Jan 28, 2025
Environmental Sampling of Freshwater Microbial Communities
- Mary Coates1,
- Alberto Scarampi1,
- Orkun Soyer1
- 1School of Life Sciences, Gibbet Hill Campus, University of Warwick, CV4 7AL, UK
- OSS Lab
Protocol Citation: Mary Coates, Alberto Scarampi, Orkun Soyer 2025. Environmental Sampling of Freshwater Microbial Communities. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldrqnxg5b/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: January 09, 2025
Last Modified: January 28, 2025
Protocol Integer ID: 118459
Keywords: Freshwater microbial communities, Environmental water samples, Water filtration
Funders Acknowledgements:
Gordon and Betty Moore Foundation
Grant ID: GBMF9200
Abstract
This protocol covers sample collection and analysis of microbial composition from lake water.
Guidelines
Associated protocols / media sheets:
OSP_37: ‘Culturing and Long-term preservation of Cyanobacterial Communities from Environmental Samples’
Qiagen PowerWater kit User Guide:
Method in a nutshell
Environmental water samples are collected to either analyze the microbial composition present or to set up laboratory cultures from. In either case, the samples are first passed through a sieve to remove large debris, then filtered through Miracloth to remove finer debris. This filtrate can be used to set up laboratory cultures and/or further filtered through a 3μm, and then a 0.22μm filter membrane. These filters can then be used directly for DNA extraction and the microbial composition analyzed using metagenomics. The two filters can be combined or analyzed separately.
Materials
• Van Dorn 4L sampling bottle with Bronze quick-opening messenger MB (450g) and sampling line (ABT-VD-04, Aquatic Biotechnology) – useful for sampling from a specific depth. For surface sampling, we have also successfully used plastic 5L bottles with larger mouth opening.
• Container, wide neckVWR International (Avantor)Catalog #BURK1406-1005
• 190μm sieve (nylon strainer, Inntech)
• Filtration unit for coarse stage (funnel with two layers of autoclaved Miracloth MiraclothMerck Millipore (EMD Millipore)Catalog #475855
• Filtration unit for fine stage, MILLICUP™-FLEX, 47mm Disposable Filtration Kit; Nonsterile, Solvent-Resistant Starter KitMerck MilliporeSigma (Sigma-Aldrich)Catalog #MCFLX4702
• 3μm filter membrane, autoclaved (Mixed cellulose esters, 47mm diameter, MilliporeSigma)
• 0.22μm filter membrane, autoclaved (Mixed cellulose esters, 47mm diameter, MilliporeSigma)
• Screw cap tube, 50 ml, (LxØ): 114 x 28 mm, PP, with printSarstedtCatalog #62.547.254
• DNeasy PowerWater Kit (50)QiagenCatalog #14900-50-NF
• 5L glass bottle (Duran) x 4, autoclaved (for collecting Miracloth filtered filtrate)
• 1L pressure resistant glass bottle (Duran pressure plus+, VWR 215-0042) x 4 (to use with Millicup-Flex)Laboratory bottles, pressure plus+, narrow neck, with thread GL 45, DURAN®VWR International (Avantor)Catalog #215-0042
• Sterile forceps for handling filter membranes
• Vacuum pump (add details)
• Environmental sensors:
- PAR light meter
- pH strips
- thermometer
- Multi-parameter sonde
- Other, as available/desired.
Step-by-Step Method
Step-by-Step Method
5L water containers are detergent/ethanol (or acid) washed prior to use and labeled with date and sample ID.
Note
Collected water volumes will depend on the project and site. For microbial focus, we have successfully collected more than enough DNA from 2-5L volumes, collected from reservoir and lake sites.
For each sample point, take a picture of it, and record environmental parameters as possible/needed.
Note
We tend to use either a multi-parameter Sonde to take measurements of nitrate, pH, light, and temperature, in a depth-resolved manner, or use pH strips, light meter, and thermometer to take surface measurements. In the latter case, the pH strip is submerged in the water collected, whilst the thermometer and light meter are submerged at the same depth where the sample was collected.
Depending on sampling site, the water containers can be directly used to collect ~2-4L surface water sample.
Alternatively, a sieve, wooden spatula, or Falcon tube can be used to collect visible cyanobacterial granules or mats directly together with some water from the same site.
Alternatively, a Van Dorn sampling container together with the bronze quick-opening messenger, can be used to collect water samples from specific depths.
The collected water samples are immediately poured through a sieve into a clean 5L collection bottle to remove large debris such as sticks and feathers.
Keep the samples cool by placing On ice or surround with ice packs and transport to the laboratory for further processing. This is to discourage any change to the microbial populations.
Note
Depending on travel distances involved or scientific questions asked, transport on ice might not be needed necessarily.
In the laboratory, each water sample is run through two layers of autoclaved Miracloth, using a funnel and sterilized bottle – autoclaved or ethanol washed - to collect the filtrate. The step removes large eukaryotic fraction and debris >20μm.
Note
Lab cultures can be initiated from this filtrate by mixing 50:50 with media – see protocol OSP37 ‘Culturing and Long-term preservation of Cyanobacterial Communities from Environmental Samples’
Each water sample from step 5 is then pulled through a stacked filter system, using 3μm and 0.22μm filter membranes to separate the microbial fraction from the filtrate and collect them on the filter membranes. To do this we use the MilliCup flex system, 47mm diameter, mixed cellulose esters (MCE) filter membranes and a vacuum pump (see figure 1).
Screw the membrane support collar onto the 1L pressure resistant, sterilized collection bottle.
Place a 0.22μm MCE filter membrane that has been pre-wetted in sterile distilled water, on the collar. Directly on top of this, place a pre-wetted 3μm MCE filter membrane.
Click on the Millicup Flex funnel and attach the vacuum pump to the vacuum port on the support collar. It is a good idea to check the integrity of your filter membranes at this point and that a good vacuum will form by turning on your vacuum pump and checking that the filters are sucked onto the collar, without wrinkles or tears.
Add your water sample from step 5 until up to 1L has been filtered.
Note
The top filter may clog quickly if the sample is particularly turbid. A fresh 3µm filter membrane can be switched in when the funnel is empty of sample. Save the removed filter membrane by placing in a 50 ml or 15 ml falcon tube using sterile forceps.
Repeat step 7.4 until the desired amount of sample has been filtered e.g. we split a 4L sample into 4 reps, usually filtering 400-500ml per rep.
Note
In our experience, a filter clogging or changing color visibly is a good sign that possibly enough microbial material has been collected. Filtering could be stopped at that point.
Place the filter membranes at -80 °C to preserve the microbial material.
Using a pair of sterile forceps, roll the filter membrane so that the collected microbial material is facing inwards (i.e. not touching the plastic walls of the Falcon tube), and place inside a 15ml/50 ml Falcon tube. Remember to keep the 3μm and 0.22μm filter membranes separate for downstream analysis.
Note
- The filtrate from go to step #6 can be kept for chemical/viral analysis.
- Other bottle top filter systems have successfully been used e.g. Sartorius, Nalgene, which can be reused, but for consistent successful filtering, the Millicup flex system was preferred.
Figure 1: Water filtration set-up using the Millicup Flex system and a vacuum pump.
Discard any remaining filtrate or sample according to your local rules e.g. down the sink/autoclaved/sterilized.
For DNA extraction of the microbial material from the membrane filters, we use the Qiagen DNeasy PowerWater Kit, following the standard protocol and including the additional heating at .
1-2 filter membranes (reps from same original sample) can be placed into the 0ml bead tube directly from the -80 °C freezer.
We elute in 50 µL Solution EB (elution buffer from the kit).
DNA samples are verified by nanodrop and sent for shotgun metagenomic sequencing.
Acknowledgements
We thank Richard Puxty and Pedro Yeves Cabello for their initial input to the sampling approach that resulted in this protocol and Marco Polin for trialling the protocol with us.