help out a noob, marmite for malt medium?

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Micky Dembek

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Jan 29, 2015, 12:07:31 PM1/29/15
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Hello
Go easy on me please! 
I'm first year Natural Sciences student. I do it with Open University as this is the only way I can stay working and keep studying, it has it ups and downs. We do mostly NOT INTERESTING STUFF, but it's not only through computer as on science courses we actually get to go for lectures every now and then. The real down side is that i'm mostly interested in biology and genetics and we don't really do a lot of it on first year. I know that next year I will have lots of microbiology etc. Anyways i've seen an article on synthetic biology in a foreign newspaper and i've got interested straight away.  I have decided to set up my own lab and do my own experiments as I have vitualy no access to laboratory on OU and any kind 'scientific investigation' we do is not related to subjects i'm interested in. 

Since i knew nothing about laboratories i've done as much research as possible and I have ended up on this group, finding many interesting DIY websites and DIY guides (which stopped me from buying lots of expensive equipment without knowledge how to use them)

So i want to start my first culture and (obviously!) decided it's going to be a P.stripticus - yes just because it glows!

now i have found one research paper on P.stripticus and it states that for best growth and luminescence malt yeast agar medium should be used. Now (and please don't laugh. There are no stupid questions!:)   ) can marmite be used in this medium? As far as i'm aware it is a malt yeast extract....anyone  can hep me on this one?:)

I will set up a website to show results of any of my endavours with biology so noobs like me can have another great starting point :)





leaking pen

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Jan 29, 2015, 12:27:58 PM1/29/15
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Hmmm, its a good thought, but the salt content in the marmite might be a bit much, they add enough salt to autolyse the yeast, will effect the growth of the bug you want. 

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Nathan McCorkle

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Jan 29, 2015, 12:35:15 PM1/29/15
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Go to a store that sells to beer brewers, there you should be able to
get various stages of malted grain and probably also food-grade
maltose. You could also malt your own grain (in general, look up more
details if interested) by sprouting grains then browning in a home
oven. You should then be able to throw this into a spice/coffee
grinder to roughly mill it, then add it to the rest of the media (PDA
works for a lot of fungi).
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Filip Hasecke

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Jan 29, 2015, 12:54:31 PM1/29/15
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Hi Micky,

welcome to the DIY community! =D

Marmite is not the conventional ingredient for your medium, but it should do the trick. You could additionally buy some malt extract, which is used by some home brewers.

Typical malt yeast agars contain:

Ingredients (g/L):
Peptic digest of animal tissue, 5.00
Yeast extract, 3.00
Malt extract, 3.00
Dextrose (Glucose), 10.00
Agar, 20.00

You might test this recipe and replace yeast extract by Marmite (probably a bit more than 3g/L) and some malt extract, maybe this one:
http://www.northernbrewer.com/shop/brewing/brewing-ingredients/malt-extract

You can buy glucose in a supermarket.

You might try some gelatin instead of agar.

Skip the peptic digest of animal tissue. I don't have a proper replacement for that in mind, but it should work without it.


I never tried a medium like that, but I'm interested whether it works, I don't know why it should not ^^


As for your glass ware: before using it, cover it with alufoil and put it into an oven for at least two hours at 160 °C.
You should also somehow sterilize your medium, propably simply by cooking? ._.

All other tools should be dipped in 70% Ethanol (if available) and burned before use. Also, you should put a bit of ethanol on your gloves and rub it dry before touching anything. ^^

Good Luck! I'm looking forward to your results! ;)

Cheers
Filip


Mega [Andreas Stuermer]

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Jan 29, 2015, 1:37:50 PM1/29/15
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I grew p. Stipticus in wood, wooden chips, straw, autoclaved grass; .... On agar it didn't work out last time, got a mold contaminant

Mega [Andreas Stuermer]

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Jan 29, 2015, 1:39:46 PM1/29/15
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Mega [Andreas Stuermer]

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Jan 29, 2015, 1:41:54 PM1/29/15
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Google Diyspartanbiotech -> you'll find some photos of P. Stypticus
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Micky Dembek

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Jan 29, 2015, 2:25:25 PM1/29/15
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Thank you all for quick reply!
So i decided to make two plates, one with PDA and one with Marmite just to check if it works!
I have applied as much safety precautions  as I could but these are my first plates so probably not enough safety :) , i will make few more since i've got plenty of agar left. I have sterelized the plates in an oven, i've got autoclave tape so i'm sure it have reached the temperature. used the alcohol burner and 95% alcohol to sterelize the spreader  and needle(syrienge with the fungus) I also have hepa filtered air purifier on the desk aimed at the plates.
 
I have placed them into my DIY incubator. cost me  £10 to make it.  I lined styrofoam box with aluminium foil and placed reptile heating pad(cheapest one with temperature control on ebay  £1.59!) and for keeping eye on temperature I have placed cheap digital thermometer! I was able to keep the temperature of 35 degrees celcius for few days without any trouble

Patrik D'haeseleer

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Jan 30, 2015, 2:59:10 AM1/30/15
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Yes, marmite can be used as a component of a growth medium for many microorganisms, but as some have pointed out, its salt content may be a problem for some organisms.

Simon Park's "Microbiology at Home: A short non-laboratory manual for enthusiasts and bioartists" has some nice DIY recipes for growth media that use marmite.

Patrik

Jonathan Cline

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Jan 31, 2015, 12:45:13 AM1/31/15
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On Thursday, January 29, 2015 at 9:07:31 AM UTC-8, Micky Dembek wrote:
 I'm first year Natural Sciences student.  ...  We do mostly NOT INTERESTING STUFF,

Depends on your perspective and your choice of side projects.  I'm not sure the long term salary potential of a natural science graduate is very interesting, that much is true.


> So i want to start my first culture and (obviously!) decided it's going to be a P.stripticus - yes just because it glows!
 
If you are successful, then send pictures.  

Perhaps you could look into nutritional yeast (a yellow powder from the grocery store): 

http://en.wikipedia.org/wiki/Nutritional_yeast

This is basically 'a yummy yeast extract'.  It is also tasty on popcorn for when you're taking a break from being a mad scientist.  Also if I recall you can add potato starch or potato flakes to make it more nutritious (for your microbes or fungi.. not for your popcorn).   You might even be super lazy and microwave the mix to sterilize though it might be best to pressure cook it.

Keep in mind though, that fungi grow on nearly everything, including many things you might find at a garden supply store, as long as you keep things sterile.  Building a properly clean hood is more important than the food/medium for a first beginner homebrew attempt.



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Nathan McCorkle

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Jan 31, 2015, 5:03:45 AM1/31/15
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On Jan 30, 2015 9:45 PM, "Jonathan Cline" <jnc...@gmail.com> wrote:
>> Keep in mind though, that fungi grow on nearly everything, including many things you might find at a garden supply store, as long as you keep things sterile.  Building a properly clean hood is more important than the food/medium for a first beginner homebrew attempt.
>

A hood is not necessary, I was 14 or 15 when I achieved sterile technique in my mother's kitchen over an open oven. 10+ years later the most I've done as far as  sterility is to make a still-air box, and to know when to be careful.

Micky Dembeck

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Feb 1, 2015, 5:31:29 PM2/1/15
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so it's been 2 days. I have incubated two petris with a medium of agar glucose and marmite  (20 grams, 10 grams, 15 grams) at 25°C. both petris a starting to get cloudy in spots that I have 'planted' a drops of fungi. I took a sample out of one to see how is it going under microscope. so here are the pictures. i'm not sure if its a fungi or not but something is growing! 
 yes i know  petris don't look great with all the bubbles but i have made new samples with different mediums today, and they are spread evenly. so I will see how it goes. 
Untitled.jpg
DSC_0003.JPG
DSC_0005.JPG

Nathan McCorkle

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Feb 2, 2015, 2:01:26 PM2/2/15
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On Sun, Feb 1, 2015 at 2:31 PM, Micky Dembeck
<genial...@googlemail.com> wrote:
> yes i know petris don't look great with all the bubbles but i have made

Wow, lots of bubbles! I wonder if you didn't cool the media long
enough after sterilizing it... did you let the pressure cooker return
from 15 PSI to atmospheric without removing the pressure weight? When
you are ready to pour the plates, the media should be cool enough that
if you spilled it on your skin you wouldn't be burned (or conversely,
that the jar containing the media is cool enough to hold with a
bare/thinly-gloved hand).

The bubbles make it extremely hard to tell if you've got sub-surface
growth happening. Were the petris also sterile?

Brian Degger

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Feb 2, 2015, 2:01:57 PM2/2/15
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If you get bubbles you can flame them quickly (under a second) with a bunsen  to pop them when poured but not set.

Micky Dembeck

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Feb 2, 2015, 4:49:33 PM2/2/15
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W dniu poniedziałek, 2 lutego 2015 19:01:26 UTC użytkownik Nathan McCorkle napisał:


Wow, lots of bubbles! I wonder if you didn't cool the media long
enough
Yes it was cool enough, the reason for bubbles was simply that agar start to solidify before i even had the chance to pout it. so in panic I have shaked  the jar well and pour it straight in. :)

 Were the petris also sterile?
Yes I have baked everything for a long time in 150 C  oven, wrapped in aluminium foil to prevent contamination when i took them out from the oven...i tried to do everything as per guides I can find in internet :)

today "IT" got a bit bigger and i can already clearly see it grows upwards...(i say 'IT" because i'm still not sure if I actually suceeded.)


Nathan McCorkle

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Feb 2, 2015, 7:15:34 PM2/2/15
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On Mon, Feb 2, 2015 at 1:49 PM, Micky Dembeck
<genial...@googlemail.com> wrote:
> W dniu poniedziałek, 2 lutego 2015 19:01:26 UTC użytkownik Nathan McCorkle
>> Wow, lots of bubbles! I wonder if you didn't cool the media long
>> enough
>
> Yes it was cool enough, the reason for bubbles was simply that agar start to
> solidify before i even had the chance to pout it. so in panic I have shaked
> the jar well and pour it straight in. :)

Ahh, so I guess 'too cool' maybe. If this happens in the future, a few
10-second bursts in a microwave should fix things up! (but definitely
only do bursts in the microwave, if the agar boils it bubbles a LOT
and can erupt out of the container, and when you realize and open the
microwave, the eruption can suck back into the container, taking
contaminants with it)

>> Were the petris also sterile?
>
> Yes I have baked everything for a long time in 150 C oven, wrapped in
> aluminium foil to prevent contamination when i took them out from the
> oven...i tried to do everything as per guides I can find in internet :)

Cool, sounds good. Did you not use a pressure-cooker then? Moisture
conducts a lot more heat, so that could be something that might cut
off some sterilization time. For things like petri dishes, you can
either bake or pressure cook them dry, but the last few times I
cleaned glass for culture, I added just a bit of water... this way I
know there is sufficient heat conduction inside them, and also I have
extra sterile water on hand in case I need it.


> today "IT" got a bit bigger and i can already clearly see it grows
> upwards...(i say 'IT" because i'm still not sure if I actually suceeded.)

Congrats! Next you might try cloning a mushroom you buy at the grocery
store, oyster mushrooms have fruited for me on PDA alone, with barely
any care provided to them (but I also never opened those to prevent
any sort of contamination).

Cathal Garvey

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Feb 3, 2015, 9:03:00 AM2/3/15
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Warning: if using a butane bunsen (campinggaz style) do not invert the
bunsen! :)

Get a culinary blowtorch for this instead, or better yet learn to avoid
making bubbles in agar in the first place; swirl, don't shake, and let
it cool a bit before pouring. Bubbles burst on their own in hot agar,
but once they form in cooling agar you're in trouble.

Another nice trick; swirling a poured plate gently will encourage the
bubbles to the edge of the agar plate, where they can be either ignored
or encouraged together with a sterile needle and popped.

On 02/02/15 19:01, Brian Degger wrote:
> If you get bubbles you can flame them quickly (under a second) with a
> bunsen to pop them when poured but not set.
>
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Micky Dembeck

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Mar 1, 2015, 4:06:23 PM3/1/15
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Hello
It's been a while and I was busy at work + GFs family came over for 2 weeks so I did not even look inside my incubator.

good news is: something has grown on each plate!
even better news: two plates looks like are  occupied by P.stripticus 
Two plates I have set up with just yeast very successful
The original two plates have grown something that looks more like lichen, they stopped their growth probably due to lack of water.
One plate is covered by something black with white 'web' growing underneath perhaps someone can identify this?

Noob question 1 : if growth have stopped can this be restarted by adding a little bit of water?

Noob question 2 :What is the best way to identify what has grown on the plates? I have access to microscope so more picture to come. 

Noob question 3: can P.stripticus be grown bigger from only what I have on plates? providing I will supply all the necesary substances, I have noticed that i can be grown on piece of wood. I want to create glowing shroom box for my brother as decoration for his new sushi place :)



DSC_0070.JPG
DSC_0071.JPG
DSC_0072.JPG

Nathan McCorkle

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Mar 1, 2015, 4:33:33 PM3/1/15
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The black stuff is probably not what you were interested in growing, it looks like aspergillus Niger possibly.

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