TITLE: Medium without CRF for Neocallimastigomycota
AUTHORS: Julia Vinzelj, Diana Young, Akshay Joshi, Sophia Strobl, Ljubica Begovic, Nico Peer, Magdalena Nagler, Rolf Warthmann, Veronika Flad, Urs Baier, Michael Lebuhn, Sabine Podmirseg

[DESCRIPTION]
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. 180 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, 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.
2. 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 20 min to 30 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.
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 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. 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.  
 


5. Once the medium turned yellowish, set the pH of the medium to pH 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. 
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. 

  

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 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.