Medium without CRF for Neocallimastigomycota

Sophia Strobl, Julia Vinzelj, Nico Peer, Diana Young, Akshay Joshi, Rolf Warthmann, Veronika Flad, Urs Baier, Michael Lebuhn, Ljubica Begovic, Magdalena Nagler, Sabine M.Podmirseg

Published: 2023-10-03 DOI: 10.17504/protocols.io.36wgq779kvk5/v1

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Abstract

This is the protocol for the standard, nutrient-rich, undefined cultivation medium without clarified rumen fluid (CRF) used within the HiPoAF project.

In previous publications this medium or slight variations of it have also been referred to as "defined medium" (since it does not contain CRF) - this, however, is not completely true, since it does contain undefined components (yeast extract, and tryptone).

If you need a completely defined medium, check out our protocol "Minimal Medium for AF".

Before start

Before you start, make sure you have prepared all necessary stock solutions, and have all chemicals needed at hand (see Materials section for details).

You will need approx. 3h 0m 0s in total to prepare the medium (depending on the volume you prepare, it can take longer or might be faster).

Safety information

Please also ensure, that all necessary safety measures are in place (see also: Safety Warnings).

Steps

Add Salt Solution I, Salt Solution II, Yeast Extract, Tryptone, Hemin Solution, Resazurin Solution, Xylan powder (as an additional C-source for AF; can be omitted), and MilliQ water to a cooking pot.

Heat the mixture and simmer it until the colour changes to dark yellow. For us with 2 L of medium in a 5 L cooking pot, this takes about 0h 20m 0s to 0h 30m 0s .

Note

Take care not to cook it too long. Otherwise you will lose a lot of water, making the medium more concentrated. You can also carefully heat the mixture in a microwave.

2.1.

While the mixture is simmering, flush a large enough Erlmeyer flask (best chose one with two times the volume of the medium) with pure CO₂for 0h 2m 0s.

Carefully pour the mixture from the pot into the Erlmeyer flask. Cool it with ice-water while agitating it by magnetic stirrer and bubble it with pure CO₂.

Once the mixture has cooled to around 39 °C, add the trace elements solution, NaHCO₃, soluble C-source (like cellobiose, glucose, etc.; if necessary) and lastly the L-cysteine-HCl. Keep gassing with CO₂, but adjust the gas flow in order to avoid bubbling/overflowing of the medium. Bubble with CO₂ until the medium turns yellow again.

4.1.

If you want to have a simple, nutrient-rich cultivation medium for anaerobic fungi without clarified rumen fluid add milled wheat straw or pressed wheat straw pellets to your serum bottles. The combination of a complex C-source like wheat straw and a less complex one like xylan helps keeping the metabolism of AF active. Using less complex C-sources like glucose or cellobiose also works well, but it might inhibit parts of the carbohydrate metabolism of AF and can give rise to bacterial or yeast contamination if you are not handling your cultures carefully.

Note

For example: for our standard culture collection medium, we use serum bottles with a total volume of 120 mL. We add 0.35 g wheat straw to those bottles, and add 45 mL medium (in step 6). We later add 0.5 mL vitamin solution only shortly before use and sub-culture 5 mL of an actively growing culture into this medium, which brings the total volume of liquids in this bottle up to 50.5 mL. Pro-tip: For culture care (not for experiments), the pressed pure wheat straw pellets (e.g. horse feed as you might find in a pet store) are way more convenient to handle! You can simply eyeball the amount needed.

Once the medium turned yellowish, set the pH of the medium to 6.9with the help of NaHCO₃ powder or 5M NaOH solution.

5.1.

While setting the pH, flush your serum bottles with pure CO₂ for 1-2 minutes, to minimize oxygen contamination when aliquotting your medium in step 6.

Aliquot the medium into your serum bottles under continuous flow of CO₂ in the serum bottles and the Erlmeyer flask filled with medium. Depending on the amount of medium prepared this step takes around 0h 30m 0s.

Dispension of the medium into 120mL glass serum bottles (max. 50mL per bottle). Picture copyright: Julia Vinzelj & Nico Peer

6.1.

Take the gas hose out of the serum bottle and immediately close it with a rubber stopper, then crimp the bottle with an aluminium cap.

Closing of the serum bottles with 3-legged butyl-rubber stoppers after the medium has been dispensed. Picture copyright: Julia Vinzelj & Nico Peer.

Autoclave the serum bottles at 121°Cfor 0h 20m 0s .

Store the bottles without regard to temperature for up to 2 months. 4°C works as well as 37°C for the duration of 2 months at least. Discard any bottles with medium that turned red.

8.1.

Before use, they should be warmed to 39°C. Also, 0.01mL vitamin solution must be added per mL of medium shortly before use by injection. If needed, antibiotics can be injected as well.