TITLE: Microalgal culture
AUTHORS: Victoria Jackson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A brief guide to subculturing marine eukaryotic microalgae such as prasinophytes, diatoms and dinoflagellates.</div></div>

[STEPS]
?. [Media preparation]
Microalgal culture media are comprised of macronutrients (nitrates, phosphates, sulphates), micronutrients (trace metals) and vitamins.
For media preparation, it is best to prepare stock solutions of the macro- and micronutrients and vitamins at concentrations higher than those needed in the final medium. This gives a consistent source for the medium and reduces the need to weigh chemicals etc.Once you have prepared your stock solutions (in deionised water), they can be stored refrigerated (between   and   ) for long periods. You can also seal the stock solutions with parafilm to prevent evaporation which may alter concentrations. Vitamin stocks can also be frozen and re-frozen without degradation.
?. [Media preparation]
Filter aged seawater using a 0.45 μm membrane filter or 0.7 μm GF/F filter and autoclave. Once you have FSW, it should be kept refrigerated and in the dark.
To make medium for marine microalgae, either use artificial seawater (ASW) or aged, filtered and autoclaved seawater (FSW).
Don't forget to loosen the lid of the bottle(s) prior to autoclaving.
?. [Media preparation]
Add the macro- and micronutrients in the required quantities for the media recipe being used to the cooled ASW or FSW (following autoclaving). Mix by inverting the bottle several times and autoclave.
Media recipes:NCMARoscoff Culture CollectionCCAP
Don't forget to loosen the lid of the media bottle(s) prior to autoclaving.
Once autoclaved, precipitation can be reduced by rapidly cooling the medium.
?. [Media preparation]
Using a laminar flow hood, pour a small amount of vitamin stock solution into a beaker. Using a syringe and 0.2 μm filter, filter sterilise the vitamins into a clean beaker. Pipette the required quantity of sterilised vitamins into the cooled medium and mix well.
?. [Media preparation]
You can check the pH of the medium using a pH meter if culturing strains that are sensitive to pH changes.Keep the medium refrigerated and in the dark when not subculturing.
?. [Preparation]
Work under a laminar flow hood.Label a new T25 culture flask (vented cap, non-treated) with strain ID, species name, medium used, temperature of incubation and date of subculturing.
?. [Subculturing]
Using a pipettor, add  of medium to the new culture flask.
20 mL
?. [Subculturing]
If required, add  of antibiotic solution to the medium for 1/1000 dilution.
20 µl
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?. [Subculturing]
Using a transfer pipette, transfer  of the old culture to the new flask and gently shake to mix.
2 mL
?. [Culture Growth]
Screw the lid on the flask and incubate the culture at the appropriate temperature and light regime.
?. [Culture Growth]
Culture growth can be monitored by microscopy, fluorescence using a plate reader, flow cytometry, or by simply observing culture colour.
?. [Culture Growth]
Repeat approximately every ~2 weeks, depending on culture growth.