Daphne Thompson
Grade 11 | Dunmore, Alberta
INTRODUCTION
Fungi are a kingdom that can be divided into five Phylums: Chytridiomycota, Zygomycota, Ascomycota, Glomeromycota, and Basidiomycota (Malloch, D. (1981). Moulds: their isolation, cultivation, and identification. University of Toronto Press.). Mould is a broad term for many species, but all mould is made up of hyphae. Hyphae absorb nutrients from their surroundings, transport nutrients, and produce spores. Specific classifications of mould can be made according to what kind of spores are present as well as the appearance and formations of the hyphae (Kaiser, G. E. (2020, September 7). Lab 10: Fungi, Part 2 - The Molds. Biology LibreTexts).
“Snow mould” becomes a problem as snow melts, leaving a damp and warm environment for mould to thrive. When snow melts quickly, the hyphae don’t have time to lose water, resulting in them freezing internally (Kiriaki, M. et al (2003, December). Antifreeze proteins from snow mold fungi. Canadian Journal of Botany. 81(12): 1175-1181). Not only does melting snow provide an ideal environment for the spores of the mould to reproduce and the hyphae to grow, it can also be a carrier of spores. Snow can offer insulation and protection to the spores from external forces. Despite possibly being an insulator, snow also creates a colder environment that prevents the spores from spreading (Malloch, D. Discussion of Winter Fungi. Winter Fungi.). However, different research suggests that the hyphae themselves of some moulds may contain an antifreeze protein that allows the spores to survive at colder temperatures (0°C≥) by absorbing ice crystals (Kiriaki, M. et al (2003, December). Antifreeze proteins from snow mold fungi. Canadian Journal of Botany. 81(12): 1175-1181). Overall, spores have been proven to survive at a variety of temperatures, including below 0°C, but more research needs to be done to determine how.
Given the presence of spores and mould in snow, individuals that suffer from allergies and asthma may react adversely upon exposure. Asthma is a condition where the airways will swell and extra mucus is produced. This can cause breathing difficulties, and ranges in severity. Exercise, allergies, and other irritants can cause asthma to flare (Mayo Clinic Staff. (2020, August 11). Asthma. Mayo Clinic.).
Many studies have already been done, but none were able to prove that prolonged exposure to mold causes asthma or any other chronic respiratory illness (Soloway, R. Mold 101: Effects on Human Health. Mold 101 Effects on Human Health.). While correlations have been identified, there is a lack of evidence speaking to any direct impact that snow mould has on these individuals (Dales, R. et al Residential fungal growth and incidence of acute respiratory illness during the first two years of life. Environmental Research. 2010;110:692-698). Additionally, it is reported that mould spores released when the snow melts agitate asthmatic symptoms (Soloway, R. Mold 101: Effects on Human Health. Mold 101 Effects on Human Health.). Understanding how the spores interact with the snow and the environment can lead to new information that will increase knowledge about asthmatics’ reactions to the environment.
METHOD
Snow was scooped into a new Ziploc bag using a spoon that was sterilized with heat. The Ziploc bags were labelled in numerical order. Variables including from where snow was taken, temperature, and the date of the last snowfall were recorded. Two different medias were made to grow snow samples on, Czapek and Bacto Potato Dextrose Agar media, created using"Difco and BBL: Manual of Microbiological Culture Media.". The collected snow sat in a fridge and was melted to liquid before it was filtered. 10ml of melted snow was filtered through a filter tower with a 0.45μl filter. The filter was then placed on a petri dish with the media. Some samples were also used to make spread plates, but this allowed for excessive bacterial growth and made assessment hard. In early experimental trials, 100ml was filtered but that also lead to an overgrowth of fungi, which was difficult to count. The samples were incubated on the petri dishes for several days at room temperature. Afterwards, individual fungi colonies were counted under a microscope. To further observe what kind of growth was on the plates, slides were prepared from different samples. Tape mounts were used by gently pressing clear tape onto the desired colony. The tape with a colony was placed on a slide, a few drops of oil was added, and cover slips were applied on top of the oil. Using a Zeiss Axioskop microscope equipped with phase objectives and guides, the genus was identified. These identifications were verified by a microbiologist. When all the data was collected, samples were placed in an infectious waste bin that was autoclaved to sterilize it. If the samples sat too long, they would grow out of the petri dish.
RESULTS
In early experimental trials, the Czapek media grew more bacteria than mold. The potato dextrose media grew more mold and less bacteria, therefore, the potato dextrose media was used. The spread plates primarily grew bacteria, not fungi (Figure 1).
A large variety and quantity of fungi was found. The two highest counts were snow samples 8 and 13 (Table 1). Both samples were taken near two different rivers with trees nearby.
Using guides and a microscope, Penicillium, Rhizopus, and Stachybotrys were identified (Figures 2-4). Other types of fungi were found but could not be identified.
Table 1: Colony growth from snow samples.
Figure 1: The two plates on the left were potato dextrose media, and the two plates on the right were Czapek media. The two bottom plates were spread plates, while the top two were filtered. The sample on all four plates was the same sample.
Figure 2: Penicillium was identified by the branches and the circles at the end, the conidia.
Figure 3: Rhizopus was identified by the sacs called sporangium.
Figure 4: Stachybotrys was identified by broccoli-like figures.
DISCUSSION
Although quantitative results in my experiment were limited due to COVID-19-related scheduling interruptions, it is unsurprising that there were a high number of fungi in snow because melting snow and the resulting release of microorganisms is known to irritate people with mold allergies or asthma. Research has suggested that many different factors may contribute to some spores unusual tolerance to temperatures below 0°C. Further work should be put into correlating the results with the environment so that there is more specific insight on the tolerance of spores in different environmental settings. To investigate whether the spores came from vegetation beneath the snow or if the spores were already present in the snow, taking ground samples may provide additional insight. Fungal spores in rain have proven to agitate asthmatics during thunderstorms (W. Anderson, et al, Asthma admissions and thunderstorms: a study of pollen, fungal spores, rainfall, and ozone, QJM: An International Journal of Medicine, Volume 94, Issue 8, August 2001, Pages 429–433, https://doi.org/10.1093/qjmed/94.8.429), so additional work could be conducted into how the spores interact with other weather events, such as rain. Any additional information about how spores interact with the environment may be beneficial.
ACKNOWLEDGEMENTS
Thank you to Dr. Wallis and Hyperion Research for helping me and giving me access to resources. Thank you to my mom for driving me.
REFERENCES
Dales, R. et al Residential fungal growth and incidence of acute respiratory illness during the first two years of life. Environmental Research. 2010;110:692-698
Geunhwa Jung, Ph.D. Seog Won Chang, Ph.D. Young-Ki Jo, Ph.D. A Fresh Look at Fungicides for Snow Mold Control
Jackson Kung'u, Penecillium, Mold and Bacteria consulting Laboratories
Kaiser, G. E. (2020, September 7). Lab 10: Fungi, Part 2 - The Molds. Biology LibreTexts. https://bio.libretexts.org/Bookshelves/Ancillary_Materials/Laboratory_Experiments/Microbiology_Labs/Microbiology_Labs_II/Lab_10:_Fungi,_Part_2_-_The_Molds.
Kiriaki, M. et al (2003, December). Antifreeze proteins from snow mold fungi. Canadian Journal of Botany. 81(12): 1175-1181
Malloch, D. (1981). Moulds: their isolation, cultivation, and identification. University of Toronto Press.
Malloch, D. Discussion of Winter Fungi. Winter Fungi. http://website.nbm-mnb.ca/mycologywebpages/Checklists/WinterFungi/WinterFungiDiscussion.html.
(Mayo Clinic Staff. (2020, August 11). Asthma. Mayo Clinic. https://www.mayoclinic.org/diseases-conditions/asthma/symptoms-causes/syc-20369653.)
Soloway, R. Mold 101: Effects on Human Health. Mold 101 Effects on Human Health. https://www.poison.org/articles/2011-oct/mold-101-effects-on-human-health.
Richard, A. Humber. Fungi: Identification Ch. 5-1
W. Anderson, G.J. Prescott, S. Packham, J. Mullins, M. Brookes, A. Seaton, (2001, August). Asthma admissions and thunderstorms: a study of pollen, fungal spores, rainfall, and ozone, QJM: An International Journal of Medicine, Volume 94, Issue 8, Pages 429–433, https://doi.org/10.1093/qjmed/94.8.429)
Zimbro, Mary Jo.Power, David A. (Eds.) (2003) Difco & BBL manual :manual of microbiological culture media Sparks, MD: Difco Laboratories, Division of Becton Dickinson and Co.
ABOUT THE AUTHOR
Daphne Thompson
Daphne is a grade 11 student attending Eagle Butte High School in Dunmore, Alberta. She enjoys learning about anything and everything but takes a special interest in the sciences. Outside of academics, Daphne uses her time to read, practice the violin, and participate in sports such as snowboarding and cross country running.