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Rosebud95
Stranger
Registered: 05/13/19
Posts: 33
Last seen: 3 years, 14 days
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First time Agar - Suggestions?
#26435118 - 01/15/20 02:53 PM (4 years, 14 days ago) |
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Hey everyone,
I did a lot of research to ensure I would be taking the proper steps beforehand, but I still didn't have any luck with my Agar plates. Now I realize it takes practice, but I want to know if anyone can pinpoint where I went wrong.
So I will start by letting you know how I went about this.
I had ordered 20 sterilized Petri dishes from Walmart, and the bag I had received had a hole in it. So I kept it in the box in my sterile room until ready for use.
The MEA powder I am using is already pre-mixed with Agar powder, light malt extract, and a few extra additives to assist the growth (such as antibiotics). I use 16.5 grams for 300ml of distilled water and mix it in little bits at a time to avoid clumping.
Once it is all mixed, I insert the polyfill to let it breathe and then I cover it with foil before inserting it into my presto 23-quart pressure canner. 15 - 16psi for 45 minutes. and then I let the pressure release at its own pace with a 91% alcohol towel covering it when I open the pressure canner.
Then I put the jar of MEA solution in the SAB while it hot (unfortunately I do not have the funds for a flow hood). I already have the SAB in a clean room and the inside of the SAB has been wiped down with 99% alcohol. I leave the inside of the SAB damp to pick up any dust with a damp towel on the base so that dust is not stirred up. I have a metal rack that raises the working area from the base of the SAB. I leave the SAB for only half-hour so the MEA solution doesn't cool on me. before pouring I use the flame to sterilize where the MEA solution will pour from the jar and then I pour the Petri dishes with minimal movement (lid is always left facing down). I have watched many videos to ensure I make the minimal movement needed with proper stacking methods.
I stored the plates in a plastic container that has been cleaned before use, for storing the plates in the fridge.
I also didn't store them upside down which lead to a lot of condensation on the lid when working on them, but I fixed that and placed them upside down after inoculation.
Once the Petri dishes had been cooled for more than an hour in my fridge. I put them in the SAB with the syringe. (everything had been cleaned and sterilized). I used 1cc for each dish but I feel that was too much because I got a splash effect which made it harder to identify what was spore growth and what was contamination.
Now I did use glad cling wrap when wrapping them up, but I bought some parafilm for next time. I feel that contamination was still able to get through because it wouldn't stay tight.
I always wear non-latex gloves, long sleeves (freshly washed) and damp my sleeves with Lysol, and soak my gloves with 91% alcohol when doing any work even when it is unnecessary some say.
I stored the plates at room temperature, but because its winter where I am I feel that it might have gotten too cold during the night times. I will start to monitor the temperature more and make sure it does not drop more than 10 F in a 24 hour period.
I am sorry for the rant and I hope you can appreciate my interest. Now I have read many of the threads/posts on here and I found them to be very helpful. But I wanted to reach out to see if anyone could assist me with their expertise.
I have attached pictures of the plates after 10 days. They all gave off a strong odour that did not smell good when I took the wrap off.
PS I did label them but the marker was used on the wrap.
Thank you for your time to read this and I hope you have a wonderful day.
Please leave your thoughts, comments or concerns.
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InfiniteDreams


Registered: 10/25/19
Posts: 1,224
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Re: First time Agar - Suggestions? [Re: Rosebud95]
#26435246 - 01/15/20 04:11 PM (4 years, 14 days ago) |
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70% iso alcohol is recommended. Yes it works better than 90% or 99%.
Many people recommend making your own agar solution rather than a premix.
Not sure putting the plates in the fridge is needed before inoculation. Pretty sure Bod's tek said just to wait 5-10 minutes for it to settle. Actually, was this a dedicated fridge? Because it may have picked up the contam from the fridge if it is your normal one.
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FreeCanadianHugs
Stranger

Registered: 08/07/19
Posts: 67
Last seen: 1 year, 10 months
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Re: First time Agar - Suggestions? [Re: Rosebud95]
#26435250 - 01/15/20 04:13 PM (4 years, 14 days ago) |
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Very possible your syringes are contaminated. Also you are most likely adding far too much to your agar when you should add a drop or two. Make some clean plates, let them sit on a counter for a week and see if they remain clean and then use them. If plates are clean until you use the syringe that is most likely the source. If you plates are already contaminated after a week then you need to work on making clean plates.
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Rosebud95
Stranger
Registered: 05/13/19
Posts: 33
Last seen: 3 years, 14 days
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Thank you very much. How come 70% is better? too many fumes and not enough oxygen?
And I store the plates in a designated fridge and even cleaned it beforehand with Lysol wipes and 90% alcohol. I don't put the Petri dishes directly in the fridge. I put them in a plastic container beforehand, and the plastic container has been sterilized.
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Rosebud95
Stranger
Registered: 05/13/19
Posts: 33
Last seen: 3 years, 14 days
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Good idea thank you. I will leave one of the 10 that I have left from the first time doing it, and see if any contamination grows. If it is clean then I know its from too much syringe solution. I already figured the syringe was contaminated, that's why I am using the agar method. So that I can work away from the contamination and create a nice and healthy culture after a few transfers. Thank you very much. I will put one of the unused plates on the counter now and see if anything grows within the next few days.
Does anyone have a preferred cheap Canadian petri dish source (sterilized, and not to big)
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InfiniteDreams


Registered: 10/25/19
Posts: 1,224
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Re: First time Agar - Suggestions? [Re: Rosebud95]
#26435540 - 01/15/20 07:31 PM (4 years, 14 days ago) |
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Rosebud95
Stranger
Registered: 05/13/19
Posts: 33
Last seen: 3 years, 14 days
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That's incredible and very helpful. Thank you very much. It makes a lot of sense to when you think about it. cells are so cool.
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Rosebud95
Stranger
Registered: 05/13/19
Posts: 33
Last seen: 3 years, 14 days
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Re: First time Agar - Suggestions? [Re: Rosebud95]
#26436638 - 01/16/20 11:26 AM (4 years, 13 days ago) |
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Another question for anyone ......... Do you think inserting the polyfill before the tin foil cover is necessary when P.C the MEA solution?
I was looking at the unused plate that I left out yesterday and it looks like little strands of polyfill might have fallen into the MEA solution.
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Baeomaze



Registered: 11/02/16
Posts: 179
Loc: PNW
Last seen: 8 months, 1 day
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Re: First time Agar - Suggestions? [Re: Rosebud95]
#26436749 - 01/16/20 12:29 PM (4 years, 13 days ago) |
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I think getting a “sterilized” bag with a hole in it is your problem it is no longer sterile if it has a hole in it. My 2 cents
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Rosebud95
Stranger
Registered: 05/13/19
Posts: 33
Last seen: 3 years, 14 days
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Re: First time Agar - Suggestions? [Re: Baeomaze]
#26437156 - 01/16/20 05:01 PM (4 years, 13 days ago) |
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lol this is true.
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