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Janonymous
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Registered: 03/30/22
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Solving the agar bacteria contamination problem
#27815776 - 06/12/22 09:04 AM (2 years, 7 months ago) |
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For few months I am unsuccessful having clean agar dish. It is getting more than frustrating especially when I have three years of growth with high yields. I'm doing something wrong and I can't figure out what. I keep detailed record of everything I do so I would be easier backtrack the problem. I couldn't find out anything by trial and error, so here I am going to put down whole process and I'm hoping that someone will point out in what part of process is it wrong. I have left three fully colonized jars with oats in fridge. I plan to do grain to agar process as I did last time. This has to work. I had huge loses and if I loose this I have nothing left...π Here is the whole process of agar making, sterilization and inoculation: -I boil out about 6 potatoes in pot. Let the water from it to chill in fridge. -I use plastic pp5 dishes. I wash them in soap water. Then dry and soak in sterilizing fluid used for baby bottles for 15 minutes. After that dishes are dried with paper towel. The lids will have stick new micropourus tape over holes. -agar is made from 550 ml of potato water mixed with 10 grams of agar powder. I cook it and let agar dissolve. I add squeeze of honey (about tablespoon) and bring to boil. I simmer it for few minutes. -ready hot agar is poured in dishes. I close them with lids, wrap in tin foil and put to pc. -when I cook I have pc without rocker. As soon as the steam will go out, I let it on for 10 minutes and then put rocker on -after the pc reach 15 psi, I cook it for 40 minutes -I leave pressure cooker unopened from 12 to 24 hours until Inoculation process
-I take shower and use clean clothes -Kitchen is used for inoculation. Put everything out of the place but kitchen table and things I need for inoculation. -The kitchen door is closed tight to prevent draft. -I clean the counters and table with dishwasher soap, new sponge, then spray the surface with solution of water and bleach. Finally I wipe it with paper towel. -SAB is sprayed with air duster. I open the pc unwrap agar dishes, place them in Sab. The colonized jar is sprayed with air duster too and put to sab -SAB is heavily misted with distilled water. I set the timer for 10 minutes to settle down particles. -the tweezers are burned under gas cooker to sterilise and put to sab to cool down until the timer is off -I wash the hands with soap. Use vinyl gloves and wipe them with sanitizer. -as soon as the timer is off I start the work in Sab. -I open colonized jar. With tweezers I pick one oat grain, slightly open the lid of agar dish. Drop in the grain. Close the lid and reapet the process. Now it's all done. Please do tell me what I'm doing wrong and how to fix it.
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SirPsycho
Purple Belt in Google-Fu



Registered: 01/01/20
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Re: Solving the agar bacteria contamination problem [Re: Janonymous] 3
#27815790 - 06/12/22 09:17 AM (2 years, 7 months ago) |
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Quote:
Janonymous said: -the tweezers are burned under gas cooker to sterilise and put to sab to cool down until the timer is off
This is your problem. Your tools are supposed to get flame sterilized right before the transfer and in between each transfer.
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PBJ710
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Re: Solving the agar bacteria contamination problem [Re: SirPsycho] 1
#27815899 - 06/12/22 10:45 AM (2 years, 7 months ago) |
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Are the oats bacterial to start with? How about some pictures of the oats and agar contams?
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Janonymous
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Re: Solving the agar bacteria contamination problem [Re: SirPsycho]
#27815943 - 06/12/22 11:19 AM (2 years, 7 months ago) |
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So are you saying I should invest to kitchen blow torch? How long should I wait after burning tool to transfer?
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SirPsycho
Purple Belt in Google-Fu



Registered: 01/01/20
Posts: 7,340
Loc: Rent free in your head
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Re: Solving the agar bacteria contamination problem [Re: Janonymous]
#27815946 - 06/12/22 11:21 AM (2 years, 7 months ago) |
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Yes, I use a "dab torch" 
You don't need to wait at all. Cool your tool by dipping it in the agar plate you're gonna transfer into then do it transfer
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Ask about free prints: Ps tampanesis, subtropicalis, cubensis(ESS) and Pan cyanescens
Balance in life is like running on ice.
π
π
π
£π
£π
π
π
π
π
π
   
"Mist your balls and fan your asshole" - Pandaskis, 2023
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Janonymous
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Re: Solving the agar bacteria contamination problem [Re: PBJ710]
#27816816 - 06/12/22 09:20 PM (2 years, 7 months ago) |
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Oats are clean I'm pretty sure of it. Mycelium is lovely white everywhere. The agar dishes I boiled over for 15 minutes to kill bacteria right away to not get any chances of farther contam
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normalperson
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Re: Solving the agar bacteria contamination problem [Re: Janonymous]
#27816970 - 06/13/22 01:13 AM (2 years, 7 months ago) |
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i think your oats are dirty, what happens if you g2g with them? show us some pictures of your oat jars please.
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Eggsalts
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Registered: 03/22/22
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Re: Solving the agar bacteria contamination problem [Re: Janonymous]
#27817103 - 06/13/22 05:32 AM (2 years, 7 months ago) |
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Yea bro get one of those blue torches at wall mart and hit it till itβs red hot then move them directly into your sab. I also donβt even spray my sab anymore. I do after Iβm done, then put it away. I was getting alot of contam when I would iso before doing my work. Since then, Iβve started taking iso wipes, and hitting my rack in my sab and the roof of the sab, nothing else, then wiping down everything Iβm working with, then puting it in sab.then waiting for it to evaporate (15mins). Any metal tools get flame sterilized 10 seconds before use then held very still inside SAB to cool down enough to use. I donβt squelch the tools in agar cuz I feel like it causes contam too.
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Janonymous
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Re: Solving the agar bacteria contamination problem [Re: Eggsalts]
#27817160 - 06/13/22 07:05 AM (2 years, 7 months ago) |
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Thank you for advice
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Janonymous
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Re: Solving the agar bacteria contamination problem [Re: normalperson]
#27817161 - 06/13/22 07:05 AM (2 years, 7 months ago) |
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I will send it when I'm home
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Janonymous
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Re: Solving the agar bacteria contamination problem [Re: normalperson]
#27817542 - 06/13/22 12:16 PM (2 years, 7 months ago) |
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Sorry. I have no idea how to put image in here π
Edited by Janonymous (06/13/22 01:45 PM)
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PBJ710
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Re: Solving the agar bacteria contamination problem [Re: Janonymous]
#27817710 - 06/13/22 02:15 PM (2 years, 7 months ago) |
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How to upload images
I suggest using a seperate tab to manage your images from so you can copy/paste the URLS into your thread using the image icon above your text.
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Janonymous
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Re: Solving the agar bacteria contamination problem [Re: PBJ710]
#27818876 - 06/14/22 06:03 AM (2 years, 7 months ago) |
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I came to conclusion it has to be not flame sterelising my tools considering I had contamination even when doing spores inoculation or g2g.
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