Monitoring Growth

Bacterial growth was monitored using viable counts and for axenic bacteria optical density measurements at 600nm were also obtained. Algal growth was monitored using both coulter counter measurements and viable counts. 100µl culture sample was diluted with isoton buffer solution to a total volume of 10ml, which was then gently mixed and particle density counted using a Beckman Coulter counter, aperture size 100µm, calibration constant (Kd) 60.05 and size range 3.5-12 µm. For viable counts a series of 10x dilutions were done with 20µl added to 180µl sterile water. Appropriate dilutions were then inoculated on TY agar plates by placing a 20 µl drop on one end and tilting the plate to create evenly distributed streaks. For viable counts of algae, the plates were incubated at 25⁰C in constant light and for viable counts of bacteria the plates were incubated at 30⁰C in the dark.  

Sample Preparation for SIMS Analysis

To prepare samples for SIMS (Secondary Ion Mass Spectrometry) first the sample was fixed with 2% formaldehyde, the samples were then washed and resuspended in a 1:1 by volume mix of autoclaved 1xPBS buffer and 96% ethanol solution, then stored in at 4⁰C until required. The samples were stained using SYTO9 nucleic acid stain and then the cells were deposited, by vacuum filtration onto filters with 0.2µm pores (GTTP…) that had been pre-sputtered with gold. The filters were then imaged using an Olympus Fluoview Laser Scanning Confocal Microscope to confirm the even distribution of cells on the filter before sub samples were cut out. The sub-samples were then marked with a laser and imaged with a Zeiss laser micro-dissection microscope using a x20 objective. These images were used to determine the areas of the filters to analyse and confirm which algal cells were analysed by SIMS. The sub samples were then placed on conductive adhesive tape, coated in gold and mounted onto a … sample holder.

Secondary Ion Mass Spectrometer (SIMS) Analysis

SIMS measurements were carried out at the Nordsim facility at the Natural History Museum, Stockholm, Sweden. Laser marks were used to identify areas of interest. The run routine used was a 10 second pre-sputter across a 45x45 µm area followed by the measurement collection of 12C14N and 13C14N for 100 planes over a 35x35 µm area. The mass resolution used was 7000 and the primary ion beam was about 60-80 pA.
The SIMS images were analysed using the WinImage software. The 100 planes were collected accumulated and the ROIs for the bacteria cells were selected manually as ellipses in the 12C14N image. For algal cells a slightly different analysis procedure was used because they become much more highly labelled and the carbon-13 is not homogenously distributed across the cell. Therefore to minimise the bias when selecting ROIs for the algal cells after accumulating the 100 planes the 12C14N and 13C14N images were summed (in a 1:1 ratio) to create a linear combination image showing the distribution of total carbon across the sample. Using fluorescence images as a guide the algal cells were then manually selected in this linear combination image. The carbon-13 isotope ratio was then calculated for each ROI as the mean for the 100 planes using the on pixel correction mode to account for the lag time in switching between detection of different masses. For algae, three sets of 100 planes were analysed (unless otherwise stated) and the mean of these three measurements calculated to obtain an estimate of the carbon-13 isotope ratio of the algal cell.
After SIMS analysis the analysed algal cells were imaged using an Olympus Epifluorescence Microscope.
Data processing and analysis was carried out in Matlab.

Mathematical Modelling