Culturing cyanobacteria - urgent advice please!!

[jmjabt]

Hello,

 

I am currently trying to start a series of trials using Oscillatoria tenuis, Anabaena circinalis and Planktothrix aghardiulture the. I have obtained my cutures from CCAP in Cambridge, and am starting to try to culture them. However, I can find very little information on-line on culture techniques, advice, etc.

 

Three weeks ago, I inoculated the cultures at a ratio of 1:10 with UTEX medium as advised by CCAP. I was not sure on light levels so I put them in a slightly shaded window with very gentle aeration.

 

After two weeks I placed them on a light bench with around 5000 lux of standard white fluorescent light (this as the only value I could find on-line). I have since removed the aeration in case this was having an effect. I now have very slightly green water, a little cliudy, but no major growth. I was advised that after 4 weeks I should inoculate again at 1:10 when there was a dense culture. I am nearly at that point and I cant't see any filaments or anything of the sort.

 

I really need someone's help as this is an expensive project, and they are expensive cultures, and I cant get them to do anything... If required I can add pictures.

 

Thanks in advance,

 

Jack

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[CharonY]

There are a lot of papers around describing culture conditions, although there are apparently variations. One of the older ones is Stanier RY, Kunisawa R, Mandel M & Cohen-Bazire G (1971) Purification and

properties of unicellular blue-green algae (Order Chroococcales). Bacteriol. Rev. 35: 171-205.

 

5000 lux appears a bit on the high side, (most I see is around 2-3k) but that should not be a fundamental issue. Did you maintain day/night cycles?

 

It depends a bit on the form and size of the tank and whether it really gets enough light. I.e. is the vessel suitable for cultivation? If the vessel is sealed and does not have a lot of headspace your culture may run out of CO2 without aeration.

 

Another factor may be errors in media preparation, of course. And finally, what temperature do you have and how is it maintained (this is a major factor)? Putting them near a window does not sound like a very controlled condition.

[Moontanman]

I wish i could be of help, I culture unicellular algae regularly as food for daphnia and fairy shrimp. I've been doing this for many years but the result is never a mono culture because I use naturally occurring algae spores. One of the most important things in culturing algae I've found Is phosphorus, i use "Navel Jelly" a rust remover as a source of phosphorus. approximately one tea spoon per 70 gallons of water. I know this is probably not much help but culturing algae is as much a skill set as it is science.

 

What i am trying to say is it took me a long time to figure out how to consistently come up with "green water" on demand. Of course when you don't want it unicellular algae is everywhere you don't want it to be. Practice makes perfect....

[StringJunky]

Here's a page with cyanobacteria media recipes...if you study the makeup of a bunch them you might see some commonality between them that is lacking in yours.

 

http://www-cyanosite.bio.purdue.edu/media/table/media.html

 

On light levels:

 

Illumination

 

Generally, cultures should be grown in a 16-hour light period alternating with an 8-hour dark period. Ideally, the cultures should be illuminated by 40-watt cool-white fluorescent tubes on a timer. A 40-watt fluorescent tube at a distance of about 15 cm will provide roughly 500 foot candles of illumination. At a distance of approximately 50 cm, the illumination will fall to approximately 200 foot candles. An inexpensive light meter calibrated in foot candles can provide a simple and accurate way to regulate the light intensity in your lab.

 

Freshwater algal cultures should be grown under a light intensity of 400 to 500 foot candles. At this light intensity, cultures will reach optimum growth in 7–14 days, depending on the species and condition of the initial algal inoculum. After this period, reduce the light to 50 –100 foot candles.Marine algae grow best in slightly lower intensities than those required by freshwater algae — 200 – 300 foot candles.

 

The light cycle and growth period are the same as those recommended for freshwater cultures.

 

http://openwetware.org/images/8/85/Working_with_algae.pdf

Edited June 20, 2012 by StringJunky

[jmjabt]

Hi,

 

Thanks for the comments. The medium is UTEX as advised by the Culture Collection of Algae and Protozoa. They are no longer in the window and are in a stable temperature of around 28, on 16 hour day and 8 hour night.

 

Based on the advice I will put them a little further from the bulb to give around 3000 lux in the centre of the vessels. These are early in the scale up process and as such are only 120 ml in volume, so light penetration isn't an issue. Upon swirling, there are some 'flakes' for want of a better word, but I cant see any filaments.

 

I have got the paper you mentioned above, but my alga are not in this order, and it seems as it mentions that some species require low light (500 lux) even in the same order, it is hard to know what conditions my species require!!

 

I am still trawling the web for free access literature... I was led to believe I should have dense cultures after 4 weeks to go to the next scale up stage, but this is not the case, hence my worry...!

 

Cheers all,

 

Jack

[CharonY]

Well for the genera you mentioned I generally found values around 2-3k (you may also want to check the ATCC website, I assume they should also have info on it). I remember at least on continuous culture approach with Oscillatoria (I think) that was even down to 500. But I do not remember details on growth kinetics (and the goal is obviously different from batch).

[absegretin]

Hi! I'm working with cyanobacteria at the moment and I'm having similar problems with my samples. I wanted to know if you coud finally find what was happening with the cultures and made them work...
Thank you

[StringJunky]

Hi! I'm working with cyanobacteria at the moment and I'm having similar problems with my samples. I wanted to know if you coud finally find what was happening with the cultures and made them work...

Thank you

 

The OP has not been here for over a year so it is unlikely you will get response.

[jmjabt]

Basically, I switched to BG11 from the UTEX, and they grew from the tiny amount of innoculum I had left after all else failed! I increased light from 2000 lux to 5000 over a couple of weeks as the cultures grew. The project is over now so the cultures have been taken down, but they did grow very well on home made BG11.

 

Hi! I'm working with cyanobacteria at the moment and I'm having similar problems with my samples. I wanted to know if you coud finally find what was happening with the cultures and made them work...
Thank you

[StringJunky]

Forgot about notifications.