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Author: Subject: Simple and cheap Agar media for home microbiology
BlackDragon2712
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[*] posted on 1-10-2016 at 14:03
Simple and cheap Agar media for home microbiology


Lots of interesting reagents can be obtained through the metabolism of bacteria and they have been increasingly getting more and more importance in industrial processes for the same reason.

They are everywhere, and they grow everywhere. Most of common bacteria do not require specialized growth medium and they are actually very easy to grow even in a home-made set up.

Most homemade mediums described online promotes the growth of fungus over bacteria and some lacks of any aseptic technique at all.

I just want to share my experience making a decent enough medium for bacterial growth that avoids to an extent the growth of fungus in the medium. I will update this post when I apply improvements in the method described below.


-> Materials:

1-) 0.8g of Peptone *
2-) 2g of Agar Agar
3-) 100ml of water
4-) (>2) Petri dishes
5-) Filter paper
6-) Microwave
7-) 500ml Erlenmeyer flask

*NOTE 1 see below


->Procedure:

100ml of distilled water was heated close to ~80°C in a 500ml Erlenmeyer flask and 0.8g of peptone (beef bouillon in this case) was dissolved on it. The mixture was filtered through normal coffee filters and then 2g of Agar powder was added to the filtrate. The flask was sealed with plastic wrap and paper in the top. Immediately after, the agar and the two petri dishes were put in the microwave and heated for 1 1/2 minutes. (It's important to consider that the volume of the Erlenmeyer should be at least two times bigger than the volume of its content) After this, 2 petri dishes were filled with about ~25 ml of the media each.
For the cultivation procedure a sample of dog faeces was obtained and suspended in distilled water. The work up was performed under an open flame in order to avoid contamination of flying microorganisms. The cultivation procedure was performed in only one small area of the plate and in only one of the two petri dishes, I made it this way to test the sterilization effectiveness of the microwave. The plates were incubated in hot water for 24hr maintaining a temperature of 38°C constant (Alternatively a light incubator can be design) (Most bacteria will not be harmed by temperature shifts, but their growth will be slowed down to 48h or more) (Some bacteria can even grow at room temperature)


Culture plate before cultivation:
Culture media clean.jpg - 315kB

->Results:

White, wet, big colonies were detected on the cultivation zone. No bacteria growth was detected in any other side of the same plate neither on the second petri dish prepared. Recognition of the bacterial culture was not performed.

Culture plate after cultivation and incubation:
Bacterial Colonies.png - 5MB

->Discussion:

The method described is a very easy and cheap way of producing bacterial colonies but it definitely can be improved. For future experiments maybe adding a sugar like sucrose may promote the growth of nonspecific bacteria, the problem would be that the growth of fungus would be greatly increased as well, the use of an improvised glove box would solve this (between other) problem. Another problem is to find easy and cheap sources of peptone, for this the enzymatic digestion of soy powder with papain shows promising potential for the home scientist but experiments are yet to be performed.
A replacement for the Agar powder could be unflavoured gelatine, the problem with this is that fungus growth is favoured in this medium and some bacteria can easily dissolve the gelatine.
Despite the fact that only a small group of bacteria grew in the medium, re-cultivation by following the same procedure but modifying the cultivation method by cultivating the entire plate this time and not only a small area should produce a much large amount of bacteria in the plate (Remember to make a control plate in order to ensure the sterilizing method because sometimes it can fail)

*NOTE 1: Peptone is the result of protein digestion. It can be obtained from beef bouillon that specifies the content of peptone or meat digestion products. It can also be made by enzyme digestion of meat with peptones or papain (obtained from meat tenderizer powder). It can be made by acid hydrolysis of meat but the problem with this is that the final product needs to be further purified due to the low pH and the salt content of the hydrolysate produced. They can also be made by enzymatic digestion of soy powder but the amino acids in the media may vary from those obtained from meat and it doesn't contain cystein or tryptophan whose absence could inhibit the growth of certain bacteria.

->Links:

http://www.solabia.com/solabia/content/NT000043E6.pdf
http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1982....


[Edited on 05/12/2013 by BlackDragon2712]
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Metacelsus
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[*] posted on 1-10-2016 at 16:23


Nice work. I'm surprised the microwave treatment was enough to prevent contamination of the plates. Now, can you determine the CFU/mL of your dog poo?

Here's the LB agar recipe I've used:
Per 100 mL volume (makes ~4 agar plates):
1 gram tryptone
0.5 gram yeast extract (dry powder)
1 gram sodium chloride
2 grams agar
This is then autoclaved (pressure cooker, basically).

There are many, many other kinds of solid media, and each one is best for certain kinds of microbes.

[Edited on 10-2-2016 by Metacelsus]




As below, so above.
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BlackDragon2712
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[*] posted on 2-10-2016 at 13:30


Thanks!

No, I cleaned the plate after the experiment because I was in a rush by the time...

Interesting recipe... it looks very simple to make, did you buy the tryptone or made it yourself?
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