Professional Documents
Culture Documents
7 - 11 2013, Baltimore
Set up a sampling station to collect airborne pollen and fungal spore Recognize the most common types of pollen found in the atmosphere Recognize the most common types of fungal spores found in the atmosphere
No conflicts to disclose
Aerobiological Sampling
Sampling plan or objective Choosing samplers: Rotorod, Burkard Spore Trap, Lanzoni, Allergenco One day head or 7-day head for Burkard or Lanzoni Location Preparing the samples Slide Analysis and Identification Data Analysis
Sampling Objective
Time commitment
Rotorod Samplers
Rotorod Samplers
Models most often used by allergists have retracting rods for intermittent operation Standard is 10% sampling time Head rotates at 2400 rpm Leading edge of rod coated with grease Pollen and spores impacted on greased surface Efficient for pollen and spores >10 m
Rotorod Samplers
Rotorod Analysis
Collector rods placed in a special adapter for microscopic examination Rods stained with Calberlas pollen stain Entire surface of each rod counted unless pollen/spore load very high (then a subset of the surface is analyzed) Atmospheric concentrations determined
Rotorod Calculations
C=N/V
C is concentration, N is the total number of pollen or spores counted on both rods, V is the volume of air sampled by the rods
Concentration = N/3.226 m3
With a 10% sampling time (144 min) V = 6.452 m3
Concentration = N/6.452 m3
Allows for greater accuracy for small fungal spores Permits analysis for diurnal rhythms
Time discrimination
Location
Roof of a building - ideal 3 to 6 stories above ground (30 to 60 ft) Not close to overhanging vegetation Air flow not obstructed by nearby buildings or other structural features
Parapet around roof requires platform to elevate the orifice above the wall
Standard is the 7-day sampling head Sampler drum mounted on 7-day clock Drum moves by orifice at 2 mm per hr Melenex tape mounted on drum and greased (Lubriseal, High Vacuum Grease, other) Air is brought in at 10 l/min and impacts on greased Melenex tape Drum changed each week
Melenex tape removed from drum Tape cut into seven 24 hour segments each 48 mm long Segments mounted on microscope slides in 10% gelvatol (polyvinyl alcohol) and dried Glycerin-jelly mounting medium added and a 50 mm cover slip Mounting medium contains pollen stain - either basic fuchsin or phenosafarin
Alternate head is the 24 hour head Standard glass microscope slide is greased and placed on the head
Slide is changed daily, carrier realigned Mounting medium with stain and coverslip are added
Analysis
Microscopy - 400X for pollen; 1000X for fungal spores Different methods of microscopic analysis are used to obtain
Average daily concentration - Single longitudinal traverse Hourly or bihourly concentrations which can then be averaged to obtain a daily average - 12 transverse traverses
Comparison of methods
12 Transverse Traverses
Quicker Produces average daily concentration Good for routine monitoring 3 or 4 longitudinal traverses can increase accuracy
Takes longer Can determine diurnal rhythm of airborne allergens All traverses can be averaged to determine average daily concentration
Conversion to Concentrations
Microscope counts are entered into a database such as Excel Formulas added to convert counts into concentrations Information needed
Field diameter of objective lens - Variable Flow rate (10 liters/minute) and exposure time (normally 24 hrs) for a total volume of air sampled of 14.4 m3
C = Concentration - pollen grains/m3 N = number of pollen counted on traverse W = Width of entire sample - 14 mm F = field diameter of objective lens - 0.48 mm V = total volume of air sampled- 14.4 m3 C = N x W/F x 1/V
Identification
AAAAI and ACAAI Aeroallergen courses Other aerobiology courses Reference slides
NAB/AAAAI Pollen Slide Library Reference slides from local specimens Consult a botanist at a local university
Identification Manuals
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Identification Manuals
Grant Smith. 2000. Sampling and Identifying Allergenic Pollens and Molds, AAAAI, Milwaukee R.O. Kapp, How to Know Pollen and Spores - originally published in 1950s - new edition Richard Weber. 1998. Pollen Identification Ann Allergy Asthma Immunol 80:1417. Lewis WH, Vinay P, Zenger VE. 1983. Airborne and Allergenic Pollen of North America. Johns Hopkins University Press, Baltimore, MD. Aeroallergen Photo Library, Steve Kagan, http://allernet.net/ Lacey M and West J. 2006. The Air Spora: A manual for catching and identifying airborne biological particles. Springer.
Essential Reference
Sample Pages
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Average daily concentrations can be graphed to look at the seasonal and yearly pollen levels Develop regional pollen calendar Data can be compared with patient symptoms, peak flow readings, office visits, emergency room visits Prepare for peak seasons - staffing, etc
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Data from several years can be averaged to produce a graph of the pollen season Smoothing techniques such as 5 day running mean can be used to generate a smoother curve and better estimate of the typical peak period
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Pollen grains/m 3
400
300
200
100
0
5Se p 22 -A ug 12 -S ep 15 -A ug 29 -A ug 19 -S ep 26 -S ep 3O ct 17 -O ct 24 -O ct 10 -O ct 31 -O ct
Five day running mean of airborne Ambrosia pollen in Tulsa: 20 year mean
Peak on or about Sept 10
500 450 400 350 300 250 200 150 100 50 0
31 -A ug 7Se p 24 -A ug 14 -S ep 17 -A ug 21 -S ep 28 -S ep 5O ct 12 -O ct 19 -O ct 26 -O ct
Conclusion
Air sampling allows the allergist to get a first hand understanding of the local aeroallergens, their concentration, and season occurrence Several years of sampling will allow for the development of a pollen calendar which can benefit the physician and his or her patients
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Set up a sampling station to collect airborne pollen and fungal spore Recognize the most common types of pollen found in the atmosphere Recognize the most common types of fungal spores found in the atmosphere
No conflicts to disclose
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Fungi are eukaryotic organisms that are neither plant nor animal
Mushrooms
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Puffballs
Bracket Fungi
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Most of the common airborne fungi are saprobes naturally occurring on leaf surfaces, decaying plant material, or in soil
Mycelium
Mycelium
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PASSIVE: Frequently related to wind speed and turbulence include members of the Dry Air Spora which peak in the afternoon ACTIVE: Generally require moisture common mechanism for ascospores and basidiospores
Basidiospores most abundant in predawn hours Ascospores most abundant during or following rain; however, a number of ascospores only require high humidity and are abundant in predawn hours
Conidium
Conidiophore Sporangiophore
Rhizopus Sporangium
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Chains of Conidia
Fungal Classification
Mycologist recognize 8 phyla in the Kingdom Fungi based phylogenetic analysis of DNA Three of these phyla contain important allergens
ZYGOMYCOTA -- Zygospores
ASCOMYCOTA -- Ascospores
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Most are members of the Ascomycota with a small percent members of the Basidiomycota Formerly called
Also called
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Ascomycota
Many members of the Ascomycota develop asci within a fruiting body Asexual spores are called conidia Fruiting bodies often called ascocarps Ascocarps can be
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Basidiomycota
The most conspicuous fungi in the environment Basidiospores are typically produced in a large fruiting body such as
Characteristic spores are basidiospores and four basidiospores are produced externally on basidia
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Basidia line the pores of bracket fungi and the gills of mushrooms
Basidiospores are small and single-celled often with an asymmetric attachment peg
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Basidiomycota also includes two groups of fungi that lack fruiting bodies Rust fungi and smut fungi Important pathogens on both native and cultivated plants
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Set up a sampling station to collect airborne pollen and fungal spore Recognize the most common types of pollen found in the atmosphere Recognize the most common types of fungal spores found in the atmosphere
No conflicts to disclose
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Spore size Spore shape Number of cells Attachment Scars Wall characteristics Spore color
SIZE: 2m to 100 mm SHAPE: Globose, elliptical, fusiform, asymmetric, lemon-shaped, barrel-shaped, curved SEPTATION: Non-septate (one cell), single septum, transverse septa, transverse and longitudinal septa, random septa, pseudoseptate
Other Characteristics
ATTACHMENTS: Attachment scars, attachment pegs APPENDAGES WALL CHARACTERISTICS: Smooth, granular, reticulate, spines, warts, wall thickness COLOR: Hyaline (colorless) to deeply pigmented
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Globose
Barrel-shaped
Non-septate
Random septa
Lemon-shaped
Club-shaped
Transverse septa
Attachment scars
Elliptical
Curved
Ornaments: spines
Cylindrical
Pseudoseptate
Appendages
Spore color
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Asexual Spores
Typically the most abundant spores in the atmosphere Asexual stage of ascomycetes Conidia often formed on specialized hyphae called conidiophores Look for attachment scars where the spores were attached to the conidiophore
Cladosporium
Cladosporium
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Alternaria
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Alternaria
Curvularia
Nigrospora Curvularia
Drechslera
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Drechslera-type spores
Drechslera-type spores
Pithomyces
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Epicoccum
Penicillium species
Produce distinctive conidiophores (spore bearing structures) Spores are usually spherical to oval and form in chains
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Aspergillus species
Produce distinctive conidiophores (spore bearing structures) Spores are usually spherical to oval and form in chains
Fusarium
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Botrytis
Oidium
Nigrospora
Nigrospora Culture
Air Sample
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Periconia
Cercospora
Polythrincium
Peronospora
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Stemphylium
Torula
Tetraploa
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Spegazzinia
Stachybotrys
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Ascospores are produced in an ascus. Eight ascospores are found in each ascus without any attachment scars
Chaetomium ascospores
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Leptosphaeria ascospores
Pleospora ascospores
Diatrypella ascospores
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Sporomiella ascospores
Venturia ascospores
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Phylum Basidiomycota
Basidiospore
Basidium
Ganoderma basidiospores
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Coprinus
Agrocybe - type
Psathyrella
Psathyrella velutina
Russula
Stropharia
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Lycoperdon
Calvatia
Pisolithus
Scleroderma
Mixed Basidiospores
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Puccinia uredospores
Puccinia teliospores
Smut Spores
Smut Spores
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