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 ) .
