Comparison between the counting methods used by two aerobiology networks in southern Europe (Spain and Italy)

Aerobiology studies the biological particles suspended in the atmosphere in order to know what types are present in the air and how their concentrations vary spatially or seasonally. The measurement of airborne pollen concentration requires a standardized methodology. Normally, the analysis of the slides obtained after samplings is carried out according to national network procedures. Different networks use different slide counting methods (longitudinal sweeps, transverse sweeps or random fields), and a different number of lines/fields are routinely read. In this study, we compared two slide counting methods adopted by two monitoring centres belonging to different aerobiology networks that operate in southern Europe, the University of Malaga (Spanish Aerobiology Network, the REA) and the University of Roma Tor Vergata (Rete Italiana di Monitoraggio in Aerobiologia, the RIMA). For that, the same samples were counted following the two methodologies consisting on the reading of four (REA) and six (RIMA), respectively, longitudinal sweeps at a magnification of 400X (REA). Statistical analysis was performed in order to search the degree of association between the two series of data obtained, as well as whether or not there were significant differences between them. The results showed highly significant correlation and regression coefficients. On the other hand, Wilcoxon tests, in general, did not showed significant differences between the series for the total pollen as well as for the major pollen types. This confirms that the two methods can be used indistinctly although it would be convenient for the different networks to standardize and unify methodologies in order to follow the same operating procedures.     

Quality control in aerobiology: comparison different slide reading methods

The use of standard methodology allows us to compare and to evaluate the data generated from samplers under general use. In aerobiological pollen monitoring, the most frequently slide sampling methods used are based on the selection of different number of longitudinal or transverse traverses and random fields, which represents a small proportion of the entire slide. The aim of this study is to evaluate possible methodological errors produced when the different pollen count methods are used in relation to the results obtained if the whole slide area was quantified. Moreover, the optimization of the counting method by selecting the best longitudinal or transverses traverses that reflect the more accurate counting in relation to the total pollen obtained when the total tape surface was done. Therefore, 113 slides recorded in 2008 at Ourense (NW Spain) differing in its pollen content, recollected time of the year and representation of the different pollen types were selected in this survey. A comparison between the 4 longitudinal traverses, the 12 transverse traverses, and the 493 random fields methods was evaluated. The average relative error and squared error were calculated for both, longitudinal and transverse traverses and the most accurate lines number for counting were selected. Finally, the three counting techniques were compared and significant differences were detected.     

Comparison of two pollen counting methods of slides from a hirst type volumetric trap

Two of the most frequently used methods of pollen counting on slides from Hirst type traps are evaluated in this paper: the transverse traverse method and the longitudinal traverse method. The study was carried out during June–July 1996 and 1997 on slides from a trap at Worcester, UK. Three pollen types were selected for this purpose: Poaceae, Urticaceae and Quercus. The statistical results show that the daily concentrations followed similar trends (p < 0.01, R-values between 0.78–0.96) with both methods during the two years, although the counts were slightly higher using the longitudinal traverses method. Significant differences were observed, however, when the distribution of the concentrations during 24 hour sampling periods was considered. For more detailed analysis, the daily counts obtained with both methods were correlated with the total number of pollen grains for the taxon over the whole slide, in two different situations: high and low concentrations of pollen in the atmosphere. In the case of high concentrations, the counts for all three taxa with both methods are significantly correlated with the total pollen count. In the samples with low concentrations, the Poaceae and Urticaceae counts with both methods are significantly correlated with the total counts, but none of Quercus counts are. Consideration of the results indicates that both methods give a reasonable approximation to the count derived from the slide as a whole. More studies need be done to explore the comparability of counting methods in order to work towards a Universal Methodology in Aeropalynology. This revised version was published online in June 2006 with corrections to the Cover Date.     

Sampling in aerobiology. Differences between traverses along the length of the slide in Hirst sporetraps

Two years of data from four longitudinal traverses along each day's slide prepared from a continuously running Burkard sporetrap have been analyzed statistically. Using the Friedman test, a statistically significant difference was found between the four traverses, with a greater than 7% loss of pollen grains in the two outer traverses in relation to the inner. Four slides were then selected for more detailed analysis, using 18 longitudinal traverses with a 1-mm separation from the upper to the lower edge of the Melinex tape. There was found to be a progressive decline from the centre to the outside, and more than 4% of pollen grains were found outside the typical 14 mm width of the impaction orifice. There was no correlation between pollen grain size and the decline in counts from the centre to the outside. For the complete data set, there was a general rise in the diversity of pollen types with increasing sample counts, but above about 1000 pollen grains per sample there were no more than 27 pollen types found, often even fewer. A discussion is presented of whether four traverses really should be a fixing sample size, or whether it might be better to fix the total pollen count beginning with a traverse in the middle of the slide and ending with a variable number of traverses when that count is reached.     

Sampling in aerobiology. Differences between traverses along the length of the slide in Hirst sporetraps

Two years of data from four longitudinal traverses along each day's slide prepared from a continuously running Burkard sporetrap have been analyzed statistically. Using the Friedman test, a statistically significant difference was found between the four traverses, with a greater than 7% loss of pollen grains in the two outer traverses in relation to the inner. Four slides were then selected for more detailed analysis, using 18 longitudinal traverses with a 1-mm separation from the upper to the lower edge of the Melinex tape. There was found to be a progressive decline from the centre to the outside, and more than 4% of pollen grains were found outside the typical 14 mm width of the impaction orifice. There was no correlation between pollen grain size and the decline in counts from the centre to the outside. For the complete data set, there was a general rise in the diversity of bollen types with increasing sample counts, but above about 1000 pollen grains per sample there were no more than 27 pollen types found, often even fewer. A discussion is presented of whether four traverses really should be a fixing sample size, or whether it might be better to fix the total pollen count beginning with a traverse in the middle of the slide and ending with a variable number of traverses when that count is reached.     

Comparison between the allergenic airborne pollen in Trieste and at Lozzo di Cadore (Italy) in 1989

Summary A comparative study was carried out between aeroallergenic pollen spectra in Trieste and at Lozzo di Cadore. The two localities were found to be different on a five-day running mean basis as to the 8 pollen taxa monitored by the National Aerobiological Monitoring Network managed by the Italian Association of Aerobiology (AIA). In the zone of Lozzo the pollination curve of the spring flowering taxa showed a lag of about one month due to the colder climate. Lozzo di Cadore showed a poor airborne pollen content,Corylus and Graminaceae being prevailing. Trieste has a higher airborne pollen diversity and longer pollination times because of its higher floristic and vegetational complexity. In Trieste the allergenic pollen spectrum showed great quantities ofOstrya, Quercus, Oleaceae, Graminaceae and of ruderal taxa, widespread over the area, due to man's influence.     

The aerobiological results from the 1994 cruise of the Urania (cnr) on the Adriatic. I. Pollen and spore counts on the Mediterranean sea as compared to mainland Italia

In July 1994, we were able to collect airborne fungal spores and pollen grains over the Adriatic Sea from the upper deck of the Oceanographic Ship Urania (CNR). The biological particles were collected using a modified Lanzoni VPPS 1000 sampler (operating at a flux of 10 LPM), on glycerine-gelatine coated microscopic slides. Not only were the airborne concentrations of different organisms estimated, their viability was also tested with a 1% TTC solution. Particles were collected for 60 min (i.e. a volume of 600 liters of air sampled) at every 2 h from 0600–2100 h. Up to 689 pollen grains/m³ and an impressive 48 990 spores/m³ were collected daily. Forty-two fungal taxa were identified and the most abundant spores collected were Cladosporium (82.6%), Smuts (4.8%), Ascospores (2.8%), Basidiospores (2.1%) andAlternaria (1.7%). 20 pollen taxa were identified, and the dominant pollen were Urticaceae (57.9%), Graminaceae (20.7%), Fagaceae (2.4%), Plantaginaceae (1.4%), Pinaceae (1.3%) and Eucalyptus (1.1%). The most abundant captures were done at 0800 and 1000 h (17.8 and 16.7% respectively) and at 1400 and 1600 h (13.2 and 13.8% respectively). Pollen viability per species ranged from 0 to 100%, but for the most abundant taxa, it ranged from 3.8 to 75%, and averaged 27.7%. Maximum viability was found at 0800 and 1200 h. Pollen concentrations were of the same order of magnitude as the ones found on the mainland (Brindisi, Chieti, Matera). However, its specificity was evident. Future work should therefore look more at the pollen transport process which should account for this different assemblage of pollen.     

The aerobiological results from the 1994 cruise of the Urania (cnr) on the Adriatic. I. Pollen and spore counts on the Mediterranean sea as compared to mainland Italia

In July 1994, we were able to collect airborne fungal spores and pollen grains over the Adriatic Sea from the upper deck of the Oceanographic Ship Urania (CNR). The biological particles were collected using a modified Lanzoni VPPS 1000 sampler (operating at a flux of 10 LPM), on glycerine-gelatine coated microscopic slides. Not only were the airborne concentrations of different organisms estimated, their viability was also tested with a 1% TTC solution. Particles were collected for 60 min (i.e. a volume of 600 liters of air sampled) at every 2 h from 0600–2100 h. Up to 689 pollen grains/m³ and an impressive 48 990 spores/m³ were collected daily. Forty-two fungal taxa were identified and the most abundant spores collected were Cladosporium (82.6%), Smuts (4.8%), Ascospores (2.8%), Basidiospores (2.1%) andAlternaria (1.7%). 20 pollen taxa were identified, and the dominant pollen were Urticaceae (57.9%), Graminaceae (20.7%), Fagaceae (2.4%), Plantaginaceae (1.4%), Pinaceae (1.3%) and Eucalyptus (1.1%). The most abundant captures were done at 0800 and 1000 h (17.8 and 16.7% respectively) and at 1400 and 1600 h (13.2 and 13.8% respectively). Pollen viability per species ranged from 0 to 100%, but for the most abundant taxa, it ranged from 3.8 to 75%, and averaged 27.7%. Maximum viability was found at 0800 and 1200 h. Pollen concentrations were of the same order of magnitude as the ones found on the mainland (Brindisi, Chieti, Matera). However, its specificity was evident. Future work should therefore look more at the pollen transport process which should account for this different assemblage of pollen.     

An abbreviated method for the quality control of pollen counters

We present an abbreviated method for conducting large scale quality control (QC) exercises over limited time periods, which was used for examining the proficiency of technicians involved in the electronic Pollen Information Network (ePIN). The goal was for technicians to have their analysis skills evaluated at least twice: (1) by having at least one of their slides successfully checked by other counters in the ePIN network and (2) by successfully examining at least one additional slide from other sites. Success was judged as a relative difference (RDif %) ≤ 30% between the two daily average pollen concentrations. A total of 21 sites participated in the ePIN QC exercise. All of the results for total pollen had RDif % < 30%. Only five results had RDif > 30%, three for Betula and two for Poaceae pollen. Of these, three were slides containing < 40 pollen/m³ daily average and two were for sites that had microscopes with small fields of view and examined < 10% of the slide surface. More than 80% of the participants had at least two slides successfully checked by someone else in the network, and all of the participants had one slide successfully examined. The latter is comparable to a traditional ring test where only one slide is sent to participating sites. The method described here enabled a large number of technicians to be examined in a short period of time and represents a viable alternative to other approaches that can take many months to complete.     

An evaluation of two methods used for microscopic analysis of airborne fungal spore concentrations from the Burkard Spore Trap

The Burkard Volumetric Spore Trap is a common and efficient instrument used to collect outdoor air samples. In North America, two slide counting methods have been widely used by aerobiologists: the single longitudinal traverse method and the twelve transverse traverse method. The purpose of this study was to compare the two counting methods by assessing fungal spore concentrations of ascospores, basidiospores, smut teliospores, Cladosporium, Alternaria, Epicoccum, Curvularia, Drechslera, Pithomyces, other spores, and total spores at two metropolitan Tulsa, Oklahoma sites (Tulsa and Hectorville) during September 1996. Results showed that both methods were sensing parallel fluctuations in average daily spore concentration, although the twelve transverse traverse method usually resulted in higher concentrations. At the Tulsa site, the twelve transverse traverse method gave statistically higher concentrations than the single longitudinal traverse method except for Epicoccum, Pithomyces, smut teliospores, and other spores. At the Hectorville site, however, only Cladosporium and basidiospores showed that the twelve transverse traverse method was statistically higher than the single longitudinal traverse method. Comparison with concentrations obtained by counting the total slide surface of two slides indicated that neither method was equivalent to the total slide spore count, although the twelve transverse traverse method gave a lower absolute percent difference from the total slide surface concentration. While the twelve transverse traverse method gave slightly better approximations of the spore concentration, the increase in accuracy may not justify the extra effort required to analyze with this method.     

Gravitropic microtubule reorientation can be uncoupled from growth

The causal relationship between gravitropic growth responses and microtubule reorientation has been studied. Growth and microtubule reorientation have been uncoupled during the gravitropic response of maize (Zea mays L.) coleoptiles. Microtubule orientation and growth were measured under three different conditions: (i) a gravitropic stimulation where the growth response was allowed to be expressed (intact seedlings were displaced from the vertical position by 90°), (ii) a gravitropic stimulation where the growth response was suppressed (coleoptiles were attached to microscope slides and kept in a horizontal position), (iii) suppression of growth in the absence of gravitropic stimulation (coleoptiles were attached to microscope slides and kept in a vertical position). It was found that (i) gravitropic stimulation can induce a microtubular reorientation from transverse to longitudinal in the upper (slower growing) flank of the coleoptile, and an inhibition of growth; (ii) the reorientation of microtubules precedes the inhibition of growth; (iii) the gravitropic response of microtubules is weaker, not elevated, when the inhibition of growth is artificially enhanced by attaching the coleoptiles to a slide; and (iv) artificial inhibition of growth in the absence of gravitropic stimulation cannot induce a microtubular response. Thus, the extent of microtubule reorientation is not correlated with the extent of growth inhibition. Moreover, these findings demonstrate that microtubules do not reorient passively after growth changes, but actively in response to gravitropic stimulation. Received: 23 November 1999 / Accepted: 10 May 2000     

European allergenic pollen types

Summary Pollen data have been collected from 66 stations, situated in 19 European countries thanks to the initiative of both the Working Group «European Aeroallergen Network» (of the Int. Assoc. of Aerobiology) and the Subcommittee «Aerobiology of inhalant allergens» of the European Academy of Allergology and Clinical Immunology. Volumetric data over periods of 3 to 5 years (up to 1988) were included, and 15 both aerobiologically and allergologically relevant taxa were selected. Geographically adjacent stations were grouped together into regions looking at the peak period of the airborne grass-pollen presence. The result is a collection of 21 regional European pollen calendars, some of which are covering several countries (e.g. «Western Europe»), others are applicable only to a part of one country (e.g. «Southern Italy»).Comparing the calendars, general features are the South-to-North delay shift in the appearance of tree-pollen types in Spring, and the reverse shift for the weed-pollen types in late summer. The presence ofAmbrosia pollen in the air in several parts of Central Europe is clearly shown.     

Automated Instrument for the Fluorescent Treponemal Antibody-Absorption Test and Other Immunofluorescence Tests

An automated diagnostic test instrument and its development program are described. The instrument automates the fluorescent treponemal antibody-absorption test for syphilis to the extent that only 4 hr of technician time is required to conduct approximately 200 tests daily. Evaluation to date suggests its efficacy. In addition, preliminary studies indicate the feasibility of detecting antibodies to Toxoplasma gondii, Plasmodium malariae, and nucleoprotein (antinuclear factor). The instrument would seem to have broad application for routine and research immunofluorescence testing. Two elements comprise the instrument: a slide processor and a microscope attachment. The slide processor is an electro-pneumatically actuated device which automatically feeds special laboratory slides, on which antigen or other reagents are prefixed, through a series of operations which provide reagent application, incubation, washing, drying, and stacking of the finished slides for readout. The instrument provides flexibility in that incubation time and temperature as well as point, sequence, and duration of reagent application can be varied to accommodate a variety of immunofluorescence techniques. The microscope attachment can be fitted to all conventional dark-field fluorescence microscopes and makes possible the reading of three to six slides per minute. The reacted slides from the processor are injected sequentially onto the stage of the microscope by movement of a lever. As injected, slides are automatically in visual focus; fine focus is occasionally required. Scanning of the reacted field is accomplished by means of the normal microscope controls. A buffered glycerol coupling is maintained between the darkfield condenser substage lens and the slide cover glass by means of a pushbutton-actuated feed system.     

Monitoring of pollen rain in moss polsters and mats in view of their suitability as controls for determining the air-borne component of pollen spectra in herbivore faeces

Pollen has been extracted from six bryophyte and two lichen taxa, sampled from three habitats: upland heath, lowland bog and mixed woodland, in both west and east Scotland. The highest number of pollen taxa was recorded in bothSphagnum andThuidium; the lowest numbers of pollen taxa were recorded in two of the mosses and both lichens. For pollen rain control purposes in herbivore faecal studies, woodland species of bryophytes are not suitable due to the sheltering effects from rain provided by the overhead canopy. Open habitats such as moorland heath and bog provide polsters/mats which normally trap pollen rain more effectively for control purposes. The aim of this study was twofold: to observe the effectiveness of selected polsters and lichens as traps for pollen rain and to establish eventually, whether or not such vegetative traps could be used as controls in pollen spectra analyses of herbivore faeces for the determination of nutritional regime and habitat. From a comparison with recent studies in north west Spain of three taxa in common with Scottish samples,Polytrichum demonstrated a consistently higher number of pollen taxa trapped.Thuidium andCladonia all yielded between one and a half and three times the number of pollen taxa as in the same three bryophyte/lichen taxa sampled in Scotland. Investigation relating the pollen rain trapped in polsters to that in faecal material are continuing, and will be reported in due course.This paper is based on one given by E.C. at the 5th International Aerobiology Conference, held at Bangalore, India, August 1994.     

Airborne pollen in the El-Hadjar town (Algeria NE)

The diversity of airborne pollen grains in El-Hadjar town (northeast Algeria) was measured for 1 year, from July 1, 2012 to June 30, 2013, by means of the gravimetric method using Durham apparatus. The total number of pollen grains/cm² was calculated from slides that were changed daily. This aerobiological study documented the air concentration of pollen from 50 taxa, where 28 belonged to arboreal and 22 to non-arboreal taxa. The percentage of pollen from arboreal and non-arboreal taxa was 56 and 44 %, respectively. From the list, the major collected taxa causing allergy in humans dominant in the Mediterranean area were Cupressaceae (14.86 %), Olea sp. (7.18 %), Casuarina sp. (6.44 %), and Fraxinus sp. (3.83 %) among arboreal plants, whereas for the non-arboreal plants Poaceae (23.20 %), Mercurialis sp. (12.58 %), Plantago sp. (1.69 %), Urticaceae (0.95 %), and Chenopodiaceae (0.85 %). The highest pollen counts occurred in the period from February to April. The pollen calendar for the region presented in this paper may be a useful tool for allergologists and botanical awareness.     

Improvement of silver impregnation technique (protargol) to obtain morphological features of protists ciliates, flagellates and opalinates

The research on ciliates, flagelates and opalinates have been widespread by the utilization of techniques employing silver impregnation (protargol), modified by several authors. However, these are time consuming and its results are variable. The present work is a variant of the technique described by Tuffrau (1964, 1967) showing some adaptations made in our laboratory. The organisms can be preserved by different fixatives (alcoholic Bouin, Stieve's fluid, 2.5% glutaraldehyde and others) and then rinsed in destilled water followed by a fast clarification by 3% sodium hypochloride. If the organism is very sensitive to hypochloride, 4% sodium lauryl sulfate may be used and then washed 3 times in distilled water. The protista can be adhered to the glass slides with Mayer's glycerinated-albumin (1 glycerin vol. to 1 or 2 albumin vol.), diluted in water at a proportion of 1:10 Cv/v., or with 1% polylysine followed by fast washes with distilled water. After the slide preparation, they were covered with a layer of 0,8% Silver proteinate. Right after that, the slide has to be placed in a glass tray lined with moist tissue and covered to prevent the proteinate to dry. The tray was placed in a incubator at 40 degrees - 50 degrees C for 30 minutes. The slides are rinsed for 1 minute. with warm (35 degrees C) distilled water. The development of the material should be done with 0.4% hydroquinone with a maximum incubation time of 1 minute. It should be developed gradually, controlling the silver impregnation intensity by observation under optical microscope. Next, rinse in distilled water for 1 minute, and then, fix in 2,5% Sodium thiosulfate. Rinse the slide for two minutes before dehydrating it in an alcoholic serial 50-100 degrees. Finally rinse the slides in xylene. Mount the slides with Entellan MerckTM or Canada balsam.     

三种寡核苷酸芯片片基的比较

将荧光标记的寡核苷酸打印在Corning、多聚赖氨酸包被的DAKO玻片和多聚赖氨酸处理的一种显微镜载玻片上,并按常规方法进行洗脱,通过GenePix4100A扫描并以Genepix6．0软件分析点的大小、荧光强度和背景荧光强度来衡量3种片基的均一性和固定效率,并通过不同浓度梯度的60bp寡核苷酸探针的杂交实验来衡量片基对杂交效率的影响．结果显示,3种片基的均一性都较好,而多聚赖氨酸包被的DAKO和Cornning芯片的固定效率和杂交效率优于国产芯片．     

Monitoring of pollen rain in moss polsters and mats in view of their suitability as controls for determining the air-borne component of pollen spectra in herbivore faeces

Pollen has been extracted from six bryophyte and two lichen taxa, sampled from three habitats: upland heath, lowland bog and mixed woodland, in both west and east Scotland. The highest number of pollen taxa was recorded in bothSphagnum andThuidium; the lowest numbers of pollen taxa were recorded in two of the mosses and both lichens. For pollen rain control purposes in herbivore faecal studies, woodland species of bryophytes are not suitable due to the sheltering effects from rain provided by the overhead canopy. Open habitats such as moorland heath and bog provide polsters/mats which normally trap pollen rain more effectively for control purposes. The aim of this study was twofold: to observe the effectiveness of selected polsters and lichens as traps for pollen rain and to establish eventually, whether or not such vegetative traps could be used as controls in pollen spectra analyses of herbivore faeces for the determination of nutritional regime and habitat. From a comparison with recent studies in north west Spain of three taxa in common with Scottish samples,Polytrichum demonstrated a consistently higher number of pollen taxa trapped.Thuidium andCladonia all yielded between one and a half and three times the number of pollen taxa as in the same three bryophyte/lichen taxa sampled in Scotland. Investigation relating the pollen rain trapped in polsters to that in faecal material are continuing, and will be reported in due course. This paper is based on one given by E.C. at the 5th International Aerobiology Conference, held at Bangalore, India, August 1994.     

Collodion membrane secures cells sorted by flow cytofluorometry onto microscope slides

Quantitative microscopic cytology of cells previously sorted by flow cytofluorometry has been hindered by the loss of cells from the microscope slide during staining procedures. The simple application of a semi-permeable membrane of collodion over fixed or unfixed cells sorted directly onto a microscope slide secured virtually 100% of the cells onto the slide. Cells covered with the collodion membrane studied with Papanicolaou's stain as well as routine clinical cervical cytologic preparations. In contrast, fewer than one half of the cells sorted onto uncoated or albumin coated slides were retained after staining.