Nutrient deplete/replete algal culture for elemental analysis
Culture Preparation/GrowthComparative measurements depend on having well-characterized culture growth.
Initiate an experimental culture to allow for at least 7 generations of characterized exponential growth (10 generations is the gold standard) prior to centrifugation and resuspension.
Monitor growth daily, including transfer/dilution to pre-determined mid-exponential density (i.e., semi-continuous culture) until target biomass is reached.
A safe estimate for the target biomass is to assume ~10% recovery of cells after centrifugation and washing (this is strain dependent).
Ensure cell density remains within the exponential range by increasing culture volume (not cell density) when ramping up to target biomass.
Example Ostreococcus lucimarinus (CCMP2972A) culture preparation:
Growth conditions: 18°C, 14:10 hour light:dark cycle, light irradiance of ~100 µE m-2 s-1.
Growth media: L1 with natural seawater base.
Exponential range: 7x105 – 2x107 cells/mL Day12345678910.
Initial density (mL-1)3x107Skip1.5x1071x1079x1068x1061x1079x1061x1071.4x107.
Growth Rate   (d-1)NA0.5270.6280.5580.3840.7200.6390.5540.261.
Dilution/ TransferTransDilDilTransDilDilDilNA. 
Final density (mL-1)5x1065x1065x1065x1065x1065x1065x1061x107. 
Volume (mL)30x386x3172x380x9125x9257x9401x9401x93.6 L (use!)~7 generations of growth prior to centrifugation.
NOTE: Extra-large volumes used for omics experiment Concentration and Resuspension.
When target culture biomass has been reached, split volume evenly among 2 or 4 acid-cleaned and autoclaved centrifuge bottles.
Weigh and balance bottles by removing volume until bottle pairs are within 0.01 g of each other.
Only open bottles in hood to maintain axenicity!
Carry bottles over to Sorvall RC 26 Plus centrifuge.
Fit centrifuge with large rotor (accomodates 4 centrifuge bottles).
Make sure pins are offset when rotor is placed into the centrifuge.
Place paired bottles opposite of each other in the rotor and tighten the lid.
Spin at the following settings: Rotor = GS-3 (choose option code “03”), Speed = 7300 RPM (equivalent to 10,000 rcf with this large rotor), Time = 0:32 minutes (includes 2 minute ramp up), *strain specificTemp = +20/+25°C, (will try to maintain lower temp, and shut down at max temp).
After the spin, carefully transport the bottles back to hood, taking care not to resuspend the cell pellets/smears.
Gently pour off the supernatant into a waste container, trying to disturb the cell smear as little as possible.
Resuspend/wash cells with a volume of nutrient deplete medium equivalent to the initial culture volume in each bottle.
Repeat 2-8.Resuspend cells with a volume of nutrient deplete medium that is ~5% of the initial culture volume in each bottle.
Run a sample of the cell concentrate in the Accuri to determine density.
Store cells at normal growth conditions during Accuri run and calculations.
Experimental Culture Initiation.
Calculate appropriate volume based on Accuri measurement to inoculate experimental nutrient replete and deplete culture flasks from the cell concentrate at a starting density corresponding to early-exponential growth.
NOTE: It helps to have some amount of media pre-aliquoted to experimental flasks.
Adjust as needed to achieve target density and volume.
Once all experimental cultures have been inoculated, mix and sample for FCM and Accuri measurements to monitor growth daily.
When the average daily growth rate of the nutrient deplete cultures (GRDEP), is reduced to half or less of the growth rate of the replete cultures (GRDEP/GRREP < 0.5), collect samples for FCM (i.e., cell counts) and elemental analyses (i.e., particulate carbon/nitrogen and phosphorus, as well as dissolved nutrients).
Sample CollectionFlow Cytometry (FCM, for cell counts).
Transfer 1 mL of culture to a sterile 1.2 mL cryovial tube.
Add 10 µL 25% glutaraldehyde (0.25% final conc) and gently vortex to mix.
Aliquot 500 µL to a duplicate cryovial.
Snap cryovials into cryocanes and incubate at 4°C for 30 minutes in the dark.
Flash freeze in liquid nitrogen.
Store at -80°C until analysis.
Dissolved and Particulate Elemental Analysis (EA, for cellular elemental composition & dissolved nutrient concentrations).
NOTE: Separate filters are needed from each sample for POC/N and POP analyses!
Additional filters must be collected if you want technical replicates for each sample type.
Set up an acid-cleaned and autoclaved glass filter unit with pre-combusted 25mm Advantec glass fiber filter.
*Use ethanol-cleaned forceps in hood!
Apply 20-40 mL of media or culture to the filter funnel and turn on the vacuum until the filter is dry.
Use ethanol-cleaned forceps to fold filter in half and collect in a 12-well plate or piece of combusted aluminum foil.
Pour filtrate into an acid-cleaned 50 mL conical tube.
Repeat for sample duplicate.
Rinse filter tower with MilliQ between samples.
Store filters and filtrate at -20°C until further processing.
DAPI (for microscopy to assess axenicity).
Transfer 1 mL of culture to sterile 1.2 mL cryovial tube.
In the chemical hood, add 100 µL of 37% formaldehyde (3.7% final conc) and mix by inverting.
Do not vortex.
Snap cryovials into cryocanes and incubate at room temp for 15 minutes (in the dark).
Store at 4°C until analysis (can be stored for several days or flash frozen in liquid nitrogen and stored at -80°C).
Sample Processing.
Samples for elemental analysis should be sent to Analytical Services at Horn Point Laboratory (University of Maryland Center for Environmental Science, UMCES).