1. FUNGAL SPORES Michel Thibaudon, Samuel Monnier, Julien Clertant
In collaboration with Bernard CLOT and with slides from
Dr. M. Muilenberg, Cambridge U., USA
Dr. D. Job, U. of Neuchâtel, CH
Inspired from « Mycology of the air »
Michel Thibaudon – March 2018

2. Disclosure
In relation to this presentation, I declare the following, real or perceived conflicts of interest:
Type
Company
Employment
full time / part time
Resau National de Surveillance Aérobiologique
Spouse / Family member
employment / engagement
None
Research Grant (P.I., collaborator or consultant; pending and received grants)
Other research support
Speakers Bureau / Honoraria
Ownership interest (stock, stock-options, patent or intellectual property)
Consultant / advisory board
A conflict of interest is any situation in which a speaker or immediate family members have interests, and those may cause a conflict with the current presentation.
Conflicts of interest do not preclude the delivery of the talk, but should be explicitly declared. These may include financial interests (e.g. owning stocks of a related company, having received honoraria, consultancy fees), research interests (research support by grants or otherwise), organisational interests and gifts.

3. Michel Thibaudon – March 2018
Classification
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6. Michel Thibaudon – March 2018
Eukaryotes: 5 kingdoms
MONERES
PROTISTS
PLANTS
FUNGI
ANIMALS
Fungi: eukaryotes, heterotrophic, filamentous (hyphae  mycelium), cell wall made from chitin, sexual and asexual reproduction by spores.
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Fungi
~ species, or more
~ species described
Saprophytic or parasitic
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CLASSIFICATION
Domain
Kingdom
Divisions
Subdivisions
Basidiomycetes
Eumycètes
Eucaryote
Fungi or
Mycetes
Ascomycetes
Myxomycètes ??
Deuteromycetes
Zygomycetes
Mastigomycetes
Michel Thibaudon – March 2018

10. Fungal spores primary functions:
to tide over unfavorable periods
to disperse the fungus to new food sources
(meiospores) genetic recombination
Fungal spores take-off is often an active mechanism
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11. Michel Thibaudon – March 2018
Basidomycetes
Basid  4 basidiospores meiospores, exospores
never septate
never branched
bilateral symetry
apiculus (attachment point)
usually short (lenght / width ratio smaller than 4:1)
sometimes a pore
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12. Basid and basidiospores
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13. Basid and basidiospores
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Ascomycetes
Ascus  8 ascospores meiospores, endospores
septate or not
groups of 8 (sometimes)
often long (more than 4 times longer than wide)
never birth scars
never germination pores
never branched
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16. Ascospores No attachment points; variable size, shape, septation
B.Kendrick drawing
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17. Michel Thibaudon – March 2018
asci
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18. (Deuteromycetes) = (Fungi imperfecti) = Mitosporic fungi (~moulds)
mitospores = conidia (sing. conidium) (asexual)
produced at the end of specialised hyphae
always a central birth scar
frequently septate
can be very large (or very small)
can form chains
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mitosporic fungi often responsable of:
biodeterioration
biotechnology (enzymes, hormones, etc production)
food industry (beer, wine, cheese, …)
pharmaceutical (allergen extract., antibiotic prod., …)
mycoses
mycotoxins
allergies
infections (A. fumigatus)
sick building syndrom often associated with moisture and molds
plant pathogens
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20. Michel Thibaudon – March 2018
Fungal allergens
not enough data and research
one problem: lack of standardized extracts
well known:
Cladosporium (in particular children)
Alternaria (and other cross-reactive)
Stachybotris (indoor, big story in the USA)
occupational allergies in the mushroom industry (eg Pleurotus)
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22. How to collect fungal spores Material and method2
How to collect fungal spores Material and method
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23. Today, we will discuss two very important topics in aerobiology:
viable spores sampling, problem of the substrate (growing medium) …
indoor and outdoor aerobiology
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Outdoor fungal spores: Hirst method - Continuous detection
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25. vocal recognition system1 1
Outdoor fungal spores: Hirst method - Continuous detection
Dividing the band
into daily sections
1 slide by day
Counting with a
vocal recognition system
Collection of the
drum band
spores count
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26. 1 Cyclonic method Coriolis Specific or non-inert particles
optical
microscopy
Coriolis
Coloration / filtration
Easy and rapid method
Air
Liquid
Particles
300 l/min
3 m3 in 10 min
Liquid sample for rapid analysis
PCR
Method for specific identification
Chemscan
Transfert of biological particles from the air to a collect LIQUID medium BROADBAND
LAL
Endotoxins
Mycotoxins
Method with high sensibility
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Impaction on culture medium
Culture
Culture
Culture
Be careful to the quality of culture medium
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28. 3 Filtration method Incubation
Transfer gelatin membrane on petri dish
Air filtration on dense gelatin membrane
Incubation
Be careful to the quality of culture medium
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Continious impaction : pollens and molds trap : LANZONI VPPS 2000
Impaction on culture medium : MAS 100
Filtration on gelatin membrane : AIRScan
Cyclonic sampler : Coriolis®µ
Equipment used for this study:
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Analysis methods used:
Culture medium :
Trypto casein soy,
Sabouraud
MEA
Incubation temperatures between 20°C and 25°C.
Identifications :
Macroscopic
Microscopic by transfer on transparents supports, coloration and placed on glass slides.
Numeration realised when it is possible (invasions limiting quality counts in some samples)
Michel Thibaudon – March 2018

31. 4 Analysis methods used: Detection method by optical microscopy
(for liquid samples from cyclonic sampler and gelatin membranes membranes gélatines whole or fractionned) :
Analysis by optical microscopy, magnification x400 4
Analysis methods used:
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Spore Identification Important Characteristics
Color
Size
Shape
Septation
Attachments
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Study period:
Sampling from 2010 to 2012 in very different environments (during 3 years):
Indoor air
Industries facilitise
Offices
Hotel
House
Laboratories
Clean room
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Results
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35. 3 Results Moulds Hirst trap MICROSCOPIC OBSERVATION
Impaction / filtration on dense gelatin membrane
Culture on :
Cyclonic trap/ filtration on dense gelatin membrane
MICROSCOPIC OBSERVATION
TSA
Sabouraud
MEA
Absidia + ++
+++
Acremonium
Alternaria
Ascospores
Aspergillacea
Aspergillus
Penicillium
Aureobasidium
Basidiospores
Botrytis
Cercospora
Chaetomium
Cladosporium
Didymella
Entomophthora
Epicoccum
Erysiphe
Fusarium
Fusicladium
Helicomyces
Helminthosporium
Mucorales
Myxomycetes
Easy detection
Possibility of detection
No possibility of detection
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+++ ++ +

36. 3 4 Results Moulds Hirst trap MICROSCOPIC OBSERVATION
Impaction / filtration on dense gelatin membrane
Culture on :
Cyclonic trap/ filtration on dense gelatin membrane
MICROSCOPIC OBSERVATION
TSA
Sabouraud
MEA
Peronospora
+++
Pithomyces + ++
Pleospora
Polythrincium
Sporidesmium
Sporobolomyces
Stemphylium
Stachybotrys
Taeniolella
Tilletiopsis
Torula
Trichoderma
Trichothecium
Uredospores
Ustilago
Easy detection
Possibility of detection
No possibility of detection
Michel Thibaudon – March 2018
+++ ++ +

37. 3 4 Results Air contamination Culture Optical microscopy detection
Spores small quantity ++
+++
Detection +
Counting
Identification
Spores high quantity
Détection
++ (invasion problem)
Mycelium small quantity
Mycelium high quantity
Easy detection
Possibility of detection
No possibility of detection
Michel Thibaudon – March 2018
+++ ++ +

38. Michel Thibaudon – March 20183 4
Results
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39. Michel Thibaudon – March 20183 4
Results
Moulds
Infectious impacts
Allergic impacts
Alveolit
Toxic impacts
Absidia corymbifera
YES
Acremonium
NO DATA
Alternaria alternata
Aspergillus flavus
Aspergillus fumigatus
Aspergillus glaucus
Aspergillus niger
Aspergillus versicolor
Aureobasidium
Chaetomium
Cladosporium sphaerospermum
Epicoccum
Fusarium
Mucorales
Penicillium sp.
Stachybotrys chartarum
Trichoderma
Trichothecium
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40. Michel Thibaudon – March 20183 4
Results
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41. Michel Thibaudon – March 20183 4
Results
Outdoor fungal spores 2017
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42. Michel Thibaudon – March 20183 4
Results
Outdoor fungal spores 2017
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43. Michel Thibaudon – March 20183 4
Results
Outdoor fungal spores 2017
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44. Michel Thibaudon – March 20183 4
Results
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45. Example of recommendation4
Example of recommendation
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46. Michel Thibaudon – March 20183 4
Results
We must destroy the wall
Pollens
Moisissures
Bactéries
Virus
Air intérieur
Extérieur
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47. Michel Thibaudon – March 20184 4
Seasonality and risk of contamination
Implentation of
Action plan
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Conclusion
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49. Michel Thibaudon – March 2018
All methods of sampling and analysis based on detection thanks to culture of molds viable and culturable, leave aside a considerable number of non- culturable fungi.
It is important to associate classic analysis culture and methods of analysis by direct detection (optical microscopy) and qualitative methods (PCR…)
The future development of standards, including the ISO 14698, must consider these issues on the mastery of fungal contamination in cleanrooms
Michel Thibaudon – March 2018

50. Thanks for your attention
Happy end
Thanks for your attention
Michel Thibaudon – March 2018