TITLE: Culturing Chlorella Vulgaris and Desmodesmus Quadricauda
AUTHORS: Jakub Nedbal

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes the steps required for maintaining a healthy culture of </span><span style = "font-style:italic;">Chlorella vulgaris</span><span> and </span><span style = "font-style:italic;">Desmodesmus quadricauda</span><span>. It uses ½ SŠ Growth Medium (0.5 g/l). The cells are grown until saturation or when a new split is needed. At the lowest light level, the cell culture split should take place at least once every two weeks.</span></div></div>

[STEPS]
?. [Peparatory Steps]
Warm-up ½ SŠ Growth Medium (0.5g/l) to room temperature.Leave it outside of a fridge for at least an hour, or leave it in a 37 °C water bath for 10 minutes or so.
?. [Peparatory Steps]
Pre-label Erlenmeyer flasks with the culture type, date, own initials, passage number or other pieces of information as required.For sterilizing the Erlenmeyer culture flasks, see Autoclaving Erlenmeyer Flasks for Sterile Microalgae Cultures
?. [Peparatory Steps]
Turn on and wipe clean the sterile hood.
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Take out previous culture passage from the shaker and bring it to the tissue culture room.
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Dissociate cell aggregates on a shaker.Use low power setting to prevent the suspension splashing onto the cotton wool plug..Desmodesmus quadricauda cells do not require as much vortexing. Five seconds is enough.Chlorella vulgaris, when overgrown, equires much more vortexing. The more shaking the better. Long vortexing is especially important when cells are deposited along the flask sides and thus form clumps. A minute or two of shaking is not excessive!
?. [Inoculating Fresh Culture Medium]
Count the microalgae cell culture densityHandle the cultures inside sterile hood. Pipette  of the dissociated cell culture suspension into the hemocytometer.For detailed protocol see Counting Microalgae Culture Density.Count the culture density using hemocytometer and keep the tally.With Desmodesmus quadricauda, count each cell, not just the coenobia, i.e. one coenobium with four cells counts as four cells, one coenobium with eight cells count as eight cells.Calculate the cell culture density:D. quadricauda  \rho_{Ddq}\ \left[\textrm{ml}^{-1}\right]C. vulgaris  \rho_{Chl}\ \left[\textrm{ml}^{-1}\right]
10 µl
?. [Inoculating Fresh Culture Medium]
Calculate the dilution factors for inoculating fresh medium.The seed density in the freshly inoculated medium is:\rho_{Ddq}^{seed} = 2\times10^5\ \textrm{ml}^{-1} for D. quadricauda\rho_{Chl}^{seed} = 1\times10^6\ \textrm{ml}^{-1} for C. vulgarisThe volume of culture V=20\,\textrm{ml}  is same for boths species.The cell density of the existing culture has been calculated in the above step.\rho_{Ddq}\ \left[\textrm{ml}^{-1}\right] is the existing cell culture density for D. quadricauda\rho_{Chl}\ \left[\textrm{ml}^{-1}\right] is the existing cell culture density for C. vulgarisThe inoculation volume of the existing culture is calculated as follows:V_{Ddq}^{culture} = V \times \frac{\rho_{Ddq}^{seed}}{\rho_{Ddq}} = 20\,\textrm{ml} \times \frac{2\times10^{5}\ \textrm{ml}^{-1}}{\rho_{Ddq}} inoculation volume of existing D. quadricauda cultureV_{Chl}^{culture} = V \times \frac{\rho_{Chl}^{seed}}{\rho_{Chl}} = 20\,\textrm{ml} \times \frac{10^{6}\ \textrm{ml}^{-1}}{\rho_{Chl}} inoculation volume of existing C. vulgaris cultureThe volume of the fresh medium is then calculated by subtracting the inoculation volume (calculated above) from the total volume of the culture V .V_{Ddq}^{medium} = V - V_{Ddq}^{culture} = 20\,\textrm{ml} - V_{Ddq}^{culture}  volume of fresh medium for D. quadricauda inoculation.V_{Chl}^{medium} = V - V_{Chl}^{culture} = 20\,\textrm{ml} - V_{Chl}^{culture}  volume of fresh medium for D. quadricauda inoculation.
?. [Inoculating Fresh Culture Medium]
Dispense the right volumes of the fresh medium into the pre-labeled Erlenmeyer flasks:V_{Ddq}^{medium} volume of fresh medium dispensed into the flask for D. quadricauda cultureV_{Chl}^{medium} volume of fresh medium dispensed  into the flask for C. vulgaris culture
?. [Inoculating Fresh Culture Medium]
Inoculate the medium in the Erlenmeyer flasks with the existing cultures:V_{Ddq}^{culture} volume of fresh medium dispensed into the flask for D. quadricauda cultureV_{Chl}^{medium} volume of fresh medium dispensed  into the flask for C. vulgaris culture
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Check that the fresh medium is not too cold, if it was taken out of the fridge only a short while ago. If it is cold, leave it standing for a few minutes, it will warm up quite quickly in the small volume standing on the metal surface of the laminar flow hood.
?. [Inoculating Fresh Culture Medium]
Place the cells back on the algal shaker.
?. [Inoculating Fresh Culture Medium]
Clean up the sterile hood and the lab space. Sterilize old cultures by adding bleach or Virkon overnight.
?. [Inoculating Fresh Culture Medium]
Counting Microalgae Culture Density protocol​Autoclaving Erlenmeyer Flasks for Sterile Microalgae Culture protocolPreparing 1/2 SŠ Algal Inorganic Nutrient Medium.​1/2 SŠ inorganic nutrient medium: Recipe from the Culture Collection of Autotrophic Organisms​​Zachleder & Šetlík, 1982: The original article introducing this medium