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GOAL:
The goal of this hands-on project was to make and validate microbial growth plates with ingredients and tools commonly found in a kitchen.

STUDENTS:
I performed this with my first year bachelor students in dietetics and nutrition.

LEARNING OBJECTIVES:
By making these plates the students get a better understanding of what microbial organisms need as an energy and nutrient source.
Also will they have to perform standard methodes and practices (working sterile, waste managment,...) applied to microbiology labs in order to achieve the goal.
By interpreting the results students will learn to analyze and be critical. They will also learn a bit more about differential (growth) behaviour of microbes.
By using common kitchen ingredients and tools, microbiology is taken out of the lab and placed into the dietician's world. I hoped by doing this microbiology would become less abstract and more fun to them.

THOUGHTS:
I tried this project with bachelor students in dietetics and nutrition, but I guess it is suited for other students as well.
This is a low-cost project that can be performed in any basic kitchen.
My students were enthousiastic, it was fun to do!


Step 1: Ingredients

We decided to make a microbial growth plate based on the growth plate YPD routinely used to grow yeast in research labs.
YPD consists of 1% yeast extract, 2% peptone and 2% glucose.
Therefore we used 1% dry yeast, 3% milk and 2% sugar in our growth medium.

As solidifying agent we used gelatin (10%) instead of agar.
We also added salt (0,9%) since we want to grow bacteria on our growth medium as well.


Step 2: Mixing the Ingredients

- Weigh precisely, for a final volume of 150ml:
      salt:             1,35 grams
      sugar:         3,0 grams
      dry yeast:   1,5 grams
      milk:            4,5 milliliters (mls)

- Put these 4 ingredients in a beaker (or alternatively a pan) and add 100 mls of water.
- Heat the mixture (we used a bunsenburner, but alternatives are a kitchenburner or a microwave oven) to better homogenize everything.

=================
- Weigh 15 grams of gelatin leaves and soak 1 minute in cold water.
- Take the soaked gelatin leaves out of the water
- Make sure to thoroughly wringe the gelatin leaves
- Add the gelatin leaves to the mixture

You will notice that by adding the gelatin to the mixture, its volume will increase with 50%, from 100 mls to 150 mls.





Step 3: Pour the Mixture in Jars

We used cleaned jam jars to which we added about 40 mls of our growth medium mixture.

Step 4: Sterlize the Growth Medium

For sterilization we used an old high pressure pan.

- First, pour 500 mls water, preferably destilled, into the pan
- Next, place the closed jars with medium into the pan. CAUTION: do NOT close the lid of the jar tightly! Otherwise the jars may explode while cooking!
- Additionally: put a piece of sterilization indicator tape on the lid.
- Steam the jars for 20-25 minutes into the high pressure pan.

Step 5: Let the Growth Medium Solidify

After sterilization, the growth medium is liquid. In order to become solid it has to be put into the fridge.
This will take at least 30 minutes.
To increase the solidification process, the jars can be placed into an ice bath.

Make sure not to open the lid, the medium is sterile now.

Step 6: Inoculate the Solidified Growth Medium

The growth medium is sterile and since we want to avoid contamination, the inoculation should be done in the vicinity of a bunsenburner. Alternatives for a bunsenburner are a camping gas burner or a kitchen torch. If none of these are available, just rapidly inoculate and rapidly close the lid afterwards.


We inoculated the growth media with different microbial sources:

- One jar was inoculated with baker's yeast, E. coli and Staphylococcus aureus.
        *This was done by plunging a sterile (ear) swab into a tube with either baker's yeast, E. coli or Staphylococcus aureus and next      
          to "draw" a straight line with the soaked swab on the growth medium.
        *(Ear) swabs can be sterlized in an closed jar using a high pressure pan as well.
         
- Another jar was inoculated with the residual yeast found in (Belgian) beers.
        The bottle was slowly decanted, the residual yeast was poured into a beaker and part was pipetted on the growth medium

- An additional jar was inoculated with a swab that was used to sample the region behind one's ear. But you could also swab a nose,  
  a doorknob, ... or put a finger directly on the medium.

- Finally, one jar was not inoculated, this was our negative control to verify the sterilization process.

Step 7: Incubate the Jars

Incubation was done for 4 days at 25 degrees Celsius.

Make sure to place the jars for 4-5 days into a closet, or whatever, with a constant room temperature (20-25 degrees C).

The temperature may not exceed 28 degrees C!
Above 28 degrees C gelatine will become liquid again!

Step 8: Check Results

Our experiment was successful!

Picture 1:
We clearly see that the bacteria E.coli and S. aureus grow well on our growth medium. 
S. aureus is known to digest gelatin and this is also noted: the gelatin becomes liquid again at the place where a line was drawn with a swab containing S.aureus.

Yeast however grows poorly. We were also unsuccessful in our attempt to isolate yeast from beer.
Next school year I will organise another attempt to successfuly grow yeast with my students, I'll let them try other nitrogen sources than milk, plus we will cut out the salt.

Picture 2:
The medium was inoculated with the swab sampling the ear.
Little colonies are seen to grow on the medium!
Additionaly we notify the unpleasant odour of the lactose in the milk being fermented to lactate

Picture 3:
No growth on the negative control!

<p>However even M(EDTA)-1(aq) would enter the bacteria cell like a negative charge salt Cl-(aq). So some Metal EDTA-1(aq) would enter the cell and damage the DNA and the cell wall structure (my Theory).</p>
<p>Fe+2 + H3EDTA &lt;&lt; &gt;&gt; Fe(EDTA H)-1(aq).</p><p>The Fe(EDTA H)-1 when exposed to the cell wall of a gram positive bacteria would REPEL due to the negative charges of both the cell wall of a gram positive and the EDTA H Fe molecule.</p>
<p>However cobalt, manganese and iron if an<strong> oxidizer</strong> like H202 is added for pH 7.0 to 7.8 would act like Co3+, Mn3+ and Fe3+.</p>
<p>All metals I work with expect aluminum would have a +2 charge. They include iron, copper, manganese, magnesium, calcium, zinc, etc. EDTA at ph 6.2 to 10.3 would produce Al(EDTA) with no charge since Al+3 would react with the oxygen bonds of the three EDTAs. Pratically inert in terms of reaction.</p><p>Al3+ (aq) + H3EDTA-3(aq) &gt;&gt;&gt; Al(EDTA). most likely a colliod or something.</p>
<p>Here is a picture of EDTA charged with a Metal with +2 state. Most of the metals I work with expect Aluminum have a +2 charge.</p>
<p>I was wrong about the Fe.</p><p>Here is the equation</p><p>Fe+2 + H3EDTA &lt;&lt; &gt;&gt; Fe(EDTA H)-1(aq).</p><p>At pH 6.0 to 10.5 the H3 EDTA dominates and forms a weird complex.</p><p>Picture below.</p><p>Here is the reference too.</p><p><a href="https://www.researchgate.net/post/Can_someone_explain_why_EDTA_needs_basic_condition_for_dissolving" rel="nofollow">https://www.researchgate.net/post/Can_someone_expl...</a></p>
<p>Here is a theory on how EDTA works with Iron.</p><p> Fe+2 (aq) +H4EDTA(aq) &lt;&lt; &gt;&gt; Fe (EDTA)-2(aq)<br>+ 2H+(aq).</p><p>This is an equilibrium chemical one. When acid is added the equilibrium shifts left producing more Fe+2 ions which if they are inside the cell produces H202. The Hydrogen peroxide (H2O2) damages cell structures, etc including DNA.</p>
<p>I plan to work with sodium EDTA and the following salts.</p><p>Iron sulfate</p><p>Copper sulfate</p><p>Magnesium sulfate </p><p>Zinc sulfate</p><p>Aluminum sulfate </p><p>Manganese sulfate</p><p>Calcium chloride</p><p><em><strong>Warning: Manganese salts are considered potent neurotoxins if inhaled, swallowed or absorbed through the skin, Always wear PPE plus a gas mask to avoid inhaling the dust. Always wear gloves (Nitrile) and goggles or face shield before using this chemical to avoid toxicity to the brain, kidneys and liver.</strong></em></p>
<p>I am continuing a project with EDTA of iron, copper, manganese, magnesium, calcium, zinc, etc. I am testing bacteria growth with fructose sugars.EDTA salt and the compound require backwashing of a filter etc (0.1 micron) recording Time (Hours) vs pH.</p>
I have pulled yeast from beer multiple times. Your best bet though is to make a yeast starter first, than pull from the starter to the growth plates. Of course you have to use beers that have been bottle conditioned( bottle with yeast to carbonate it.) <br><br>Lots of tutorials from us beer makers if you look up making beer/yeast starters.
how about SBA? ;) Just kidding. thanks for the tutorial and I am planning on saving this for my future kids!!! First to try this with my 9 y.o. sister <3
Very cool...will help me to be more cultured...haha get it...culture.
i've been looking for a way to make petri dish medium at home, this process seems a lot easier than buying the actual agar and cooking it up. what i want to do is keep a running colony of penicillium. my hypothesis is that keeping a running colony will allow the penicillin made from the mold to be effective against newer strains of bacteria like staphylococcus. <br> <br>i don't have a pressure cooker though, would it be possible to sterilize the jars in a regular boiling pot of water over the stove? <br> <br>i also have a propane stove at my house so it seems like i could inoculate the dishes there in the presence of the flame however i was wondering what being in the presence of a flame actually does? does it just move the air away from where you're working or does it actually sterilize the air from the heat? <br> <br>finally your method for getting yeast from the beer won't work because the alcohol in the beer has already killed off the yeast. it's shed it's mortal coil and now it's body adds a grainy flavor to the beer that it made.
Hi Waldosan, <br> <br>I've a protocol for the isolation of Streptomyces from mud. Streptomyces is known to synthesis antibiotics as well. I tried it out last year with students, and it worked out fine. I'll try to post it later (busy period right now: exams). <br> <br>If you don't have a pressure cooker, you can try the process called tyndallization. http://en.wikipedia.org/wiki/Tyndallization. I've never tried it, but it would be great to know your experience if you decide to try it out! <br> <br>The flame is necessary to create a sterile environment. This will prevent contamination with other micro-organisms. <br> <br>I've already isolated yeast from Westmalle, but by using YPD plates. Though it didn't always work. Most of the yeast in the beer is indeed dead.
tyndallization seems like the best way to go for that thankyou, I'm guessing it could be done in a big pot, those big ones that can make a noodle salad for an army. <br> <br>exams, i don't miss those i won't lie... <br> <br>do we know if the same people who are allergic to penicillin are also allergic to the streptomyces antibiotic? or are there common cases of being allergic to one and the other? <br> <br>this makes me want to get some of those immortal cells that they always use instead of human testing...

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