Problems with some botles of nan

[JulioC]

I hace a culture with a few botles of nan.
On sunday I split 2 botles into 5 new botles. 4 of them are doing great but one is geting clear. Looking at it you can see litle dots of algae. It is like it´s beeng floculated. Water culture, base culture, light and everithing is the same in the 5 botles?

Does anyone know what might be happening?

Julio

[Luis A M]

Have you seen viewtopic.php?f=145&t=324

[JulioC]

Yes, I´ve read it last year but the problem is that I´m succesful with 4 out of 5 botles. I´ll take some pictures to show. When I started It was impossible to have a crush but last year it has been more difficult.

What Id didn´t remember of your method is how you keep shock cultures just in case. I´ll do it to be prepared for a total crash!

I cleam my botles and rigid tubes with bleach and acid to be sure there are nothing in there. If I have a crush I replace airline with a new one.

[Luis A M]

Julio,I´m pasting below about the stock cultures.Let me know any question /doubts.

Flasks
Stock cultures are kept pure and possibly sterile in flasks. Each flask is filled with 60 ml of medium, cotton stopped and sterilized in the microwave oven. When cool, 10 ml of a thriving culture is seeded with a sterile pipette.
I keep three flasks for each algal species; old, medium and new. I seed new cultures 2-3 times per month, so this is not too much work. Every morning I agitate the cultures by swirling.
I need to keep stock cultures because it is very difficult to obtain them here. In places where they can be easily replaced when lost, one can do without them.

[JulioC]

I´ll start keeping sterile cultures in flasks to be safe.

I have between 5 and 12 bottles and generally don´t need a back up culture but it is good to be in the safe side. Generally 1 or 2 cultures crushes and the rest do well.

Sometime I keep 1 flask of culture and one flask of rotifers in the fridge just in case. Now I have faf disk that I´ll try to fill with algae. I´m planning to sediment some nan and place a concentrated layer of nan in the disk to preserve it. I can´t concentrate Tet because it does not precipitates because is motile.

I use inverted Coke botles so I don´t need to agitate them daily. Maybe that´s my mistake.

Julio

[Luis A M]

Julio,I use 100 ml ehrlenmayer flasks.I fill them with 60 ml of medium,cotton stop them and sterilize them in the microwave.I seed them with 10 ml of the middle age flask (30 days old) and use the older one (45 days) to start a bottle.Bottles are kept upright,aerated and used once and discarded.

[Luis A M]

double post

[JulioC]

Thanks Luis.
I used to discard botles after 2 or 3 uses but now i´m sterilizing them. I´ll start using new botles. Perhaps bleach is not enought and some spore is contaminating everithing. Yesterday I observed that the problem looks like some spores that multiply eating phyto.

[JulioC]

I used 2 ehrlenmayer flasks to try and both one with nan and one with tet. The one with nan become very dark but the one with tet not.

I also started new cultures with ne botles covered with cotton and all are doing fine. It seems I was not giving contamination enought atention...

Thanks,
Julio

[Luis A M]

Julio,you can keep stock cultures pure (unialgal) and possibly sterile,using ehrlenmayers sterilized in the microwave and sterile pipettes.and then you can always start bottles with uncontaminated algae.
Are you in B.A.?.Our tap water frequently is contaminated with Chlorella.

[JulioC]

Yes, I plan to use ahrlenmayers as stock cultures. I use RO water, I don´t know if that will separate chlorella or not.

[Luis A M]

I haven´t considered that,but yes,if RO membranes can stop ions,much more bacterial or algal cells
Chlorella are large,round cells.

[JulioC]

I think I found the problem with my phyto cultures...

Light bulbs where more than 1 year old and maybe that is why cultures were not strong enought. I noticed an improve when I started covering botles with coton but now it seems they develop faster.
Ehrlenmayers continue doing well as back up cultures!

[Luis A M]

Can´t you try natural light?

[JulioC]

Unfortunatelly not now but I´m building a new growout room...
Since reducing heating/cooling costs is my goal I´ll build it with Retak and no windows. I´m planing to add some skylights to naturalll lighting but I´ll try to include a place to grow phyto.

If I can grow phyto with leds lighting will be very cheap and long lasting but I´m just starting.