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How to extract lactic acid bacteria without nutrient broth?

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How to extract lactic acid bacteria without nutrient broth?

Postby fursona » Thu Feb 09, 2012 10:48 am

Hi I was wondering if there are any alternatives to extracting lactic acid bacteria from foods such as Japanese natto WITHOUT using nutrient broth? I'm sadly really inexperienced but would any other these methods work:

1) Crushing the natto beans and making a paste, which would then be spread over an agar plate and incubated

or

2) Centrifuging the natto and looking for pellets, inoculating the pellets on an agar plate and incubating them

Uh my apologies for being wrong... Thank you!
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Postby JackBean » Thu Feb 09, 2012 11:15 am

If you want to get bacteria from food, just wash it with liquid media and plate an aliquot on agar plate. However, what media do you mean by agar plate?
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Postby fursona » Thu Feb 09, 2012 11:25 am

Thank you for the reply! I just want to identify that natto and other fermented foods do have lactic acid bacteria via gram staining after isolating a strain.

Umm I was planning on using a normal trypic soy agar plate since lactic acid bacteria usually grows better on it.

And could you please expand upon washing it with liquid media? Does the liquid media have to be a nutrient broth or can it just be something like distilled water? And would I literally wash it or should I crush the beans and then wash it...?

Sorry for the multiple questions but I'm confused about the washing part...
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Postby JackBean » Thu Feb 09, 2012 11:58 am

I cannot really give you advice on the media, but if you say it grows better on it then it should be fine :)

By washing I mean put it into the liquid, let it shake for one hour (since you do not want to quantitate it, just like 30 or maybe even 10 minutes should be enough) and then take the liquid and plate it. Crushing the nattos is not a good idea, since the bacteria should be mainly on top and you've had to remove the debris, so you would rather loose.
The liquid should be something that the bacteria will like. Small aliquot of your media (w/o agar of course) would work best. Water should work as well, but who of us likes distilled water, right? It's pretty hypotonic, thus adding at least a bit of some sugar (e.g. sucrose) to make it almost-isotonic would be probably better (but fresh water should work as well).
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Postby fursona » Thu Feb 09, 2012 12:13 pm

Wow, thank you so much for the info!

So I should proceed by:
1) Getting a container with a solution of distilled water + ~1 teaspoon of table sugar
2) Put in the food/natto in the container and let it sit for one hour
3) Remove a small portion of the liquid from the container
4) Spread the liquid on the trypic soy agar plate and incubate at 37 C

Also, the kimchi I'm working with contains pepper flakes and other spices - would it be necessary to wash it first before dunking it in the solution of water + sugar?
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Postby JackBean » Thu Feb 09, 2012 12:40 pm

If are the bacteria able to grow on it with the spices, they should be fine ;)

Bud do not let it sit, instead keep it shaking.
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Postby fursona » Thu Feb 09, 2012 12:54 pm

Keep it shaking? So would it be fine if I continuously shook the container with the food inside for about 10 minutes then?
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Postby JackBean » Thu Feb 09, 2012 1:03 pm

Probably. Keep it at 37°C or what's the optimal temperature for lactic bacteria.

You will see, whether will you have some growth, if not, you can adjust the conditions.
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Postby fursona » Thu Feb 09, 2012 1:07 pm

Oh so just put the food in the container, shake for 10 minutes, get the liquid, and inoculate basically.

But what do you mean by growth? Do you literally mean the bacteria will grow in the container from just shaking - not from incubating on the plate?
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Postby JackBean » Thu Feb 09, 2012 2:12 pm

No, I mean on the agar plate after inoculation :) Sorry for confusion.
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Postby fursona » Fri Feb 10, 2012 5:06 am

Thank you very much for the replies! However, my teacher also mentioned something about using the solution and filtering the bacteria from the water by using paper disks - would you also recommend I try this method too?
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Postby JackBean » Fri Feb 10, 2012 7:37 am

That depends on how many bacteria you expect to grow. If the bacteria should be diluted, it is good to filter it and thus get all bacteria from e.g. 100 ml in your plate. On the other hand, if should they be abundant, only say 250 ul could work.
But I'm not able to provide real answer for this. However, from what I remember from our micro lab, only aliquot should work, if you want dilute it too much (say you will use 1 volume unit per 1 mass unit, i. e. 1 ml of your solution per 1 g of natto)
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