Isolation and isotopic labeling of bacteria
Prepare M9 media stocks .
The recipe for 1L of minimal growth medium ( M9 ) is as follows :
5x M9 salts ( autoclaved ) 200 mL ; D - glucose stock ( 20 % , 0.2µm filter sterilized ) 20mL ; Vitamins stock ( 0.2µm filter sterilized ) 0.5 mL ; Trace Metals stock ( 0.2µm filter sterilized ) 1 mL ; 1 M MgSO4 ( autoclaved ) 2 mL ; 1 M CaCl2 ( autoclaved ) 0.1 mL ; MQ H2O ( complete to 1L ) .
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 ) :
KH2PO4 15 g ; Na2HPO4 ( anhydrous ) 34 g ; NaCl 2.5 g ; NH4Cla 5 g .
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 ) .
