Why is stock solution needed to be sterilized? - Calcium chloride, and Tween 80 ok after autoclave? (Jul/16/2008 )

Hello, everyone, i have two questions.
first, why is stock solution needed to be sterilized?

My experiments require making of agar with Tween 80 and added calcium chloride to provide calcium ion in the medium.

other colleagues in my lab added the calcium chloride by just weighing 0.04g of it into 400ml H2O

and i just feel that 0.04g of calcium chloride dihydrate is really hard to weigh, so i've make 40ml stock solution 0.1M calcium chloride, and i'm thinking adding some volume of the solution instead of weighing that weight.

however, i didn't pass my stock solution through a 0.22 micrometer filter, just after making it i just store in 4 degrees
because at first i thought i was going to add it into the medium prior to autoclave so didn't really thought of the necessity to filter the stock solution.

But now it's just come to me that is it possible that it'll have problems?

Should stock solutions always be filter-sterilized?


Another problem i have is that I used to add Tween 80 into the medium after the autoclaving the medium.
however i didn't find articles on why tween 80 shouldn't be added into the medium before autoclaving

does any one know why?

i did a little experiment about this, i added tween 80 into the medium prior to autoclave
and when it came out, the solution looked cloudy, but when i was pouring it into the plates, it seemed clear again.
i didn't quite observe the process, so i am still unsure whether tween 80 can be added into the medium prior to autoclave or not.

does anyone have any idea?
i checked books, and on it says that tween 80 are added into the medium after autoclave.

thanks alot

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0.1M solution refrigerated will probably not give you as much a micro problem as chemical stability problem with Calcium carbonate or Calcium hydroxide precipitation and you may speed that with filtration. For that, advise make it fresh each time or follow the lead of your colleagues.

There is no problem with normal autoclave sterilization of Tween 80 - look up letheen agar. The cloudiness is always observed @ > 80-90C and, as you noted, disappears as it cools.

Are you using Ca ion to precipitate fatty acids from tween 80 hydrolysis?

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I would not autoclave tween to be honest!

It is not good.

I will try and find the references I have about this, however I am not sure I'll find it again.

Anyway: when I used tween I never autoclaved it, but filtersterilized it.

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I have not been able to find the reference I had, but I did find others:
http://www.mpbio.com/product_info.php?products_id=806576
(read: Solubility)

http://www.ffcr.or.jp/zaidan/FFCRHOME.nsf/...36;FILE/B26.pdf
==> they describe adding the tween after the autoclaving. (page 6)

however I must be honest and also show a topic like this one:
http://www.bd.com/ds/technicalCenter/inser...orbate_80-2.pdf
in wich they state that they have added the tween before autoclaving.

but then read this:


http://www.sigmaaldrich.com/sigma/product%...et/p5927pis.pdf
(storage and stability)
it is about tween20 not 80


anyway: maybe you can contact a firm that delivers or makes tween and ask them the question?

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I am not sure about the use of Tween in agar, but it is perfect if you autoclave a Tween soulution for making spore suspensions or dilutions....don't worry about the cloudiness!

I would prefer to autoclave Tween 80, as some bacteria can utilise Tween as carbon source (I think it were Coliforms or Enterobacteria, but I need to look this up again!). And no matter which, but some Bacteria managed to grow in our last tween stock

I think as always it depends what you want to do with it?

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Actually, i'm working on a project on staphylococcal lipases, as i know know those lipases hydrolyse tween 80 and forms halos on the agar as mentioned in an article as calcium precipitate fatty acids.
also, calcium ion is required for full activity of staphylococcal lipases.
I'm still unsured about what calcium conc. should it be, from what i've seen typical medium preparation for lipase activity assay uses CaCl2 monohydrate as 0.1g/L
but i don't know if it should be add up since calcium ion is required for full enzyme activity.
though someone pointed out to me that the calcium ions used by the lipases may only be a very little fraction of the 0.1g/L

Thank you all

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i understand your caution pito. However media houses (Difco-BBL) as well as the FDA BAM recommend autoclave sterilization. Further - you'll have to signficantly dilute Tween before the solution filters efficiently.

I've used autoclave sterilized Tween media for the purpose mentioned here (Ca as cofactor for lipase as well as counterion to precipitate fatty acids as calcium soaps. My bug was Malassezia.

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A GOOD IDEA:

If you have a reagent/stock solution that has water in it....AND you're going to keep it for a long time. (days, weeks, months, years) then it's a good idea to sterilize it...

If you have have a 0.2 micron filter then USE it...

ANY auqeous solution can become contaminated or infected with yeast, bacteria, fungus, etc....

Naturally, you will sterilize your preparation AFTER you've used an aliquot of your stock solution but this will NOT prevent all the possible damage from a contaminated stock solution.

1: Bacteria, yeast, fungus will ALL consume any sugar in your stock solution. Also, they will degrade much of the protein in your stock solution. Thier very existence will alter the pH...

so the end result is that your stock solution is no longer what you intended to be. Sterilization becomes a moot point.

2: Even thought autoclaving may kill the yeast and bacteria, they are still present... They have gummed up your mixture.... They have added their own protein, lipids, dna and rna to the mix...That MIGHT not be bad, but it certainly will not be good... Why risk it?

Play it safe, take a few minutes, filter your reagents....it's to your own benefit.

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What medium are you using the requires your addition of more Calcium ion for lipase cofactor?

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i'm using a recipe that uses yeast extract as carbon source and peptone no.3 as nitrogen source
supplied with some NaCl and bacto agar

it was a recipe that used in an article by Nostro et al. in 2001

since calcium ion is required for forming the calcium soaps of fatty acids that presents halo around the colonies and calcium ion was required to give full enzymatic activity, i was just wondering if 1mM of Calcium chloride dihydrate is enough.

today, i observed the little experiments i started two days ago(48hrs observation).
first is to observe which calcium concentration is required, i have 0.3mM 0.5mM and 1mM calcium chloride added medium
and the results are that the lipases seemed having the same activity.

also, i did a little experiment comparing the effects of autoclaving to solution with tween 80.
i did it by one group were plates with medium added tween 80 prior to autoclaving and another group with plates added tween 80 just prior to pouring the plates.

The lipase results looked the same but the calcium soaps looked a little different.
In the group that had tween 80 added and then autoclaved showed bigger soaps in halo, and the group that had tween 80 added after autoclaving showed smaller soaps.

does anyone know the reason of the difference?

another little question
my tween 80 is purchased from wako, Japan
and it requires to be stored away from sunshine i think, since it's in amber glass bottle.
Now i want to take some out and autoclave it and then store the autoclaved tween 80, can i just get another amber glass bottle from like kimble or Boeco?
is there any differences of the amber glass bottle between chemical companies and kimble or Beoco's?

thanks alot

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