Hey everyone, I have been trying to isolate Azospirillium from soil samples using this media recipe:

Ingredients Gms / Litre

Part A -

Malic acid 5.000

Dipotassium hydrogen phosphate 0.500

Ferrous sulphate 0.500

Manganese sulphate 0.010

Magnesium sulphate 0.200

Sodium chloride 0.100

Bromo thymol blue 0.002

Sodium molybdate 0.002

Calcium chloride 0.020

Agar 1.750

Part B - Potassium hydroxide 4.000

Final pH ( at 25°C) 6.8±0.2

I put the media is put into air tight tubes, innoculate, then the Azospirillium is meant to turn the solution blue as it grows. Then you can check morphology under a microscope. Ive added pictures of my tubes.

I am just not getting any growth. Even with an Azospirllium brasilense pos control. Im guessing there is a practical aspect I am missing, but cannot find enough information on it. Is the volume wrong? Am I meant to get rid of any air in the tube so it is completely anaerobic? Just not sure.

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