Dec 17, 2024

Public workspaceFeCl3 solution for NEFF Media

DOI
https://dx.doi.org/10.17504/protocols.io.bp2l6d9ezvqe/v1
  • Christoph Meister1,
  • Luisa F Jimenez-Soto1
  • 1Walther Straub Institute of Pharmacology and Toxicology, LMU - Munich
  • Luisa F Jimenez-Soto: corresponding author (l.jimenez_(at)_lmu.de)
Luisa F Jimenez-Soto
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DOI: https://dx.doi.org/10.17504/protocols.io.bp2l6d9ezvqe/v1
Protocol Citation: Christoph Meister, Luisa F Jimenez-Soto 2024. FeCl3 solution for NEFF Media. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6d9ezvqe/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: December 04, 2024
Last Modified: December 17, 2024
Protocol Integer ID: 113896
Keywords: Iron(III)chloride, NEFF, Tetrahymena, FeCl3, axenic media, stock solution, nef media with iron, iron chloride, solution for neff media, iron change, axenic growth of tetrahymena thermophila, iron, iron in the media, minimum waste, nef media, years with minimum waste, neff media, oxidation, oxidation of the solution, tetrahymena stock center, neff, tetrahymena thermophila, storage, stock solution
Abstract
For axenic growth of Tetrahymena thermophila in the lab, NEFF media requires a final concentration of 33 µM (see Tetrahymena Stock Center (RRID:SCR_008362)). To guarantee that the iron in the media is as fresh as possible, we complement NEF media with iron (resulting in NEFF) shortly before use, and store the complete medium for no more than a week, if it contains iron. With this in mind, we keep iron chloride as a stock solution 1000X concentrated. In this protocol, we describe how we prepare the stock solution and its storage. This method has allow us to store it up to 3 years with minimum waste because of oxidation of the solution. In addition, having them in small aliquots for a long time, allows us to reduce growth variability and behavior related to iron changes.
Guidelines
  1. As good microbiological practice, all containers used for components should be labeled with the name of the solution, the date of creation / aliquote, and initials of the person who did it, in order to ask questions if something is needed.
  2. If you have doubts about how to perform a step in the protocol, consult your supervisor or someone who has prepared the solution before.
  3. For any questions, feel free to email us (exotoxinslab-(at)- gmail.com). We are more than happy to help you.
  4. Some of the protocols will be posted in our YouTube channel (www.youtube.com/@exotoxinslab).
Materials
  • ReagentIron(III) chloride hexahydrate 250 gCarl RothCatalog #P742.1
  • Deionized water <= 0.05 µS
  • Conical tubes 50 ml volume and 15 ml for storage
  • Filter 0.22µM with luer connection compatible with syringe connector
  • 50 ml syringe with luer connection
  • 1.5 ml conical tubes for long term storage

Protocol materials
ReagentIron(III) chloride hexahydrate 250 gCarl RothCatalog #P742.1
Troubleshooting
Safety warnings
Check the safety information available for FeCl2 before you start to work with it.
Download Safety data sheet Iron(III)chloride hexahydrate.pdfSafety data sheet Iron(III)chloride hexahydrate.pdf297KB
Before start
  1. Be aware of your lab's safety protocols and check the attached Warnings (Guidelines and Warnings) to see what steps are necessary to protect yourself, such as protocol-appropriate personal protective gear, like lab coat, gloves and glasses.
  2. Make sure to read the whole protocol before starting and have all the materials at hand.
Procedure
1h
Weight Amount0.446 g of ReagentIron(III) chloride hexahydrate 250 gCarl RothCatalog #P742.1 and place it in a 50 ml conical tube.

Add distilled water up to the 50 ml marking (so that the concave liquid limit bottom is on the 50 ml marking)

Let the salt particles dissolve until they disappear. It should take no longer than Duration01:00:00 .

1h
Filter the solution through the 0.22 µm pore filter. See the attached protocol to see how.
Protocol
Sterile filtration of liquid solutions
NAME

Sterile filtration of liquid solutions 

CREATED BY
Luisa F Jimenez-Soto

Storage
Storage option 1: Store at TemperatureRoom temperature , in darkness if possible. In this storage, you might see a faster precipitation of iron on the wall of the tube. We do not use this method for our lab. We only do it if great amounts of culture are needed in the next 2 to 3 weeks.
Storage option 2: Make 10 ml aliquots in 15 ml sterile conical tubes (4 of them) and store at Temperature-20 °C . These tubes are for long reserve and will be thawed when the small tubes are used up. With the rest (approx. 9 ml to 10 ml) solution, aliquot 500 µl in 1.5 ml sterile tubes (around 18 to 19 tubes). Each tube should be labeled with the name of the solution (FeCl3 33 mM), the solvent (water), and date of the storage in the corresponding tube. Make sure to secure the labeling with tape, since it might fade in the freezer or during following thawing and re-freezing steps.
Protocol references
The importance of iron in axenic culture of Tetrahymena thermophila has been extensively reported by other authors. The reference included here is free access and summarizes many aspects for the growth of T. thermophila: Cassidy-Hanley DM. Tetrahymena in the laboratory: strain resources, methods for culture, maintenance, and storage. Methods Cell Biol. 2012;109:237-76. doi: 10.1016/978-0-12-385967-9.00008-6.
You can find more information available in the Tetrahymena Stock Center (https://tetrahymena.vet.cornell.edu/recipes.php)