The Meeks lab uses Hutner's medium for growth of the bryophyte Anthoceros punctatus.
Hutner's -N is used when Anthoceros is grown with associated symbiotic Nostoc since the 
symbiont provides all of the necessary nitrogen. For symbiont-free Anthoceros, we grow the 
tissues with Hutner's +N medium. Additionally, we add 0.5% glucose + 5mM Mes at pH=6.4 
into sterilized medium. For stock cultures of Anthoceros, we maintain tissues in Hutner's -N 
with 5mM Mes at pH=6.4 without glucose. Liquid cultures are incubated under cool flourescent
lights like Nostoc cultures with constant shaking at ~50 rpm at 20oC with a 14/10 light/dark cycle.


A) Hutner's Stock Solution

Add the EDTA to 75 mls glass distilled H2O. Add solid KOH pellets until all the EDTA
dissolves (this takes quite a bit of HOH). Check the pH, then add KOH until pH+6.5.
Add H3BO3 and ZnSO4, then enough KOH to bring pH back to 6.5. Add all other salts
except FeSO4 and bring pH back to 6.5. Add FeSO4 and adjust pH to 6.5 with 5M
KOH. Make up to 100mls with water. The solution will be green. Bubble filter air
(45 um or cotton plug) through the solution until it turns red. This can take a long time
(up to four days) to occur. Precipitation will occur during the bubbling. Each day during
bubbling, and when bubbling is finished, check and adjust the total volume and pH.
Store the stock with the precipitate at 4oC. When using the stock, pipette the liquid
carefully, without disturbing the precipitate.



B) To make 1 liter of Hutner's combine the following:

Put ~1/2 the water into the flask before adding stocks (they precipitate if mixed).

Bring up the volume to just below total.

Adjust pH to 6.4 with 1N NaOH.

Bring up volume to total.

Divide into flasks(50mls/125ml or 100mls/250ml flask) and aoutclave sterilize.

Add 1.2% agar (12g/liter, 6g/500mls) for plates. Do not use noble agar.