Actinomycetes isolation

[Pieter van Boheemen]

Following the instructions in the Google drive, we did step 5 "Restreaking" yesterday and we are now inspecting the results.

We picked colonies of plates like this: https://www.flickr.com/photos/pietervanboheemen/10589279635/

We tested several streaking methods, one just simple zigzagging. You can see the result here: https://www.flickr.com/photos/pietervanboheemen/10589521303/

They were streaked on two mediums.

1) including potato starch

- 1/2 teaspoon of liquid fertilizer (Easy 2 Grow)

- 2 teaspoons of potato starch

- 1 tablespoon of beef-stock

- 1 tablespoon of milk powder

- 20 gram agar agar

- up to 1 L of tap water

2) without potato starch

- 1 teaspoon of liquid fertilizer

- 1 tablespoon of beef-stock

- 2 teaspoons of glucose

- 20 gram of agar agar

- up to 1 L of tap water

As you can see there is obvious growth of bacteria after grown over night. We guess they're not Actinomycetes. We speculate that the sterilization of the inoculation wire with just a bit of alcohol and a flame might not be enough, instead we perhaps need to get it red hot each time.

Anyone who have suggestions for more robust DIY colony picking?

Overall biostrike Instructions so far: https://drive.google.com/#folders/0B3mWAVWgbuXqTVNUVDVyWVd3MUE

[Mojca Zaloznik]

Hello to all,

i put some pictures of Actinomyces i isolated on google plus and i ve shared them:

https://plus.google.com/u/0/photos/117795368412553777360/albums/5941034939734178225

i hope you can all see them..if not let me know.

They were isolated when i was studying the microbial community of Ljubljana marsh soil. I will also write a recipe of the medium. I isollted about 600 colonies and more than 100 of them were Actinomyces.

As you can see when you pick the colonies you should be aware of the dusty appearance of the colony; the diameter of the colony should be from 1mm to 3 mm, but it largely depends on how much space a colony has. The color varies from white, pale to very dark grey, even black, a lot of brown hues and even  pale pink.

When isolated as pure colony concentric circle around the colony can be seen. When you restrike them you should always sterilise the wire to the red and wait untill it cools down (if its too hot then you can kill the cells you want to inoculate, if it s not totaly sterile you can bring contaminates inside). I suggest you stike them 4 times on one plate to get single colonies. This would be important if you decide to sequence them.

I also saw on one plate when a very interesting colony appeared-it looks very hairy and the comment was if this is fungi. It is actually from the genus Bacilli- Bacillus mycoides:

http://www.google.si/imgres?imgurl=http://upload.wikimedia.org/wikipedia/commons/7/7b/Bacillus_mycoides_on_TY_agar.JPG&imgrefurl=http://en.wikipedia.org/wiki/Bacillus_mycoides&h=1496&w=2256&sz=426&tbnid=GqmNgaTNnQKv5M:&tbnh=90&tbnw=136&zoom=1&usg=__Bs5N82bmOUQ7MSm8lE6U2NyC2KA=&docid=sOGxA9Va0-oGUM&sa=X&ei=gNVyUu7gEKeV7AaBpIHgBw&ved=0CC8Q9QEwAA

so this is it for today,

Mojca

 

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Date: Thu, 31 Oct 2013 16:09:47 +0100
Subject: Actinomycetes isolation
From: pieterva...@gmail.com
To: bios...@googlegroups.com

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[Patrik D'haeseleer]

On Thu, Oct 31, 2013 at 3:12 PM, Mojca Zaloznik <mojcaz...@hotmail.com> wrote:

Hello to all,

i put some pictures of Actinomyces i isolated on google plus and i ve shared them:

https://plus.google.com/u/0/photos/117795368412553777360/albums/5941034939734178225

"You do not have permission to view this album."

You may  be able to simply Share the album with bios...@googlegroups.com to give everybody on the mailing list access.

Patrik

[Pieter van Boheemen]

Thanks for this super helpful information Mojca. I added your instructions to the guide: https://docs.google.com/document/d/1hf8miZtC13zMwe9J3R_55-6pSS1xFnbrNp9dp-NoLK4/edit#

Please accept my request to view your album. I am really eager to see what the pictures look like

--

[s.p...@surrey.ac.uk]

Anyone who have suggestions for more robust DIY colony picking? Sterilized wooden toothpicks (in your pressure cooker and then  dried)  are very good if you're doing a lot of colony picking

[Mojca Zaloznik]

Hello,

i added the whole group, but i guess it isn t working....i accepted some requests and i hope it works individually...so if anyone would like to see it..send me a request and i would accept it...

will put a recipie of the media later on.

best

Mojca

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Date: Fri, 1 Nov 2013 10:18:13 +0100
Subject: Re: Actinomycetes isolation
From: pieterva...@gmail.com
To: pat...@gmail.com
CC: mojcaz...@hotmail.com; bios...@googlegroups.com

[Patrik D'haeseleer]

Yeah, not working for me either. I do know this trick works for some other Google platforms - e.g. to share a google doc with a google groups mailing list. But I guess they haven't put in the right integration code with Google+ photo albums yet.

By the way, I do really like the Actinomycetes isolation medium from the Zhang 2011 paper that I added to the Synbiota site: starch, potassium nitrate (saltpeter), and soil extract! Easy to DIY, and should be more specific than the generic medium you're using now (and customizable to each specific soil!).

The fungicide page on Wikipedia provides some ideas on what to add to this recipe to select against any pathogenic fungi you might isolate from the soil. Easy to source fungicides could include powdered sulfur, tea tree oil, cinnamon, oregano or rosemary oil, milk, and kelp.

Patrik