Isolation and isotopic labeling of bacteria
Prepare M9 media stocks.  
The recipe for 1L of minimal growth medium (M9) is as follows:   
After cooling of the autoclaved mix, add the rest of the components by 0.2 µm filter sterilization.
In order to prepare solid media for plating, add 1.5% agar (15 gr agar per 1 L of media) prior to autoclaving.
Allow cooling of the media prior to add the 0.2µm filter sterilization components.
Pour the media in Petri dishes and allow solidification of agar.
To prepare 1 L of the 5x M9 salts stock (autoclaved). 
For isotopic labeling, use D-glucose-1-13C (99% atom 13C) and ammonium-15N chloride (98% atom 15N)
Vitamins and trace metals stock used were the same as for algal DY-V media.   
Bacterial plating.   
Use a small volume (i.e. 0.1 mL) of the sample you want to isolate your bacteria to inoculate M9 agar plates.
Ensure correct striking and dispersal of the inoculum to facilitate picking of individual colonies afterwards.
Bacterial growth  Incubate bacteria at a temperature of choice.
Bacteria isolated from the algal cultures were chosen to grow at 30 ºC.
Inspect plates after 24h until big enough colonies are visible to pick.
Picking and transfer of colonies.
Using sterile picks, transfer a single colony of bacteria into M9 liquid media.
Recommended to do a first transfer into a small volume (~ 25 - 50 mL).
Prepare a battery of tubes beforehand with M9 liquid media and pick a variety of colonies.
Production of labeled bacterial biomass.  
In order to produce a large ammount of biomass, transfer some of the bacteria from the M9 cultures tubes to a larger volume (i.e. 1L).
Let bacteria grow until media is completely cloudy and a maximum yield is achieved of biomass (can be 3-4 days until C and N is consummed).
NOTE: for isotopic labeling, M9 media used in this step will contain 15NH4Cl and 13C D-glucose.
Production of HKB.  
Once you have enough biomass, follow up with the protocol for making heat killed bacteria (start at the 3rd step).
