Making heat - killed bacteria for feeding axenic phagotrophic protists
Obtaining a clonal bacteria strain ( Skip to step 2 if one is already available ) Prepare 1.5 . % agar plates with 0.5 % yeast extract and 0.5 % trypton .
Use the salinity that the protist culture in growing in i . e . 0.5 g yeast extract + 0.5 g trypton + 1.5 g agar in 100 ml of MiliQ water ( MQ ) / Filtered sea water ( FSW ) .
Heat to dissolve .
Autoclave to sterilize .
Pour into medium sized petri dishes .
When the agar plates are cooled and set , streak some liquid from a bacterized culture of the protist on the agar plates .
Incubate the agar plates at the same conditions of the protist culture
When bacteria colonies form , use a flame sterilized metal loop to pick one clonal strain and dip the metal loop into a nutrient broth ( 0.5 % yeast extract + 0.5 % trypton in MQ / FSW ) .
Let the bacteria grow in the same incubating conditions .
Grow a large amount of the bacteria .
Prepare 1 L of bacteria nutrient broth ( Or however much depending on how much bacteria you want to grow ) 0.5 % yeast extract + 0.5 % trypton in MQ or FSW and autoclave to sterilize .
Transfer 0.1 ml of the bacteria culture into the broth .
Let the bacteria grow for ~ 4 days .
Heat - killing the bacteria filled 250 ml centrifuge bottles entirely with the bacteria culture .
Place in water bath at 70oC for 30 min
Centrifuge and wash the bacteria .
Centrifuge the heat - killed bacteria at 10 k g for 20 min .
Decant supernatant .
Add ~ 25 ml of sterilized MQ / FSW ( depending on the salinity of your culture ) in the bottle .
Vortex / shake to resuspend pellet .
Add MQ / FSW to fill to 200 ml and centrifuge again at 10 k g for 20 min .
Repeat this step until you have washed the bacteria 3 times .
After washing for the last time , centrifuge and decant supernatant .
Add as little MQ / FSW as possible and vortex / shake to resuspend pellet .
You may also sonicate the concentrated bacteria if a sonicator is available .
Test the sterility of the bacteria .
Test the sterility of the bacteria by placing a dilute aliquot of the heat - killed bacteria in 5 ml of 0.5 % yeast extract + 0.5 % trypton broth .
Make sure that the liquid is still clear after adding the bacteria so that it will be possible to tell whether the liquid became cloudy if there was bacterial growth .
Incubate the sterility test for several day .
The bacteria is sterile if there is no cloudiness in the test after 5 days .
