Nov 12, 2019
Seagrass Microbiome Sample Collection and Preservation
- Jonathan JA Eisen1,
- Jonathan Eisen Lab2
- 1UC Davis;
- 2Eisen Lab, UC Davis
- EisenLab
- Seagrass Microbiomes
External link: https://seagrassmicrobiome.org/
Protocol Citation: Jonathan JA Eisen, Jonathan Eisen Lab 2019. Seagrass Microbiome Sample Collection and Preservation. protocols.io https://dx.doi.org/10.17504/protocols.io.fxzbpp6
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: September 30, 2016
Last Modified: November 12, 2019
Protocol Integer ID: 3801
Abstract
This collection protocol was developed as part of the Seagrass Microbiome Project. This was originally a collaboration among Jonathan Eisen and Jay Stachowicz at the University of California, Davis and Jessica Green at the University of Oregon, with funding from the Gordon and Betty Moore Foundation.
Guidelines
Collection: Seagrass Microbiome Tissue Samples
Collection: Seagrass Microbiome Tissue Samples
Collection: If possible, wear gloves whenever you handle the plants. Pull up one plant including the roots. Gently swish the plant in the water to remove loose sediment from the roots. Process these samples in the field according to steps 2 and 3.
Note
If you cannot process them in the field, cut the plant into two pieces (above-ground leaves and below-ground roots/rhizomes) and place in two separately-labelled clean bags on dry ice to transport to shore/lab; these samples must be processed as soon as possible and no longer than 5 hours from the time of collection.
Processing Roots: Pull off root hairs with tweezers and place them in a labelled microcentrifuge tube. If you are preserving the samples in a buffer, make sure the roots are fully submerged.
Processing Leaves:. Cut a 2 cm section from a healthy, green section of an outer leaf blade. Put the leaf section into a labelled microcentrifuge tube. If you are preserving the samples in buffer, make sure the leaves are fully submerged.
Tools: Wipe down all tools with ethanol between samples.
Collection: Sediment Microbiome Samples
Collection: Sediment Microbiome Samples
Collection: Near where you collect your seagrass tissue sample, collect a sediment sample. Insert the barrel of the syringe into the sediment and, at the same time, carefully and slowly pull back on the plunger so that the sediment surface remains intact and in place while the syringe barrel is pushed into the sediment.
Note
Each sediment sample should be collected using one 6cc syringe with the bottom removed (see a picture of the ZEN kit for an example).
Collection: Remove the syringe from the sediment and extrude the sediment until the base of the plunger is at the 3cc mark.
Use a plastic spatula to transfer approximately 0.25 grams of sediment in the syringe into a labelled microcentrifuge tube. If you are using buffer, the sediment should be fully submerged in the buffer solution.
Tools: Wipe down all tools with ethanol between samples.
Preservation
Preservation
We recommend storing samples on dry ice in the field no more than 5 hours after you collect them. Subsequently, you should store samples at -20 °C or -80 °C as soon as possible.
If you do not have access to a freezer or dry ice at or near your field site, we recommend using Zymo’s Xpedition Lysis/Stabilization SolutionTM since it is stable at room temperature for at least a month.
Note
Note: If you choose to use the Zymo Xpedition buffer be aware that it works optimally when extraction happens within a month of collection. Additionally, it forms a precipitate when interacting with the MoBio PowerSoil Extraction Kit C1 solution. See the DNA extraction protocols for the ZEN project for advice to deal with this issue.