Filters for grain spawn and liquid culture bottles....

[MagicalMedic]

I came across this offer from einseins regarding quality Culture lids and got me thinking about filters..

I recently got stuff to make some pretty good Liquid Culture lids. They have a 0.22um filter (Whatman type) and a silicone self-healing injection port

 

It seems there's heaps of options for filters on LC / spawn bottles, ranging from the pretty basic DIY like tyvek patches through to the purpose-made like the Whatman type used on these.

I was wondering how superior such filters are? I'm guessing pore size is one of the main factors; the pore size for tyvek @ 2-15 microns {http://www.materialconcepts.com/products/tyvek/uv-soft/] is pretty big, but a lot of people seem to use this product for their filters. Plain medical masks you can buy at the chemist for next to nothing are considerably better - "tested to Bacterial Filtration Efficiency 99.9%* at 3 Microns".

So obviously these Whatman ones are far superior, but are the other materials generally adequate - do people often experience contamination via poor filters?

Was also wondering about the importance of airflow - even with a very efficient filter is there still plenty of oxygen or could mycelial growth slow due to C02 buildup?

[tripsis]

I actually found Whatman syringe filters to be absolutely shit. Because the area for gas exchange is so small, it blocks up easily, leading to an anaerobic LC, which ultimately will lead to its death. Just because it looks good does not mean it is.

[MagicalMedic]

Yeah I suspected that. The filter itself & hence surface area is tiny, add to that the pore size .22um is incredibly small - seemed likely exchange would be severely restricted.

Would I be wrong in thinking that maybe these are syringe filters - made for single use sterilisation of liquid injected through them, rather than to allow gas exchange?

Edit: Several sites sell these in pre-installed in lids with self healing injection ports as if this is a great option. Evidently they are great for sterility and maintaining a vacuum seal, but for adequate oxygen.. maybe not

Does anyone have any idea what a 'minimum' surface area / pore size might be to ensure you're not restricting air too much in say a 500ml LC / grain bottle? Are we talking usually having way too much airflow when people use tyvek or similar or is it necessary to have a few good size holes in the lid?

Edited October 23, 2011 by MagicalMedic

[tripsis]

Yes, they're syringe filters. They probably work fine if they don't get wet, but I would not use them again.

0.45um is supposedly a decent pore size. Tyvek is fine. "Way too much airflow" is not an issue with LCs the way it is with grain jars. Just drill a hole around 1cm in diameter and cover that with your filter of preference.

[El Presidente Hillbillios]

What do you guys think of not having any kind of breather? Just leaving a decent air gap in the jar. I do it like that and it works. Might be faster with some kind of breather though?

[tripsis]

I'd always use something for gas exchange, but if not doing so works for you, then that's all that really matters. I expect a breather would actually lead to faster colonisation times and also prolonged life of the jars.

[MagicalMedic]

Could easily experiment to see whether it makes a difference with / without a breather - ten identical capacity jars, five half filled with grain and with no breather, five near full with grain and with a breather, and all of them innoculated (ideally) with the same clone at the same time, then stored at the same temp as they develop.

Then if the jars with roughly half as much grain were fully colonised in roughly half the time as the others you'd know it wasn't much of an issue, if they took any longer than half the time - breathers better. [also important to note if those with breathers contaminate more often though]

TBH I assumed oxygen is pretty important. But if you've been colonising the grain adequately without any breather that's pretty solid evidence that it isn't in any way critical to have gas exchange - I guess it depends on colonisation speed and contam rates.

Tripsis why is 'too much airflow'

not an issue with LCs the way it is with grain jars.

?

Also is airflow an unscientific term? it sounds a bit like it

[tripsis]

Too much much airflow/gas exchange isn't an issue the same way it is with grain due to a couple of factors. Firstly, one of the main issues with too much gas exchange in grains jars is the speed at which the grain dries out. With a liquid culture, obviously this will happen far slower due to the fact it is almost entirely water. Secondly, while it is true that you want high CO₂ levels during colonisation to promote vegetative growth rather than fruiting, you need to keep in mind water only has about 0.5% - 1% oxygen (as opposed to around 21% for air) and that oxygen diffuses into water around 1000X slower than it does in air. So even with a large hole for gas exchange, water is still limiting the amount of oxygen present in the liquid culture through its own innate nature.

You can say airflow if you want, but the term is generally referred to as gas exchange or 'fresh air exchange' (FAE).

[MagicalMedic]

Ah ok that totally makes sense. Thanks for the info man. Also I can get my mate to run that experiment, I'll update this then for interests sake; knowing breathers aren't necessary would simplify things a lot with in-jar humidity changes a non-issue and contam less likely.

[ENtiTY]

Self healing ports on LC cultures sound good but in practice only invite infection due to residue from the LC medium being trapped within the port as the needle is withdrawn. IME anyway.

[MagicalMedic]

Had not considered that, I guess it's a trade-off between the various pros and cons associated with available options. Has this happened often or just once or twice, and were you sure contamination was due to the dirty port?

Perhaps, assuming that you will only be puncturing the port a few times per grow, this problem could be solved by disposable ports - for the few times you use each port during individual grows you could make sure you puncture it roughly in a different spot each time, and when you feel the chances of contamination are rising you could inoculate a new LC in a bottle with a fresh port.

[LokStok]

This is a very interesting relevant thread to me. I agree with your "infection due to residue" theory with self healers ENt, but whats a good/best alternative.

I'm thinking for "working" LC's, possibly 5-6 seperate selfheal holes on a lid- should see it thru.

But what about master LCs?

[ENtiTY]

There is not much of an alternative unless you want to work in a glove box or flow hood. With a self healing port setup it would be best practice to make sure all the solution is clear from the inside of the needle before withdrawing, then thoroughly cleaning the port and lid surface with alcohol and then cover the port to protect it against airborne contaminants. This should greatly reduce the chance of infections being able to get at the port by reducing the amount of residue that can be potentially trapped within the port followed by removing any that may be on the external area of the port where the source of infection will come from and finally physically shielding the port from contamination sources.

The wording of my previous post kinda suggests that I think they are a bad idea which is not the case. They are not free from risk, is more what I was trying to convey.

@magicalmedic: Yeah it was def the port. You could see the contam growing through the port, grey mould it was.

Edited November 2, 2011 by ENtiTY

[Cue]

With LCs airports work the best. Tyvek may be fine but you need two layers. With only on layer it gets wet and its exposed to the bad air on the other side. To do it correctly the two layers are sepparated by a washer. Then even when the inner one gets wet from shaking the one the outside stays nice and dry.

My LCs stay clean because each time I inject into an alcohol wiped silicone port it is with a new needle. I use 60cc syringe so one iinjection into the LC I get enough solution for 12 qts of grain. I'm only paying 50cents for new needles so it just ain't worth the chance.

And for grain jars I use poly filled holes.