Apr 23, 2024
Homogenate of A. cervicornis
 Forked from Homogenate of A. cervicornis
- 1University of Miami
Protocol Citation: Stephanie Rosales, Ana M Palacio-Castro 2024. Homogenate of A. cervicornis. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l248xpg1y/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: October 14, 2020
Last Modified: April 23, 2024
Protocol Integer ID: 43191
Abstract
This protocol is to study disease in Acropora cervicornis by using a disease homogenate method. These methods are adapted from methods presented in Muller 2018 https://elifesciences.org/articles/35066.
Materials
MATERIALS
Filtered and autoclaved seawater (FSW; artificial seawater also works)
Razor blade
Falcon tube (50 mL)
RNaseZapâ„¢ RNase Decontamination SolutionThermo Fisher ScientificCatalog #AM9780
DNA/RNA ShieldZymo ResearchCatalog #R1100-50
10% bleach (1:10 dilution of commercial 5.25-6.0% hypochlorite bleach)
Airbrush
ZR BashingBeadTM Lysis Tubes (0.1 & 0.5 mm)Zymo Research
Clippers
Air compressor
Tweezers
Ruler
Slate or sterile work surface
Sterile containers for corals
Ziplock bags
Pipette (P1000) and tips
Safety warnings
Wear gloves and masks when dealing with pathogens especially those of unknown origin.
Before start
- Prepare sterilized and 0.2 micron filtered seawater (FSW).
- Collect healthy coral fragments
- Collect diseased coral fragments
- Randomly assign tanks to treatments
- Label and UV Ziploc bags and Falcon tubes and other material taht cannot be autoclaved
Obtain healthy and diseased Acropora cervicornis fragments. These fragments are preferably collected from the same area and with similar disease progression.
Note
The tissue areas should be similar between healthy and disease corals to prepare diseased and placebo blastates with similar concentrations.
Prepare placebo and disease homogenates
Grab healthy fragments from the holding tank.
Note
Sigle branch fragments are easier to blast and to estimate their tissue area.
If cutting different branches, use clean clippers.
Take a picture of every fragment with a size scale to estimate the amount of tissue blasted.
Rinse fragments with FSW, and place them in a sterile container with enough FSW to cover them.
Grab a fragment and hold it over a pre-labeled Ziploc bag (** this will require two people**).
Airbrush the tissue with an airgun and FSW.
Approximately 00:05:00 per fragment
Number of fragments to blast
Note
Work in a fume hood if possible to avoid blasting aerosols.
When working with disease fragments, remove the tissue up to 5 cm above the lesion.
5m
Pour homogenate in a pre-labeled 50mL falcon tube(s). Measure volume and store in the fridge.
Grab diseased fragments from the holding tank. And repeat steps 2.2 - 2.5.
Change gloves
Bring placebo and diseased homogenates to the same volume by adding FSW
Add 20 beads to each falcon tube containing ~ 40mL of homogenate and vortex for 10 minutes
00:10:00 each tube
10m
Save and preserve ~ 500 ul of both homogenates for sequencing.
Dose experimental corals with the placebo and disease homogenates
Sterile razor with bleach and scratch a small area of each experimental coral
Note
Create a ring around the coral located ~ 1cm from the bottom
Apply 500 uL of placebo and disease homogenate above each coral fragments in their respective treatments.
Add 16 L of seawater to each tank to completely cover the fragments
Maintained closed system for 3hours
Clean -up
Bleach tweezers, clippers, slates, airgun, surfaces
Discard bags and falcon tubes
Protocol references
Erinn M Muller, Erich Bartels, Iliana B Baums (2018) Bleaching causes loss of disease resistance within the threatened coral species Acropora cervicornis eLife 7:e35066. doi: 10.7554/eLife.35066