Feb 25, 2026
Version 2
Environmental Sampling of Lake Water and Biofilm for Microbial Analysis V.2
This protocol is a draft, published without a DOI.
- June Sales1,
- Hannah Benisty1
- 1Centre for Genomic Regulation
- PyriSentinel
External link: https://pyrisentinel.eu/the-project/
Protocol Citation: June Sales, Hannah Benisty 2026. Environmental Sampling of Lake Water and Biofilm for Microbial Analysis. protocols.io https://dx.doi.org/Version created by June Sales
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: In development
We are still developing and optimizing this protocol
Created: February 25, 2026
Last Modified: February 26, 2026
Protocol Integer ID: 243993
Keywords: Freshwater sampling, Lake water, Filtration, Biofilm, Sampling, Environmental microbiology, Environmental sampling, Lake, Pyrenees, PyriSentinel, environmental sampling of lake water, sampling of benthic biofilm, biofilm sample, biofilm for microbial analysis, benthic biofilm, microbial analysis, lake water, collection of water fraction, submerged stone, standardized sampling procedure, environmental sampling of lake water, benthic biofilm sample, biofilm for microbial analysis, benthic biofilm, water sample, microbial analysis, environmental sampling, biofilm, lakes across the pyrenee, lake water, lake, standardized field sampling procedure, comparable sample collection, submerged stone
Funders Acknowledgements:
Interreg VI-A Spain-France-Andorra Program (POCTEFA 2021-2027)
Grant ID: PyriSentinel - EFA059/01
Abstract
This protocol describes the standardized field sampling procedure implemented in the PyriSentinel project. In summer 2024, water and benthic biofilm samples were collected from 300 lakes across the Pyrenees. Water samples were sequentially filtered through 5 µm and 0.22 µm filters, and benthic biofilm was sampled from submerged stones, allowing reproducible and comparable sample collection under uniform field conditions.
Attachments
Materials
(x2) Sylphium 0.22 µm filters (filter + syringe with preservation buffer + caps)
(x1) Sylphium 5 µm filters (filter + syringe with preservation buffer + caps)
(x2) 1-liter graduated cylinders
(x2) Kitasato flask
Caps
Connector tubing
Filtration pump
Pressure gauge
Battery
Funnel
53 µm mesh
20 mL syringe
White tray
Scraping brush
Sterile plastic Pasteur pipette
(x2) 4 mL cryovials (each containing 1 mL of lysis buffer)
(x1) 15 mL Falcon tube (containing 2 drops of Lugol's solution)
Spare filters, caps, and syringes
Safety warnings
Ensure that the sampling area is left clean and free of any waste or materials brought to the site. Do not leave behind consumables, packaging, or any other residues. All equipment, used materials, and generated waste must be collected and removed to preserve the integrity of the natural environment.
Before start
Before heading to the field site, ensure that all required materials and equipment are prepared and fully functional. Verify that the filtration system, including the filtration pump, has fully charged batteries and is operating correctly. Confirm that all consumables and storage materials—such as filters (0.22 μm and 5 μm), sample containers, and labeling supplies—are available, organized, and ready for use.
Additionally, review and complete the lake information sheet for each sampling site, making sure that all relevant fields have been accurately filled in prior to departure.
0.22 µm filters
Upon arrival at the lake, identify a safe and accessible area from which to collect water samples. The sampling site should allow easy access to the water without posing any physical risk to the operator. Avoid areas with excessive vegetation, debris, or other materials that may interfere with sample integrity or represent potential sources of contamination.
Before beginning, put on disposable gloves to prevent sample contamination. Unpack and organize all necessary materials (e.g., sampling bottles, tubes, flasks, and kitasatos). Briefly rinse each item with lake water at the sampling site to equilibrate the equipment with the local microbial community and minimize external microbial interference.
Assemble the filtration system as illustrated in the following figure. Ensure all components are properly connected and secured to prevent leaks or sample loss during the filtration process. Make sure that the direction of water flow will follow the same direction as the arrow marked on the 0.22 µm filter.
With the 0.22 µm filter already connected to the tubing system, without the cap, and placed inside the graduated cylinder, position a funnel on top of the cylinder. Insert the mesh filter inside the funnel. Fill the sampling bottle with lake water and pour it through the mesh into the cylinder. Continue adding water until the cylinder reaches a total volume of 1 liter.
Note
The mesh pre-filter helps retain large particles and debris, ensuring that only the appropriate size fraction enters the 0.22 µm filtration system and reducing the risk of clogging during subsequent filtration steps.
With the filter fully submerged in the water, start suction with the vacuum pump to draw the water through the filter. Monitor the pressure gauge to ensure that the pressure applied remains constant throughout the filtration process.
Note
Important: Keep the filter continuously submerged during the entire filtration process to prevent air from entering and to avoid the membrane drying out. Allowing the filter to dry can impair its performance and may compromise the filtration efficiency.
Continue refilling the graduated cylinder with pre-filtered lake water (i.e., water passed through the mesh) as it empties during filtration. It is important to continuously monitor the total volume of water that passes through the filter. Add water gradually until the filter becomes saturated, which will be evident when the flow rate decreases and it becomes increasingly difficult for the water to pass through.
After completing the filtration, record the total volume of water that has passed through the filter on the sampling sheet.
Remove the filter from the graduated cylinder and attach a 20 mL syringe filled with air to expel any remaining water from the filter. Close the outlet end of the filter, and connect the syringe containing the preservation buffer to the other end. Introduce the buffer (following the direction of the arrow) into the filter and seal it with the remaining cap.
Safety information
Caution: When adding the preservation buffer, avoid applying excessive pressure. If the outlet cap is fully tightened, pressure can build inside the filter and cause the buffer to splash back toward the operator. To reduce this risk, keep the outlet cap slightly loosened while injecting the buffer so that excess pressure can be released safely.
Make sure the filter is properly labeled, store it in an identified bag, and repeat the same process with another 0.22 µm filter, which will serve as the biological replicate.
5 µm filter
For the 5 µm filters, repeat the same procedure as for the 0.22 µm filters, replacing all materials with clean ones. During filtration with this pore size, the mesh pre-filter should not be used, as the larger pore size prevents the filter from clogging easily. A larger volume of water will be filtered with this membrane. No biological replicate will be taken for this type of filter.
Benthos
Select a set of stones with biofilm located more than 1 m from the shoreline and at a depth greater than 20 cm. The sampling area should be approximately 40 x 40 cm.
Place each stone in the plastic tray and scrape the entire colonized surface with the scraping brush. Add a small amount of water (approximately 20 mL) to assist with the scraping.
Replace the stone and repeat the same procedure with the remaining stones.
At the bottom of the tray, all scraped material should be collected in the 20 mL of water. Using a Pasteur pipette, take 3 mL of the previously homogenized sample and transfer it into the cryovial containing the LB. Cap the vial and mix the sample thoroughly.
Pour the remaining material into a 15 mL Falcon tube containing Lugol's solution. Cap the tube and mix the sample thoroughly.