Mar 20, 2024

Public workspaceBlackwater medium V.1

This protocol is a draft, published without a DOI.
  • 1University of San Diego;
  • 2UC Irvine
Callen Hyland
Open access
Protocol Citation: Callen Hyland, Rob Steele 2024. Blackwater medium. protocols.io https://protocols.io/view/blackwater-medium-daj72crn
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: March 13, 2024
Last Modified: March 21, 2024
Protocol Integer ID: 96607
Keywords: Hydra, invertebrates, animal care
Abstract
Blackwater medium is a laboratory growth medium for Hydra that replicates the chemical composition of blackwater swamps. This medium has lower low ionic strength than traditional Hydra medium and contains humic acid. Blackwater medium is ideal for Hydra species isolated from blackwater swamps and improves the health of some transgenic Hydra lines that are prone to infection.
Image Attribution
Callen Hyland
Guidelines
Please see Low cost methods for Hydra care for general guidelines for working with Hydra.
Materials
ReagentCalcium Chloride DihydrateFisher ScientificCatalog #C79

ReagentPotassium ChlorideFisher ScientificCatalog #P217

ReagentSodium ChlorideFisher ScientificCatalog #S271

ReagentMagnesium Chloride HexahydrateFisher ScientificCatalog #BP214

ReagentHumic Acid Sodium SaltMerck MilliporeSigma (Sigma-Aldrich)Catalog #H16752

  • Lab scoops
  • Weigh boats or weigh papers
  • Electronic balance
  • DI water
  • 50 mL conical tubes
  • Syringe filter or vacuum filter
  • Adjustable volume micropipette and tips
  • Liter bottles or plastic carboy

Stock solutions
Stock solutions
Make stock solutions in 50 mL conical tubes with DI water.

ABC
ComponentStock conc.Weight for 50 mL
Calcium chloride dihydrate (CaCl2*2H2O)2.5 M18.4 g
Magnesium chloride hexahydrate (MgCl2*6H2O)1.0 M10.2 g
Sodium chloride (NaCl)5.0 M14.6 g
Potassium chloride (KCl)1.0 M3.73 g
Humic acid sodium salt20 g/L1 g
Table 1. Stock solutions for blackwater medium.

Not all of the humic acid sodium salt will go into solution. Filter the humic acid solution with a syringe filter or vacuum filter to remove undissolved particles.
Working solution
Working solution
Prepare blackwater medium by diluting stock solutions in DI water. Use a 1 L bottle, or scale up volumes for a large plastic carboy. Mix well before using.

ABC
ComponentFinal conc.Volume of stock for 1 L
Calcium chloride dihydrate (CaCl2*2H2O)0.015 mM6.0 μL
Magnesium chloride hexahydrate (MgCl2*6H2O)0.0185 mM18.4 μL
Sodium chloride (NaCl)0.125 mM25 μL
Potassium chloride (KCl)0.0075 mM75 μL
Humic acid sodium salt0.01 g/L0.5 mL
Figure 2. Chemical composition of Blackwater medium.