spring months, and in the autumn, mainly in October. A␣positive significant correlation was found between airborne spore counts and temperature and relative humidity. The results provide a picture of the spectrum of airborne fungal spores present in the atmosphere of Madrid and of the `peak' periods of their presence. Future studies will provide more detailed information on the seasonal dynamics of the spores most frequently found in the air as well as on the extent to which atmospheric conditions influence their release, dispersion and sedimentation processes.     

Ten types of microscopically identifiable airborne fungal spores at Leiden,The Netherlands

A universal method for the complete assessment of atmospheric fungal spores does not exist, which is continuous, volumetric and non-selective, and offers at the same time reliable identification of the collected spores. To perform a survey of airborne fungal spores, a choice has to be made between a viable and non-viable method. For the study carried out in Leiden, the non-viable, continuous volumetric method has been employed, showing the results over a period of 10 years, for 10 microscopically identifiable fungal spore types. Of this selection,Cladosporium spores have by far the highest airborne quantities, with an average annual total of the daily averages of over 700 000.Botrytis, Ustilago andAlternaria follow with much lower spore concentrations of between 20 000 and 30 000 as annual totals. The spore types ofEpicoccum, Erysiphe, Entomophthora, Torula, Stemphylium, andPolythrincium are represented with annual sums lower than 10 000. A spore calendar shows the overall seasonal appearance of the 10 selected types.     

Status of airborne spores and pollen in a coir factory in Kerala,India

The prevalence of airborne fungal spores and pollen grains in the indoor and outdoor environments of a coir factory in Thiruvananthapuram district of Kerala state, India was studied using the Burkard Personal Sampler and the Andersen 2-stage Sampler for 2 years (September 1997 to August 1999). The concentration of pollen grains was remarkably lower than that of fungal spores (ratio of 1:28). There was no large difference in the concentrations and types of fungal spores between the indoor and outdoor environments, with 26 spore types found to be present indoors and 27 types outdoors; of these, 22 were common to both the environments. Aspergillus/Penicillium, Cladosporium, ‘other basidiospores’ and ascospores were the dominant spore types. The total spore concentration was highest in February and lowest in September, and it was significantly higher in 1998–1999 than in 1997–1998. Twenty viable colony-forming types were isolated from inside the coir factory. The most dominant viable fungi isolated were Penicillium citrinum, Aspergillus flavus and Aspergillus niger. The total pollen concentration was higher in the outdoor environment of the coir factory than indoors, with 15 and 17 pollen types, respectively. Grass and Cocos nucifera pollen types were dominant. The dominant spore and pollen types trapped in the two environments of the coir factory are reportedly allergenic and, consequently, workers are at risk of catching respiratory/allergic diseases.     

NAGase activity in airborne biomass dust and relationship between NAGase concentrations and fungal spores

Inhalation of airborne fungal spores or fungalenzymes may cause adverse pulmonary healtheffects. The enzyme NAGase(N-acetyl--D-glucosaminidase) is achitinase presumed to be secreted by all fungi. In this study, NAGase activities andconcentrations of fungi are estimated inairborne biomass dust to acquire knowledgeabout the level of NAGase activity and therelationship between NAGase activity andconcentrations of airborne fungal spores.NAGase was sampled on both teflon andpolycarbonate filters, and polycarbonatefilters proved to be better for extraction ofNAGase than teflon filters. NAGase was foundin airborne dust at a biofuel plant and in dustgenerated from biomass. At a biofuel plant, themedian level of exposure to NAGase was 21 pmols^–1 m^–3. Significant correlationswere found between NAGase activities, totalnumber of fungal spores and CFU of fungi, withthe highest degree of correlation being betweenthe total number of fungal spores and theNAGase activity (r = 0.802, n = 76). Furthermore,when dust was stored for different periods, theculturability of fungal spores was stronglyreduced and the NAGase activity was not or onlyslightly reduced after up to 40 days ofstorage. Accordingly, NAGase activity may beused as a rapid method to get an estimate ofthe exposure level to airborne fungal spores.Whether pure NAGase or the NAGaseconcentrations observed here cause any healtheffects is not known, although it has beenshown that other fungal enzymes can causerespiratory disorders and a chitinase isdescribed as an allergen.     

Ten types of microscopically identifiable airborne fungal spores at Leiden,The Netherlands

A universal method for the complete assessment of atmospheric fungal spores does not exist, which is continuous, volumetric and non-selective, and offers at the same time reliable identification of the collected spores. To perform a survey of airborne fungal spores, a choice has to be made between a viable and non-viable method. For the study carried out in Leiden, the non-viable, continuous volumetric method has been employed, showing the results over a period of 10 years, for 10 microscopically identifiable fungal spore types. Of this selection,Cladosporium spores have by far the highest airborne quantities, with an average annual total of the daily averages of over 700 000.Botrytis, Ustilago andAlternaria follow with much lower spore concentrations of between 20 000 and 30 000 as annual totals. The spore types ofEpicoccum, Erysiphe, Entomophthora, Torula, Stemphylium, andPolythrincium are represented with annual sums lower than 10 000. A spore calendar shows the overall seasonal appearance of the 10 selected types.     

Sterilizing Effects of High-Intensity Airborne Sonic and Ultrasonic Waves

The lethal effects of high-intensity airborne sonic (9.9 kc/sec) and ultrasonic waves (30.4 kc/sec) on spores of Bacillus subtilis var. niger ATCC 9372 were determined. The spores, which were deposited on filter-paper strips, were exposed to sound waves for periods varying from 1 to 8 hr, at a temperature of 40 C and a relative humidity of 40%. Significant reductions in the viable counts of spores exposed to airborne sonic or ultrasonic irradiations were obtained. The antibacterial activity of airborne sound waves varied with the sound intensity level, the period of irradiation, and the distance of the sample from the sound source. At similar intensity levels, the amplitude of motion of the sound waves appeared to be a factor in acoustic sterilization.     

Monitoring airborne fungal spores in an experimental indoor environment to evaluate sampling methods and the effects of human activity on air sampling.

Aerobiological monitoring was conducted in an experimental room to aid in the development of standardized sampling protocols for airborne microorganisms in the indoor environment. The objectives of this research were to evaluate the relative efficiencies of selected sampling methods for the retrieval of airborne fungal spores and to determine the effect of human activity on air sampling. Dry aerosols containing known concentrations of Penicillium chrysogenum spores were generated, and air samples were taken by using Andersen six-stage, Surface Air System, Burkard, and depositional samplers. The Andersen and Burkard samplers retrieved the highest numbers of spores compared with the measurement standard, an aerodynamic particle sizer located inside the room. Data from paired samplers demonstrated that the Andersen sampler had the highest levels of sensitivity and repeatability. With a carpet as the source of P. chrysogenum spores, the effects of human activity (walking or vacuuming near the sampling site) on air sampling were also examined. Air samples were taken under undisturbed conditions and after human activity in the room. Human activity resulted in retrieval of significantly higher concentrations of airborne spores. Surface sampling of the carpet revealed moderate to heavy contamination despite relatively low airborne counts. Therefore, in certain situations, air sampling without concomitant surface sampling may not adequately reflect the level of microbial contamination in indoor environments.     

Determination of airborne fungal spores of Gaziantep (SE Turkey)

The aim of this study was to present the first airborne fungal spore research results of SE of Turkey. The presence and abundance of fungal spores were investigated with a Hirst-type volumetric spore trap (Lanzoni, VPSS 2000) for 2 years between the periods January 2010 and December 2011. A total of 211,521 spores and 47 fungal taxa belonging to Anamorphic Fungi, Phyla Ascomycota and Basidiomycota were recorded. Aspergillus/Penicillium spores type, unidentified spores, spores of Myxomycota and hyphal fragments were also recorded as groups. The relationship between fungal spore counts and several meteorological parameters was examined. Cladosporium was determined as the predominant genus (56.48 %); hyphal fragments (14.94 %), Ustilago (13.96 %) and Alternaria (5.79 %) were revealed as the common fungal aerosols of Gaziantep atmosphere. With this study, the first aeromycological survey of SE of Turkey has been conducted and new information on the field of aerobiology in Turkey has been introduced.     

Gravimetric survey of airborne fungal spores in Milan

Knowledge of airborne viable particles is basic to every study of respiratory allergies and an understanding of airborne fungal spores is particularly useful because they are harder to detect than other inhalant allergens such as pollens, which can be predicted seasonally. The gravimetric method is the simplest way to search for fungal spores.     

Gravimetric survey of airborne fungal spores in Milan

Knowledge of airborne viable particles is basic to every study of respiratory allergies and an understanding of airborne fungal spores is particularly useful because they are harder to detect than other inhalant allergens such as pollens, which can be predicted seasonally. The gravimetric method is the simplest way to search for fungal spores.     

Summer airborne mycoflora of Timişoara (Romania) and relationship to meteorological parameters

The present aeromycological investigation was undertaken to study atmospheric fungal spores in Timi?oara (western Romania). This study was carried out using a Hirst type volumetric sampler. The study revealed the existence of a rich airborne mycoflora. The atmospheric fungal spores were classified and evaluated into three groups (‘major’, ‘minor’ and ‘sporadic’) depending upon their catch percentage in the air. Cladosporium/Fusarium/Leptosphaeria-group, Alternaria, Helminthosporium airborne fungal spores and airborne fungal fragments regularly recorded (frequency 100% of days). Cladosporium accounted for 81.09% of the outdoor fungal spores. The airborne fungal fragments have been identified as abundant in our geographic area. Spearman’s correlations were applied to meteorological parameters and airborne fungal spore concentrations. In addition, correlations were calculated between the fungal spore concentrations and the meteorological variables from the previous day. A total of eleven weather factors were selected for this investigation. Following Spearman’s correlations, I identified two patterns of behaviour: most of the airborne fungal spores prefer cloudiness, lower near-surface soil temperature, lower atmospheric pressure, higher relative humidity and precipitation (pattern A) while other spore concentrations favour increased sunshine, higher near-surface soil temperature and dry conditions (pattern B). The behaviour of some fungal spores during the warm season has proven unclear (pattern C). This study demonstrates the need for investigations throughout the year and the evaluation with complementary statistical methods, regarding the correct interpretation of airborne mycoflora relationships with meteorological parameters.     

The effect of sunlight on allergen release from spores of the fungus Alternaria

Airborne fungal spores are known carriers of allergen. Correlations between spore counts and allergen concentrations are poor. It is known that germination increases allergen release, implicating spore viability as a determinant of allergen release. During aerial dispersal, spores can be exposed to prolonged periods of ultraviolet (UV) light which can reduce viability of spores. We examined the relation between spore viability and allergen release in two experiments: firstly spores from culture were treated with a UV wavelength of 254?nm (not present in sunlight reaching the earth's surface) or autoclaved, and secondly, spores were exposed to simulated sunlight over three days. In both studies viability was measured (by germination on agar and by metabolic activity with nitro-blue tetrazolium vital stain) and allergen release by the Halogen immunoassay. The UV light reduced the proportion of spores able to germinate but did not affect metabolic activity or allergen release. Autoclaving reduced the proportion of spores releasing allergen by half (p<0.0001). Three days' exposure to simulated sunlight correlated negatively with spore germination and metabolic activity (p<0.0001), but did not affect allergen release (p=0.799). In conclusion, simulated sunlight reduced the metabolic activity and germinability of spores however the proportion releasing allergen remained unaffected. These findings suggest that spore counts may reflect allergen concentrations in the air if spores are dead or dormant. The contribution of viable spores to concentrations of airborne allergen, as well as the role of germination in allergen delivery to the respiratory tract, remains to be resolved.     

Dispersal of fungal spores from three types of air handling system duct material

Exposure to airborne microorganisms in indoor environments may result in infectious disease or elicit an allergic or irritant response. Air handling system components contaminated by fungi have been implicated in the dispersal of spores into the indoor environment, thereby serving as a route of exposure to occupants. This study was conducted to provide quantitative data on the dispersal of spores from fungal colonies growing on three types of duct material. Galvanized metal, rigid fibrous glass ductboard, and fiberglass duct liner were soiled and contaminated with a known concentration of Penicillium chrysogenum spores. The duct materials were incubated in humidity chambers to provide a matrix of growing, sporulating fungal colonies at a contamination level of 109 colony forming units (CFU) per duct section, consistent for all materials. For each experiment a contaminated duct section was inserted into the air handling system of an experimental room, and the air handling system was operated for three 5-minute cycles with an air flow of 4.2 m3 min–1. The duct air velocity was approximately 2.8 m sec–1. The airborne concentration of culturable P. chrysogenum spores (CFU m–3), total P. chrysogenum spores (spores m–3), and total P. chrysogenum-sized particles (particles m–3) were measured in the room using Andersen single-stage impactor samplers, Burkard slide impactor samplers, and an aerodynamic particle sizer, respectively. The highest airborne concentrations (104 CFU m–3; 105 spores m–3; 104 particles m–3) were measured during the first operating cycle of the air handling system for all duct materials with decreasing airborne concentrations measured during the second and third cycles. There was no significant difference in spore dispersal from the three contaminated duct materials. These data demonstrate the potential exposure for building occupants to high concentrations of spores dispersed from fungal colonies on air handling system duct materials during normal operation of the system.     

An assessment of the air quality in indoor and outdoor air with reference to fungal spores and pollen grains in four working environments in Kerala, India

Qualitative and quantitative analyses of airborne fungal spores and pollen grains in four working environments (market, saw mill, poultry and cow sheds) in Thiruvananthapuram, the capital city of Kerala, India, were carried out for 2 years using Burkard Personal Slide Sampler and Andersen Two-Stage Sampler. Total spore concentration in these sites was always higher in indoor environments than in outdoor environments. Difference in concentration was not statistically significant in any of these work places except in saw mill (t test, p < 0.05). The highest spore concentration was recorded here followed by market, poultry and cow sheds. A total of 32 fungal spore types from indoor environments and 33 spore types from outdoor environments were recorded. Of them, 16 spore types were common to all the sites. Ameropsores, Cladosporium, other basidiospores, Ganoderma and Nigrospora were the dominant spore types in both indoor and outdoor environments. A total of 27 species of viable fungi from indoor and 24 species from outdoor environments were identified. Penicillium citrinum, Aspergillus flavus and Aspergillus niger were the most dominant viable fungi isolated. In contrast, total pollen concentration was always higher in outdoor environments than in indoor environments. Twenty-nine pollen types from indoor and 32 pollen types from outdoor were captured during the sampling. Poaceae, Cocos, Artocarpus, Amaranthus/Chenopodium and Tridax were the common and dominant pollen types observed in all the sites. Peak spore and pollen incidence were recorded during the late rainy and dry seasons (October–February) in both indoor and outdoor environments. The study revealed high prevalence of predominantly allergenic fungal spores and pollen grains in all the four work places. Workers/visitors are at potential risk of susceptibility to respiratory/allergic disorders.     

Natural folding of airborne fungal spores: a mechanism for dispersal and long-term survival?

Analysis of numerous air samples has indicated that dormant, viable fungal spores are highly present, which suggests that aerial dispersion is important for fungi. Whereas the majority of the spores may travel only very short distances, there is indication that a notable number of them cover much longer distances. Harmomegathy is a terminology coined by Wodehouse (1935) describing the natural folding of pollen to accommodate controlled and reversible water loss. Here, we discuss evidence that this concept may also apply to airborne fungal spores that face similar challenges and have to survive periods of drought and low temperatures while retaining viability to germinate after deposition upon a suitable moist substrate. In fact, (air)dried conidia, appear collapsed, survive for much longer times compared to spores in liquid, that deteriorate in time. This indicates that for some types of fungal spores, true dormancy is reached in the desiccated state. For these airborne spores this might be regarded as a pre-adaptation that supports long-distance transport of viable cells through air. We state that spores are naturally folded during transport in air if the humidity is low enough. We hypothesize that this is a pre-adaptation supporting release, dispersal and survival of airborne spores. Moreover, the smaller size of dry naturally-folded spores may also be relevant, e.g. for the opportunistic pathogenic fungus Aspergillus fumigatus reduced spore size supports deposition within the alveoli in the lung.     

Enumeration of airborne bacteria and fungi using solid phase cytometry

Conventional methods for the enumeration of airborne micro-organisms are inaccurate and time-consuming, hence the interest in novel approaches is increasing. In the present study, the use of solid phase cytometry (SPC) was evaluated for the enumeration of airborne micro-organisms. A 4 h SPC procedure based on viability staining was applied to samples from 50 locations and compared with an optimised culture-based method. Plate counts after air sampling were repeatable but strongly dependent on sampling volume. Samples with low or high microbial load were difficult to analyse using the culture-based method, unlike with SPC. Results show that SPC can be considered superior to the culture-based method because of its much higher dynamic range, its speed and its ability to enumerate not only culturable but all viable micro-organisms.     

Removal of Viable Airborne Fungi from Indoor Environments by Benzalkonium Chloride-Based Aerosol Disinfectants

Fungi are ubiquitous in indoor environments, and some taxa can cause clinical symptoms in humans. Thus, from the viewpoint of public health, methods to reduce indoor airborne fungi are needed. The goal of this study was to examine the efficacies of benzalkonium chloride (BAC)–based aerosol disinfectants to remove airborne viable fungi from indoor environments. The laboratory- and field-based experiments were conducted to compare airborne culturable fungal concentrations before and after the disinfectant aerosol applications. The laboratory-based experiments showed the greater efficacies by the BAC-based disinfectant aerosol than by pure-water control aerosol (p <.05, t-test). In the field study using the BAC-based disinfectant aerosols, on average a 58% reduction of total airborne culturable fungal concentrations were observed. Additionally, the significant reduction was found for a group of airborne yeasts or yeast-like organisms (p <.05, Wilcoxon signed rank test). The BAC-based aerosol disinfectants are effective when used to reduce the numbers of airborne culturable fungi, in particular yeasts or yeast-like organisms, from indoor environments.     

Relationship between bacterial counts and endotoxin concentrations in the air of wastewater treatment plants.

The relationship between bacterial counts and endotoxin concentrations in air samples was studied. Selective EMB medium favored the growth of a larger portion of airborne gram-negative bacteria than LES Endo or MacConkey medium and was a good predictor of the endotoxin levels determined with a chromogenic Limulus assay of the air of wastewater treatment plants. The bacterial counts determined with nonselective media correlated poorly with airborne endotoxin levels; however, R2A medium yielded higher viable bacterial counts than TYG medium. Direct counting by epifluorescence microscopy yielded the highest bacterial counts, but no correlation was obtained between total bacterial counts and endotoxin concentrations.