Dear Samantha,

It depends with which protists you are working and - as for any other organism - what are their growth limits. For example, if you work with marine or brackish organisms i would be surprised if they survive in LB.

In order to find a solution for your question, i guess you have two options: do some literature research and find out about the typical cultivation conditions for the protist of interest or perform experiments to find out if and how long your organism can survive in LB.

A potential additional problem is that the bacterial biomass in LB likely will be much higher than in water+fish food - this might be become critical (e.g. due to oxygen depletion, accumulation of waste products and/or secondary metabolites...)

Certain protists which usually feed on bacteria can be cultivated axenically i.e. in artifical medium without any bacteria (Paramecium tetraurelia for example). That medium is far more complex than LB.

If you want to have a simple but defined medium: many freshwater protists are happy in Volvic mineral water (of course it might grow better if you use the medium recommended for the cultivation of your protist). You can still grow your bacteria in LB, harvest and resuspend them in Volvic and add this suspension to your cells.

Martina

I agree with Martina. It very much depends on which protists and which bacteria you are workig .with For example, your protists may depend on bacteria that do not grow in LB, because the pH is wrong or a nutrient is missing, for example. I also agree that overgrowth of bacteria is a bad thing - the pH will change due to the bacterial waste products, which also may be toxic to your protists. There are broad range media that work for a lot of organisms, but one thing to try if you already have LB is to prepare a 1:10 dilution (or even 1:50) in phosphate buffered saline (or perhaps Volvic!) - this will help reduce the bacterial growth. It is a case of getting the balance correct between bacterial growth and protist growth in a medium that provides all the necessary nutrients under the correct conditions.

Start with a good spring water for freshwater bugs, artificial seawater is OK for salt but filtered seawater is better. LB is definitely too rich, you could try adding 100ul to 100ml and might get plenty of bacteria. you can also make LB agar and cut chunks out of a plate to add to your cultures to support bacteria growth. For really clean cultures though, use the LB (or other broth medium) to grow a strain of bacteria that the protists will eat, then centrifuge 10,000xg to pellet the bacteria and reususpend in the spring water or salt water, three times will get rid of metabolites and ammonium. Count the density of the final bacteria suspension and keep it at 4 deg C as a food stock. Dilute in culture fluid to about 10^7 per ml and track protist growth to stationary phase. this will give you very clean protist growth dynamics and cell preps for other uses.

Samantha: we are many protistologists even though their is no Kingdom of Protista: First of all, specify, which protist feeding upon bacteria do you want to cultivate.

Cheers

Dear all,

Thanks a lot for your help and suggestions. You confirmed my doubts about the use of LB media. It seems very nice for the design of my experiments, and I can now check different aspects of its possible limits.

We've been working on morphospecies until now so what I can say at the moment is that we have ciliates (colpidium colpoda, cyclidium) and flagellates (chrysomonads, bodo), rotifers and some that we haven't determined yet.

Indeed, as you suggested Martina, it could be interesting to have a media with no bacteria. I will read more about this possibility. I have to admit that I wasn't aware that it was feasible to avoid completely bacterial presence in protists cultures.

About the cultures in the presence of bacteria, I will perform some experiments with smaller concentrations of LB broth as you all suggested in order to get the correct balance for both protists and bacteria to develop.

The chunks of agar and the defined food stocks seems two promising ways of solving both overgrowth and the need of a clean media. I will definitely test these methods and very probably apply the defined food stock, thank you Richard.

I will continue brainstorming with all your inspiring inputs in mind.

I might get back to you if any more questions arise in the process.

Thanks again,

Samantha