basic strategy review

[shorty]

I'm brand new to all this. Occelaris started breeding, and I wanted to try my hand at raising some babies... because I think it'll be fun and a great experience. In line with that, I want to do things as inexpensively as possible, and figure I want to start out with the FULL experience in growing phyto to feed the rots, instead of using prepared/dead rot feeds. I'd considered growing phyto previously anyways, so this helps motivate me to get me going.

I would like someone feedback to my plan, and I have a few questions.
I have nanno and tetra (I think) cultures in the fridge in bottle form. I expect/hope to be set up and culturing by Sunday evening.

Setup:
* I have several 750ml (old wine bottles) that I plan to use, because I wanted to use glass vs plastic. an old 4ft T8 flourescent sitting in the garage to use for lighting.
* Washed them once with bleach, but plan to acid wash all vessels after assembled - and for future cleanings. (using muriatic acid from hardware store)
* I believe I'm planning a typical setup - with rigid airline tubing in each culture vessel - one down to bottom for air feed, a smaller hole in cork top for ventilation/equilzation.
* Air pump mounted above culture station, with airline 'manifold' to feed each valved individually
question 1) will running long vertical runs to each vessel from the manifold (within reason) reduce chances of cross contamination? In addition I've also read it's good to have an .2 micron air filter on each line. question 2) Anyone have a suggestion as to where I can find these? or make my own? I've done some quick searches without any results. What should I search for?
* Put a tap and valve near bottom of a 5 gal bucket and use it for my new water make-up (for easy despensing of water into culture vessels). Mix salt with tap water each time to fill this bucket. question 3) What specific gravity? (1.019 - 1.021?) I've read so many different specific gravity targets, what should I use? I was planning 1.019.
* add granulated pool Chlorine (figure i can find some at local store somewhere) - add to new make-up water/5 gal bucket. (about 1/8th tsp per 5 gal bucket from what I've read).
* in addition I purchased 2 part f/2 from fritzpet I intend to use as a feed.

Procedure:
1) prepair my make-up water in 5 gal bucket, add the chlorine, and then leave sit (covered) for 24 hours. question 4) Can I add the f/2 at this stage to the makeup water with chlorine in it? If not, is it necessary to filter or sterilize the f/2 media in any way? Please note: that this water could sit there for up to a month or maybe more, depending on how many vessels I get culturing. I have no idea at this point how long it will sit, but I'm assuming as long as it's chlorinated, it should be good? Is there any amount of time that it would be advised to re-chlorinate the water (meaning, after sitting x days, re-chlorinate... with ?new amount of Chlorine) ?
2) acid wash culture vessels to be used. question 5) do i rinse after wash with tap water? Do I have to let the vessel dry out? - seems like either of these could provide a contaminant.
3) fill culture vessel with prepaired water/media about 4/5 the way up. Dechlorinate. (add f/2 after this step if necessary) question 6) any amount of time to wait needed after this step?
4) add innoculant/culture - replace plug with tubing.
5) attach to air line header and apply light for 16 hours/day

Am I missing anything?

[shorty]

brand new to this forum by the way!! Just found it last night. Looks like it's gonna be a great resource with great wealth of knowledge.

[Luis A M]

Welcome Shorty!
Sounds like a plan.I assume you have read our tutorial: viewtopic.php?f=145&t=324
Some few comments/questions.
You can fertilize the medium and then sterilize with Cl.
Isn´t household bleach easier to use and dose than granulated?
I prefer SG at 1.010.Any will do,though.
I typically use all the prepared medium within a week time.Perhaps you should re-chlorinate after this time.Why not making 2 gals or one,so you use it sooner?
After de-chlorination the medium is immediately ready.
Is natural light out of question?
Do you have a microscope?

[shorty]

thanks! and thanks for the reply!
I did read your initial post. I need to read the rest of the thread. I saw your 1.010 there, but I've read so many differences here... 1.019 was what stuck out in my head when I wrote my post.

You are right, bleach is probably easier, and I already have it... I guess I'd just read a couple people's posts about how they preferred chlorine for whatever reason. So, you recommend 1.5ml/6L .. I'll start with that!

I can mix less water at a time which is a good idea - I had not really thought of the chlorine/bleach gassing off until I was actually writing my post. But the bigger the batches, the less over-all work it'd be too. So, I guess I could probably use test strips to determine the rate that I'd need to re-sterilize.

I don't have a microscope. But I'm thinking that I could bring a couple samples into work and 'sneak' into the lab to look at them. I'm assuming you are suggesting using a microscope to determine how pure my cultures are?

What about the .2 micron filter? any idea where to find these? I see that you use individual pumps for each culture vessel, but i don't think I can swing that at this point.

[shorty]

well, maybe my first attempt at this reply didn't go through... or didn't get approved... don't know.
but thanks!

I have read the tutorial! and finishing up the rest of that particular thread now.
I can use bleach, I was only thinking of pool chlorine because I'd read on a couple posts where people preferred it. I see that you recommend 1.5mL/6L of bleach to water. I'll start there!

And yes, that's a good idea to mix less media at a time - I didn't even think about the potential off-gas of chlorine until I was writing my post. But, at the same time, the more I'm able to mix at once, the less time consuming that part of it would be (... probably)... so I could always use test strips if needed to determine the rate of gass-off and how soon I'd need to re-sterilize. (and MAYBE if it's sealed, this will be minimal?) Don't know.

Hmmm.. natural light would be actually be great. I'm in the mid-west so my guess is that a South facing window would probably be best? I don't know if I'll be able to get my wife to buy into setting up green wine bottles in window cills, though. We'll seee... I'll have to put some thought into this.

I do not have a microscope, but I could probably get a couple samples and 'sneak' into the lab here at work from time to time. What should I be checking?

[shorty]

Im a little less than 3 days into the culture (started Sunday afternoon). Left is Nanno, and right is Tetra. Tetra is obviously darker green. Nanno is almost equally as ‘cloudy’/translucent, but less green…. That worries me a bit, as either something has gone wrong, or conditions are favoring the Tetra for whatever reason.

Considered factors:
1) I added more parent culture initially to my Tetra culture because it was much lighter to begin with. I used probably close to half the 4oz bottle of Phyto2, where I added more like 2/5 or even 1/3 the bottle of Nanno to get similar ‘green-ness’ to start with.
2) Temp swings – it’s been getting cool at night, so probably swinging down into the mid to upper 60s at night in the house, then up to 73-74 during the day with A/C running. In addition they are right next to a glass window, so they might feel stronger temp swings with the cool temperatures radiating through the glass at night. Would this affect Nanno more than Tetra?
3) I thought I rinsed them equally – but maybe I didn’t rinse the acid wash out of the nanno bottle as well? Would this kill, or just slow the culture?
4) De-chlorinator – I used the same amount in each, so I don’t THINK this is the issue.

At this point, I don’t know if the culture is just growing slower, or there is die-off. I have no experience.

Am I on track? Should I wait it out? Or should I re-think my procedure?
*Maybe let acid washed bottles sit and dry?
*Consider temperature factors and move them to a more temp regulated place with flourescent lighting? (i'd rather not unless absolutely necessary)
*other ideas?

[Luis A M]

You´re on track,you must wait at least 7 days to assess how it´s going.

[shorty]

Thanks. that helps me feel a little better.
i'm planning on building a 'wine' rack tonight that will hold more bottles and will allow me to neatly route out paths in the wood to run all my airline within and hold all the cultures as compactly as possible. The plan is to set that in the family room/fish tank room in front of door sized window behind a curtain. Don't worry, I won't tell my wife it was YOUR idea to use natural light instead of flourescents... lol.
For some reason she's not a fan of these things sitting in her kitchen window sill. ... She's great though, and patient with me. Hopefully the 'wine rack' will look somewhat acceptable.

For anyone interested, I am using Phyto2 cultures from aqua-tech co. Also fritzpet 2 part f/2 formula. I'll post my progress, and I'm assuming I'll have lots more questions.

Oh!, question: so in what way should I use the microscope? I had considered taking samples of my parent and child cultures of each of these for comparison. Not really sure what I'll be looking for, though. Making sure I have monocultures for one, I'm assuming, and looking for other contaminats?

[Luis A M]

Oh!, question: so in what way should I use the microscope? I had considered taking samples of my parent and child cultures of each of these for comparison. Not really sure what I'll be looking for, though. Making sure I have monocultures for one, I'm assuming, and looking for other contaminats?

You won´t need to count cells,overall darkness of the culture is enough.But you will see if you still have the intended species or some invasive allien

[shorty]

i read one source where someone continually puts in fertilizer ever couple/few days. He was using actual fertilizer instead of f/2 though. Is this necessary?/suggested?

[shorty]

Okay...I know you told me I can't judge before 10 days, BUT .. My nanno culture has no color to it any more. I think it crashed. I started another culture starting with more volume of parent culture on Wednesday, but it appears to be doing the same thing. It gets just SLIGHTLY lighter each day... Meaning cells are dying. It is hazy/cloudy like there is cell mass in there but its not green. so I assume there might be growth but its dying.

My Tetra culture is growing great. So conditions seem to be good for that guy. Because both nanno cultures seem to be following the same trend, I suspect a fundamental water parameter issue.
1) salinity too low? Started around 1.011 (potential that salt settled so it could be less-i'll measure my media batch tonight that I have left.)
2) light too low? In window sill... Natural light, south facing but partly shaded from deck lattice work.
3) temp drops next to window at night. (Although not affecting the tetra culture)
4) not enough f/2 formula/nutrients. Used fritz pet 2 part formulas recommendation on bottle- which came to about 1mL total formula per gallon.

Other suggestions? Which do you guys suggest may be the culprit(s)? And again whatever they are, might poit out differences between the nanno and tetra cultures which could be helpful to many. What sucks, is i thought nanno is supposed to be one of the easier ones to culture. I hate to change a bunch of things at once, but I don't have much starter culture left.

[shorty]

Quick update. I think my nano is finally taking off. My original two cultures were still not doing well last week. I went ahead and split the better looking one just to see what happened. Initially (this past week) it didn't look like it was going to do any better until last night - it started to get dark green and is starting to catch up to the TET cultures I have. I am thinking now that the initial parent culture had a contaminant or something that was finally out-competed in the two new bottles that I started last week. The original two bottles are still struggling.

Here's my phyto rack I made. This was last Wednesday just after splitting the cultures. Left half is TET, right half is Nano. Top and bottom nano cultures are the ones that look to be taking off now. (Top right, and bottom right).

phytoRack1.JPG

phytoRack2.JPG

[JulioC]

I like glass bottles, you can microwave them to sterilize