TITLE: Defined medium for Neocallimastigomycota
AUTHORS: Sophia Strobl, Julia Vinzelj, Nico Peer, Diana Young, Akshay Joshi, Rolf Warthmann, Veronika Flad, Urs Baier, Michael Lebuhn, Hans-Joachim Nägele, Sabine M. Podmirseg

[DESCRIPTION]
This is the protocol for the completely defined medium base for Neocallimastigomycota (anaerobic gut fungi, AGF) that was used within the HiPoAF and FUNGAS projects. 

If you add 2 g/L xylan and 3 g/L cellobiose as C-sources to this medium, Neocallimastigomycota - in our experience - can grow on it for prolonged periods (more than 18 months and running, with weekly sub-cultivation). We use this medium, however, more for short-term experiments rather than long-term culture maintenance. So far, we have tested this medium on strains of Anaeromyces mucronatus, Caecomyces communis, Neocallimastix cameroonii, Neocallimastix frontalis, Pecoramyces ruminantium and all were able to grow. However, depending on the C-source added, they might show unusual clustering behaviour (e.g., clinging onto the glass). 

This completely defined medium remains nearly colourless if no C-source is added before autoclaving the bottles, which is especially convenient for microscopy. For this purpose, a soluble C-source can be added directly to the bottles after autoclaving. 

[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. 120 min in total to prepare the medium (depending on the volume you prepare, it can take longer or might be faster). 

 


[STEPS]
1. Add salt solution I, salt solution II, hemin solution, resazurin solution, and MilliQ water to a cooking pot. 
If you want to use xylan as a C-source (or other C-sources that don't dissolve well in water), you should add it here as well. 
2. Heat the mixture and simmer until everything is dissolved and the colour starts to change to yellow. For us with 2 L of medium in a 5 L cooking pot, this takes about 20 min.

 

3. 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. This takes about 10 min to 20 min 

2.1. While the mixture is heating up, flush a large enough Erlmeyer flask with pure CO2 for 2 min. 
4. 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. Bubble with CO2 for another 10 min.

5. Set the pH of the medium to pH 6.9with the help of NaHCO3 powder or 5M NaOH solution. The medium should now be a translucent yellowish colour.

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


 

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. The medium can turn slightly pink at first upon transfer, but this usually settles quickly. If the medium turns bright pink upon transfer, however, keep bubbling it with CO2 within the serum bottles for up to 5 min before closing it up. 

 

7. Autoclave the serum bottles at 121 °Cfor 20 min .

8. 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 pink or red. 

8.1. Before use, the bottles 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, a sterilized solution of soluble C-source (e.g. glucose, cellobiose), and antibiotic stock solution can be injected as well.