Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

The overall goal of this procedure is to collect and process environmental samples in remote field locations to assess key biochemical background parameters and aquatic ecosystems. This is accomplished by first collecting adequate volumes of uncompromised sampling water from the respective locations. The second step is to set up filtration or concentration systems that allow for an efficient workflow while avoiding any extra handling steps of the sampling container as additional handling always yields potential for contamination.

Next, each sample is filtered or concentrated depending on the parameters targeted. The final step is to process or fix each sample according to the requirements of the respective analyte. Ultimately, microscopy is used to directly count viral and microbial abundance and microbial biomass and to verify the quality of the viral and microbial metagenomic samples.

This method provides the tools to assess key parameters necessary to get the minimum and information essential to characterize and understand viral and microbial community dynamics. The main advantage of this technique of existing methods is that it provides a comprehensive field tested workflow, which is independent of laboratory surroundings. Demonstrating the procedure will be Emma Church, a student in the row lab.

The collection of samples is not shown in this video, but is described in the accompanying protocol text process. The samples in the order shown here, starting with dissolved organic carbon or DOC to minimize the chance of contamination, mount one of the two previously collected hatay kin units and the respective slot of the filtration set. Connect the outlet tubing which leads to the respective filter holder.

Connect the pressurized air tubing to the kin unit. Flush all lines with 100 milliliters of sample water. Then use acid washed forceps to place a 25 millimeter pre busted GFF filter in each filter holder.

Rinse each DOC high density polyethylene bottle and cap three times with about 20 milliliters of filtered sampling water. Fill each DOC bottle with about 40 milliliters of sampling water. Collect at least duplicates of DOC samples to have a backup in case of potential contamination or loss during transport.

Freeze sampling bottles standing upright at negative 20 degrees Celsius until analysis after the DOC samples have been collected. Continue filtering until a total of 500 milliliters have passed a GFF filter, including what passed through. While collecting the DOC samples, unscrew the inline filter holder.

Use forceps to remove the filter for particulate organic matter or POM analysis and place the filter inside a square of pre busted aluminum foil. Fold the top side turned in on itself and wrap. Freeze the filters at minus 20 degrees Celsius for storage until subjected to elemental and isotopic analysis to process inorganic nutrients.

Place a 0.2 micrometer track etched filter into each filter holder. Reattach the filter cassettes, rens each 20 milliliter plastic scintillation bottle three times with sampling water. Fill each bottle to the shoulder.

Freeze at negative 20 degrees Celsius until later.Analysis. To prepare samples for microscopy, take off the filter holder and collect one milliliter of water in each of two micro centrifuge tubes. Add 66 microliters of 32%paraldehyde to one of the aliquots to be used later.

For cyber gold staining, add 12 microliters of 25%glutaraldehyde to the other aliquot for later dappy staining. Gently mix and allow the samples to fix for at least 15 minutes. At room temperature in the dark, the samples for microscopy should be processed within one hour.

To prepare samples for flow cytometry, place an eight micrometer polycarbonate filter in one of the filter holders to exclude debris and large eary cells. Pass sampling water through to fill two cryo vials for each sampling site. With one milliliter of sample, add five microliters of 25%glutaraldehyde to each cryo vial.

Invert the vials to mix and allowed the samples to fix for 15 to 30 minutes at room temperature. Subsequently flash, freeze the samples in liquid nitrogen and store at negative 80 degrees Celsius until analysis on a flow cytometer. To begin the procedure for preparing the microbial metagenomic samples, remove the inline filter holders directly.

Mount a point 22 micrometer cylindrical. Filter onto the respective line. Filter the remaining sample water of both hat skin units from each site through one filter.

After the filtration, push the remaining water out of each filter by using a clean 10 milliliter syringe filled with air. Place the filter back into its original packaging and seal the package with lab tape. Store the individually packed filters at negative 20 degrees Celsius.

Next, prepare the viral metagenomic samples. Transfer the samples immediately into the washed buckets to ensure no sample is lost or contaminated by the environment. Pre-filter using a large pore size nylon mesh to remove debris and cellular material prior to concentration.

Set up the tangential flow filters or TFF as shown in this diagram. Place a delivery line in a sample bucket and leave the return and filtrate lines running into a sink. Turn on the peristaltic pump and flush the lines with one to two liters of sample water.

Place the return line in the sample bucket to complete the cycle and add 0.7 bar of back pressure while concentrating the sea water. Top up the sample reservoir as the level drops. When the water level drops below the line intake in the bucket, transfer the concentrate into a bleach washed triple rinse trip poor beaker, and continue concentrating.

If the reservoir is empty, remove the back pressure, increase the pump rate, and push the entire sample through the lines, recovering it in the tripo beaker. Pass the concentrate through point 45 micrometer cylindrical filters to remove most bacteria without discriminating against any viral lineages. Collect the point 45 micrometer filtered viral.

Concentrate in 50 milliliter tubes. Change the point 45 micrometer cylindrical filters. After every 150 milliliters after the filtered viral concentrate has been collected, add 250 microliters of chloroform to each 50 milliliter aliquot to eliminate residual bacteria invert to mix.

Store the tubes upright at four degrees Celsius until subsequent processing. Lastly, dry the 0.45 micrometer filters and store them as shown for the microbial metagenomic samples. The extraction of genomic DNA from the microbial and viral samples will not be shown in this video, but the procedure is found in the accompanying protocol.

Text representative analysis of microscopy samples are shown here. Dappy stained and cyber gold stain slides are examined to measure the abundance and size distribution of bacteria and viruses. Collected samples are further assessed for the ratio of autotroph te heterotrophic microbes via flow cytometry to determine the number of total bacteria cells samples are stained with cyberg green one, A channel for chlorophyll and FICO eryn is used to count the abundance of autotrophs in unstained samples.

The autotroph counts from the non-ST stain portion are then subtracted from the cyber stained total count to determine the abundance of heterotrophic microbes following isolation and purification of viral like particles or vlp epi fluorescence microscopy is used to verify the presence and purity of the viral particles. These plots show representative microbial metagenome data. The relative abundance of sync.

Occus SPP is positively correlated with the percent cover of coral. In contrast to Ella Backer, SPP, the metabolic pathways for conjugated transfer was positively correlated with nitrate concentrations. While the metabolic pathway for DNA repair was consistent between all Metagenomes, this table presents data characteristics of two viral genomes generated from two islands.

This second table shows water chemistry data from various sites during one expedition. After watching this video, you should have a good understanding of how to comprehensively assess the organic and the inorganic nutrient availability and the abundance and structure of viral and microbial communities in remote field locations.