Rhodomonas salina

Q:       I am having difficulty growing CCMP1319. I've been following your protocol to make the F/2 medium but the water colour is not red/maroon as it's supposed to be. But rather it's green, which indicates  that the algae are not doing well.  Could you please tell me how I can make them return to their normal colour? Do you know the reason why they might be green?  Also, there are clumps of white solid forming at the bottom of the Erlenmeyer Flask and I don't know if this is supposed to happen.

A:       You are correct that CCMP1319 should be red in color.  In order to know if you can recover it, I would first look at a few drops with a compound microscope and see if there are any living cells in your medium.  I do not know anything about CCMP1319 and silica and selenium.  CCMP1319 does not need added silica to grow.  How did you make up your medium? Did you autoclave it to sterilize, and if so, what volume did you autoclave

Q:       I looked the salina under the hemocytometer yesterday and they're alive and moving. They're certainly changing colour.  I autoclaved 1 L of the trace metal solution and the seawater separately for the vitamin and added the vitamin in under a laminar flow hood after.  Do you know if it will eventually regain its red colour?

A:       As long as there are swimming cells, it may recover.  How much light are you giving your cultures?  The green color maybe a stress reaction.  Autoclaving 1L volume of seawater without added nutrient salts or vitamins can precipitate. 

Q:       So does that mean we're supposed to autoclave the vitamin solution with biotin, thiamine HCl and cyanocobalamin? Wouldn't doing this denature the proteins?  Also I am giving them 16 hrs light/8 hours dark. If there is an external source of lighting, will that affect the algae? Also what light fixture do you suggest me using? I am using 40 W fluorescent bulbs.

A:       We use cool white fluorescent bulbs.  I was thinking in terms of the intensity of the light- 80-120 uE is reasonable.  As far as making medium, I am a little confused by your description, so let me start from scratch. I am sorry if I am redundant.  If I was trying to avoid precipitate, I would autoclave the seawater separately.  As I mentioned, in >250mL volumes, filtered seawater can have precipitation issues.  We autoclave all of our nutrients to sterilize them, even the vitamins. I am told that autoclave sterilizing can cause the vitamins to break into sub-units but the algae can put the vitamins back together.  I would take the individually sterilized f/2 nutrients and add them, aseptically to your autoclaved seawater- 1mL@ N,P, TM and 1/2mL vitamins.