Jun 01, 2023
Abundance of fungal hyphae in seawater by epifluorescence microscopy using Calcofluor White stain method
- M.H. Gutiérrez1
- 1COPAS Sur-Austral, Universidad de Concepción, Chile
Protocol Citation: M.H. Gutiérrez 2023. Abundance of fungal hyphae in seawater by epifluorescence microscopy using Calcofluor White stain method. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq933klk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 02, 2018
Last Modified: June 01, 2023
Protocol Integer ID: 17442
Abstract
Protocol to stain fungal hyphae from seawater with Calcofluor White.
Attachments
Guidelines
References
Damare S, Raghukumar C (2008) Fungi and macroaggregation in deep-sea sediments. Microb Ecol 56:168–177
Jebarag CS, Raghukumar C (2009) Anaerobic denitrification in fungi from the coastal marine sediments off Goa, India. Mycol Res 113:100-109
Newell, S.Y., Statzell-Tallman, A. (1982) Factors for conversion of fungal biovolume values to biomass, carbon and nitrogen: variation with mycelial ages, growth conditions, and strains of fungi from a salt marsh. Oikos 39: 261-268
Rasconi S, Jobard M, Jouve L, Sime-Ngando T (2009) Use of calcofluor white for detection, identification, and quantification of phytoplanktonic fungal parasites. Appl Environ Microbiol 75:2545-2553
Safety warnings
See SDS (Safety Data Sheet) for hazards and safety guidelines.
Collection
Collection
Collect samples in sterile 50 mL polypropylene tubes and fix with formaldehyde or glutaraldehyde (2% final concentration). Store samples at 4 °C in the dark.
Filtration
Filtration
Filter 5 mL to 30 mL (depending on the environment) of seawater by 0.22-µm mesh black 25 mm diameter polycarbonate filters (Millipore Corp.).
Stain
Stain
Stain filters with the retained material from filtration by applying 600 µL of aqueous 0.1% Calcofluor White directly to sample and filter, making sure to cover the entire area of the filter.
Allow 00:05:00 to 00:10:00 then use vacuum to remove the excess stain from the filter. Avoid any light source.
Abundance
Abundance
Place the filter (sample side up) onto a slide and add 1 drop of non-fluorescent immersion oil to the top of the filter, then cover with a cover slip.
Count slides immediately on epifluorescent microscope or store at -20 °C .
Use an epifluorescence microscope equipped with UV filter (used for DAPI, eg. filter set 49 Carl Zeiss Ltd., 365 nm excitation and 445-450 nm emission band pass) to examine at 1000X the entire effective area of the filters.
Count all hyphae identified and record their length and width. Use cylinder volume as a morphological approximation to estimate the biovolume of fungal filaments.
Note
Biomass can be estimated based from biomass: biovolume ratios described from fungi (Newell and Statzell-Tallman, 1982).