Isotopic labeling of freshwater mixotrophic algae using isotopic labeled heat - killed bacteria
Prepare modified DY - V media .
Media used for Ochromonas growth was a modified version of DY - V media .
Modifications were : No MES was used , as it can be used as a source of carbon ( bicarbonate and heat - killed bacteria are the only sources of carbon used in this media ) .
No Na2SiO3 was used ( Ochromonas does not need silica ) .
No NaNO3 ( ammonium and heat - killed bacteria were the only sources of nitrogen used in this media ) .
Sodium bicarbonate added at a final concentration of 95 µM .
Bicarbonate addition is done before algal inoculum by 0.2 µm filtration of a stock solution ( do not autoclave ) .
Media inoculation with labeled HKB .
Add 15N 13C labeled heat - killed bacteria ( HKB ) to the media ( isolation and labeling of HKB was done following this protocol ) .
Add an inoculum of the mixotrophic algae ( volume of inoculum should be low as to avoid dilution of the isotopic label and carry over of nutrients ) .
NOTE : as an example , values commonly used were ~ 5x103 Ochromonas mL - 1 and ~ 5x107 HKB mL - 1 as starting concentrations for the cultures .
Mixotrophic growth of the algae Let the algae grow so it incorporates the isotopic signature in its biomass .
Track the algae growth through microscopy ( live samples and / or fixed samples ) and the decline of heat - killed bacteria ( fixed samples and staining with DAPI to assess HKB concentrations through epifluorescence microscopy .
Sampling for assessment of algal isotopic signature Ochromonas was allowed to grow for 2 - 3 generations before sampling for isotopic signature .
Two kind of samples can be collected :
Bulk measurements : filter 30 - 50 mL of the cultures onto pre - combusted glass fiber filters and dry at 60 ºC over night to stop all biological activity .
Afterwards , filters can be stored in glass vials at room temperature .
Further processing of the sample included an acidification step with HCl to remove inorganic carbon and the C - and N - isotopic composition of the sample was determined by an isotope rato mas spectrometer ( IRMS ) .
Cell - specific measurements : collect 2 mL of sample and fix with 2X EM - grade glutaraldehyde .
Sample can be stored in the fridge at 4 ºC .
Further processing of the sample involves the deposition of cells onto sylicon wafers , wash with MQ - water and drye ; map cells on the waffer using microscopy and analysis of single cells using a Cameca NanoSIMS 50 instrument .
( NOTE : actual manipulation of the machine will be done by an expert user ) .
