Penicillin to "Clean" algae cultures?

[Conner Berthold]

Hi,

Was wondering if anyone had tried to use antibiotics to make bacteria free, axenic algal cultures? If found this paper on it, and the main problem looks like the need for a mixed antibiotic mixture to effectively remove the bacteria.

-Conner

[Patrik D'haeseleer]

Interesting. I've been wondering about that as well, since we often get cyanobacteria contamination in our dinoflagellate cultures.

Might be feasible to isolate our own dino cultures from seawater when there's bioluminescent waves at the beach. The problem is that the dinoflagellates grow much slower than cyanobacteria, so you'd need some way to give them a leg up...

Patrik

[Mega]

In theory, it should also work with a filter. Dinos are eukaryotes, meaning they're up to 10 times bigger than bacteria. 

Cyanobacteria are big bacteria though. 

from 0.5 to 60 micrometres 

Dinos (here http://en.wikipedia.org/wiki/Noctiluca )

"200 to 2,000 µm in diameter"

So if you choose a 100um membrane, that should allow the bacteria to pass through but leave the dinos behind. Some bacteria may still stick to the dinos in the sieve. 

[Xabier Vázquez Campos]

Penicillin won't work. It is active vs.  Gram+ and Cyanobacteria are Gram-

There is no perfect antibiotic that kills all bacteria. Even using "powerful" broad spectrum antibiotics eg. chloramphenicol 100ppm you may not remove all the contaminant bacteria from your culture.

I had that problem with some fungal isolates contaminated with bacteria in the lab. I finally used Streptomycin sulphate (15 mg l-1, Sigma), Gentamicin sulphate (15 mg l-1, Sigma) and Tetracycline hydrochloride (12 mg l-1, Sigma). It should work too for your algae. There are other combinations but I have them in the lab. I can check on Monday if you want

[Conner Berthold]

Thanks for everyone's input! Sounds like agar plates would be the way to go.

Xabier, I will look and see if I have any of those chemicals available in our lab, otherwise I can get them from the university.

-Conner

On Saturday, November 17, 2012 9:37:21 AM UTC-5, poli wrote:

Yes antibiotics can be used to get a new culture but I wouldn't do it in the liquid phase. Make F/2, TAP, or whatever media you're using agar plates with antibiotics. Spread your culture on it and wait for single colonies. Aseptically transfer a single colony to new sterile media.

[Xabier Vázquez Campos]

OK. I'm back in the lab so... another combination can be (per L)
- Penicillin G (100 mg), Streptomycin sulfate (50 mg), Chlortetracyclin HCl (aureomycin) (10 mg)
- Streptomycin sulfate (100 mg), ampicillin (100 mg)

This two combinations are cited in

Damm, U., Crous, P. W. & Fourie, P. H. (2007). Botryosphaeriaceae as potential pathogens of Prunus species in South Africa, with descriptions of Diplodia africana and Lasiodiplodia plurivora sp. nov. Mycologia 99, 664–680.

Though they are used to clean fungal cultures, it should work with your algae.

[Patrik D'haeseleer]

Hm - wonder whether that would work for our dinoflagellates. I've never heard of growing them on a solid medium but I have to admit I haven't really looked into how they were originally isolated. They do replicate very slowly - every ~5 days - so we'd have to grow them for a couple of weeks on agar before they would form noticeable colonies. Not sure they would be very happy with that.

Patrik

On Saturday, November 17, 2012 6:37:21 AM UTC-8, poli wrote:

Yes antibiotics can be used to get a new culture but I wouldn't do it in the liquid phase. Make F/2, TAP, or whatever media you're using agar plates with antibiotics. Spread your culture on it and wait for single colonies. Aseptically transfer a single colony to new sterile media.

On Friday, November 16, 2012 11:27:17 AM UTC-5, Conner Berthold wrote:

[Conner Berthold]

I would imagine the Dinoflagellates would fair ok on agar. I keep Chlamydomonas on agar and it doesn't seem to mind with it's flagella. Otherwise, only one way to find out!

-Conner

[poli]

Chlamy is actually a soil isolate, but marine algae grow fine on agar from what I have seen. It's a little more important to keep the agar moist by wrapping the plate in parafilm the day after plating, especially for slow growers that will be on the same plate for a month or two.

On Sunday, November 18, 2012 8:48:24 PM UTC-5, Conner Berthold wrote:

I would imagine the Dinoflagellates would fair ok on agar. I keep Chlamydomonas on agar and it doesn't seem to mind with it's flagella. Otherwise, only one way to find out!

It's standard procedure to keep a culture on solid media because it harder to contaminate, easier to see if it does get contaminated, allows one to go back to a parent stock easily, etc. Liquid cultures are really for experiments/production and solid should be maintained for storage. Restreak once the solid culture gets dense and go back to frozen parent stock after 3 restreaks (cell passage). 

Slow growth does put dino at a competitive disadvantage especially if you are trying to isolate it from the wild. I would try to plate varying amounts environmental samples with a variety of different antibiotics. If the strains you are after is a autotroph and can fixate nitrogen only include NaHCO3 and KNO3 to starve organisms that can't reduce its own C+N, I'm not sure if dinos can do both, one or the other, or neither. You can also examine the plate under a microscope before colonies are well formed and poke to start a small liquid culture (I'm impatient too). If you're interested in going further with the isolation aspect it may be worth your time (not sure about money) to do a kill curve and see how much antibiotics dino can take. And of course always dispose of your biohazard material (anything cells touched) according to standard safety protocols.