Dec 13, 2024

Public workspace1 M Tris-HCl pH 7.5 for Tetrahymena mating (Stock solution)

DOI
dx.doi.org/10.17504/protocols.io.3byl4wb92vo5/v1
  • 1Walther Straub Institute for 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: dx.doi.org/10.17504/protocols.io.3byl4wb92vo5/v1
Protocol Citation: Luisa F Jimenez-Soto, Christoph Meister 2024. 1 M Tris-HCl pH 7.5 for Tetrahymena mating (Stock solution). protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4wb92vo5/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: November 28, 2024
Last Modified: December 13, 2024
Protocol Integer ID: 113065
Abstract
In this protocol we specify the steps needed to create a 250 ml stock solution of 1M Tris-HCl solution used for the preparation of Tetrahymena thermophila cells for mating. The final concentration for inducing mating behavior is 10 mM (see Bruns PJ and Brussard TB, 1974 and Cassidy-Hanley, 2012). For general information about Tris based buffers, check 1 2. It buffers in a range from pH 7 to 9.
CITATION
Cassidy-Hanley DM (2012). Tetrahymena in the laboratory: strain resources, methods for culture, maintenance, and storage.. Methods Cell Biol. 2012;109:237-76. .
LINK
https://doi.org/10.1016/B978-0-12-385967-9.00008-6

CITATION
Bruns PJ, Brussard TB. (1974). Pair formation in tetrahymena pyriformis, an inducible developmental system.. J Exp Zool. 1974 Jun;188(3):337-44..
LINK
doi: 10.1002/jez.1401880309

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
  • ReagentTris Puferan >99.3%Carl RothCatalog #AE15.3
  • Reagentdouble distilled water (ddH2O)Contributed by users
  • HCL

Protocol materials
ReagentTris Puferan >99.3%Carl RothCatalog #AE15.3
ReagentTris Puferan >99.3%Carl RothCatalog #AE15.3
Reagentdouble distilled water (ddH2O)
ReagentTris Puferan >99.3%Carl RothCatalog #AE15.3
ReagentTris Puferan >99.3%Carl RothCatalog #AE15.3
Safety warnings
The process of autoclaving requires the use of high temperatures and specialized equipment. Make sure you have been instructed in the use of the autoclave.
When handling recently autoclaved liquids, their temperatures can be above 60°C, and can cause severe burns. Follow the safety protocols of your institution and use always personal protective equipment.

In case HCl 37% solution is used, use only under the chemical hood, use lab coat, gloves and goggles.

Also consult the attached data safety sheets:
Download tris-safety-data-sheets.pdftris-safety-data-sheets.pdf225KB


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.
Buffer preparation
Buffer preparation
35m
35m
Wash the flask twice with 500 ml of distilled water to remove ANY trace of detergent, which is harmful for Tetrahymena thermophila
In a calibrated cylinder, place 1 magnetic stir bar.
Weigh Amount30.28 g of ReagentTris Puferan >99.3%Carl RothCatalog #AE15.3

Place the weighted ReagentTris Puferan >99.3%Carl RothCatalog #AE15.3 in the calibrated cylinder and add no more than Amount200 mL of distilled water.

Place the cylinder on a magnetic stirrer and dissolve the ReagentTris Puferan >99.3%Carl RothCatalog #AE15.3 .

While under agitation of the magnetic stirrer, measure the pH. To adjust it to Ph7.5 , add Concentration1 Molarity (M) HCl drop wise with a pasteur (glass) pipette, all under the chemical hood.

Add distilled water close to the 250 ml mark. Verify the pH is still Ph7.5 . Fill to the final 250 ml with distilled water.

Transfer the solution to the 500 ml pre-cleaned bottle (as described in step 1)
If the solution needs to to be autoclaved, do so at Temperature121 °C for Duration00:15:00 to Duration00:20:00 . Careful! Do not transfer the magnetic stir bar. Make sure you know how to use the autoclave before you use it.
35m
Temperature
If the solution does not need to be autoclaved, transfer the 250 ml to a 250 ml pre-washed (see step 1) flask and stored at TemperatureRoom temperature .

Protocol references
https://www.dcfinechemicals.com/en/blog/trishydroxymethylaminomethane-tris-buffer/ (accessed November 2024)
https://sigmaaldrich.com/DE/en/products/chemistry-and-biochemicals/biochemicals/biological-buffers/tris (accessed November 2024)
Bruns PJ, Brussard TB. Pair formation in tetrahymena pyriformis, an inducible developmental system. J Exp Zool. 1974 Jun;188(3):337-44. doi: 10.1002/jez.1401880309. PMID: 4209195.
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/B978-0-12-385967-9.00008-6. PMID: 22444147; PMCID: PMC3608402.

Citations
Bruns PJ, Brussard TB.. Pair formation in tetrahymena pyriformis, an inducible developmental system.
doi: 10.1002/jez.1401880309
Cassidy-Hanley DM. Tetrahymena in the laboratory: strain resources, methods for culture, maintenance, and storage.
https://doi.org/10.1016/B978-0-12-385967-9.00008-6