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Offlinemea8
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Registered: 02/16/24
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High Contamination Rate despite best efforts
    #28661612 - 02/16/24 05:40 AM (4 months, 6 days ago)

I am a new mycologist and have been trying my hands at agar cultivation in a SAB for the last couple of months. But despite my best attempts my contamination rate is high (between 65-95% of dishes contaminated). So I am going to explain my transfer technique here in some detail in the hopes that some of you more experienced growers can give me some pointers as to where I am going wrong:

  • I clean my bathroom thoroughly with bleach.
  • I open up my SAB and clean that with bleach, then wipe it down with 70Iso
  • I clean my glass petri dishes by hand, then wrap them in aluminium foil and bake them in the oven for 25min at 200°C
  • I mix my agar ingredients (MEA) in a 500ml bottle, shake thoroughly, unscrew the lid half a turn and pressure cook the bottle at 1.8 Bar for 30 min. I then close the bottle properly before removing it from the pressure cooker.
  • I put on a face mask and some used rubber gloves that I wash with bleach. The gloves also cover a portion of my arms.
  • I place the still wrapped (and hot) petri dishes into the SAB and spray everything down with 70Iso.
  • I let the agar bottle cool outside the SAB until I can hold it to my bare skin without it hurting.
  • I spray the bottle with 70Iso and place it into the SAB.
  • I unpack the dishes, unscrew the cap of the agar bottle and pour the agar from the bottom dish to the top dish, taking care to crack the dish open as little as possible and letting only the mouth of the bottle be over the dish.
  • I remove the bottle and let the agar cool.
  • I add in the first dish that I want to take a transfer from. I spray it with 70Iso then unwrap the parafilm.
  • I flame sterilise my scalpel outside the SAB for about 10s then bring it into the SAB and dip it into the new agar dish. Again only barely opening the dish.
  • I cut a piece of agar with mycelium from the transfer dish and place it in the middle of the new dish. All the while doing my best to let only the scalpel be above both plates.
  • I wipe down the new plate with kitchen roll and holding it shut, take it out of the SAB and immediately wrap it in parafilm. I then label the dish and store it outside the SAB.
  • I repeat the last four steps for as many transfers as I want to make.


This Procedure resulted in 8 successful transfers from 4 dishes. 5/8 dishes show mould or bacteria or both after 1 week. I have attached a picture of my SAB as well as a picture of one of the petri dishes. Any pointers would be much appreciated :heart:


Edited by mea8 (02/16/24 05:42 AM)

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Offline10k
KingOfQuestions
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Registered: 02/12/24
Posts: 36
Loc: Eastern seaboard between Maine... Flag
Last seen: 3 months, 8 days
Re: High Contamination Rate despite best efforts [Re: mea8] * 1
    #28661636 - 02/16/24 06:14 AM (4 months, 6 days ago)

Is your environment drafty? Or have a high level of mold or other kinds of bacteria? Some houses just have a MOLD problem the air can be SATURATED with spores and fungus, I’ve been in houses that REAK OF MOLD and anything that stays slightly damp over night starts to grow mold, not saying your house is dirty but on the microscopic level you may have a very high parts per million of particulate matter there are devices you can buy to measure that

maybe run a TRUE HEPA air purifier in the room for a day before hand, turning it off a few hours before you start your work

Also UV-C lights kill bacteria, mold, viruses and are 100% safe for mycelium so you could buy a few bulbs, run one in the room for a few hours before you work, after you shut off the hepa fan, surface the light touches ( for a set amount of time idk how long) will be sterilized

You could also put one of these UV-C lights on the roof of your SAB , just wear sunglasses when your working you can get sunburn on your eyes, and try not to stay too too long ( over an hr) in the light it’s just like strong sunlight you might get a little sunburned

They make these UV-C lights in many shapes, regular bulbs, long handheld devices, fluorescent tube lights,

Also when you use alcohol it needs to evaporate to be effective If your haveing that many problems with a good standard operating procedure like you are then I would spray with alcohol once let dry then again (hands included)

When your heating your scalpel it’s not about time in the flame it’s about the temp you get the blade up to, you want it to be red as can be (you need to be out of direct sun to see it) the hotter the better you want the redness to go like half an inch past were anything would be touching you agar, then when it’s red hot still it instantly goes in the SAB and is used to cut you agar, it will cool down and won’t hurt your transfer, you may get a little carbon/black stuff but that’s ok better then contaminated stuff

Here is a video that shows how to make a laminar flow hood for under 100$, now I have not made one yet but this man explains the concept INCREDIBLY WELL and when I do decide to upgrade I will make one useing his techniques just modify the, a smidge, I found a blower fan to run it that was equips with a true HEPA filter + I might get a thicker HEPA filter and maybe build the box myself out of wood so it looks nice….. OH AND add a UV-C light to the inside chamber behind the filter and the top above the work station

si=JCGk502X61rmHgSb

… possibly get new gloves for every use just to be safe

If your environment does have a lot of spores/ contains in it your cloths will have them on them too, if you jostle around they can dislodge and start to fly around you SAB so maybe go with no sleeves or shirt, 70% iso you let dry them do again

Or if it’s not your PPM for contamination maybe you should just scrap all your gear and start again

And your doing agar to agar right not some LC or prints right? Because if so then it’s just that’s that’s contaminated I would think… altho if it was infected LC or a Spore Syringe ANY CLEAN PLATES would be a feat deserving of songs to be written about your glorious feat! Haha I’m not sure but I hope this is a little helpful


--------------------
Much love y’all, may everone have a blessed day, may we all learn as much as we can, may we evolve and grow this hobby and help bring our wonderful fungi friends into the mainstream so their love, medicine, and beauty can be brought to the world….. THE JOURNEY IS THE DEFINITION MY FRIENDS ENJOY THE RIDE

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OfflineMwj12977
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Re: High Contamination Rate despite best efforts [Re: 10k] * 3
    #28661639 - 02/16/24 06:19 AM (4 months, 6 days ago)

In my opinion you’re shooting yourself in the foot doing anything in a bathroom. Even if you soak it top to bottom in bleach for a year straight there will still be mold spores everywhere. Shower curtains, doors, the lock mechanism on the door, towel bars, inside of drains. Anyway there are way too many cracks and crevice's that will hold spores regardless of how clean you think it is.

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Offline10k
KingOfQuestions
I'm a teapot
Registered: 02/12/24
Posts: 36
Loc: Eastern seaboard between Maine... Flag
Last seen: 3 months, 8 days
Re: High Contamination Rate despite best efforts [Re: Mwj12977]
    #28661643 - 02/16/24 06:23 AM (4 months, 6 days ago)

Maybe suran wrap over the window and rolled up towel under the door (suran wrap over the ceiling vent as well OR ductape), And Lysol the room excessively, start high and let it drift down ….

You know maybe the contams are comeing from the drain too

But the more I think about it it’s most likely you need to heat the scalpel way more and again after ever single use, as red hot as you can get it

Actually Mwj12977 is right, find another room and or location if it keep happening


--------------------
Much love y’all, may everone have a blessed day, may we all learn as much as we can, may we evolve and grow this hobby and help bring our wonderful fungi friends into the mainstream so their love, medicine, and beauty can be brought to the world….. THE JOURNEY IS THE DEFINITION MY FRIENDS ENJOY THE RIDE

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OfflineAspectOfTheCreator
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Re: High Contamination Rate despite best efforts [Re: 10k]
    #28661661 - 02/16/24 06:38 AM (4 months, 6 days ago)

I found that spraying everything so much can be counterproductive. I just mist the walls of my SAB and go to work.


--------------------





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OfflineGiermmo
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Re: High Contamination Rate despite best efforts [Re: AspectOfTheCreator]
    #28661750 - 02/16/24 07:42 AM (4 months, 6 days ago)

Is that your toilet on the right next to your SAB?

Try changing rooms like others mentioned and i'd also recommend to let the air settle for few hours IN THE WHOLE ROOM before doing anything.

So for example:
Clean and set everything up at the morning, go to work and then right after you come from work, clean your hands and go strainght to work in SAB without any rapid movement around the SAB so you won't create air currents.


Good luck


--------------------
Don't worry mate, what you're gonna feel is a slight burning in your heart, your lungs, your brain, your kidneys, your liver, bottom of your feet, your eyeballs, then you start bleeding from your nose and ears, then you start tripping out and seeing everything in the form of an anime cartoon which is really fucking cool.

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OfflineVP123
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Re: High Contamination Rate despite best efforts [Re: mea8] * 2
    #28661783 - 02/16/24 08:17 AM (4 months, 6 days ago)

Welcome to the shroomery. I am going to add a few suggestions in addition to the good advice you were given already.

Potential sources of contamination could be 1) your sterile technique, 2) contaminated materials or improper sterilization, 3) a contaminated environment.

1) If you can, have someone with experience check your sterile technique. Gallons and gallons of alcohol and bleach will not compensate for an improper sterile technique. If you can't get anyone to check your technique, you may want to film yourself while inoculating plates. Something I learned a long time ago doing cell culture is that your hands are the dirtiest thing you put inside a laminar flow hood. After you videotape yourself check the video. A little scratch on your head, nose, or even touching (even if only briefly and inadvertently) your clothes, chair, etc. can transfer contaminants. Examine also what your tools touch before transferring to the SAB or flow hood. You may want to use disposable nitrile gloves, it is not necessary to have gloves all the way to your elbows. Thick rubber gloves often make for clumsy handling of materials and this facilitates contamination. Thinner gloves allow you to feel what you handle and facilitates dexterity, which is often necessary for a proper sterile technique.

2) Have some controls such as agar plates that do not get inoculated. If the agar was not properly sterilized of if the pouring of agar resulted in contamination, this will tell you. You may even want to do "dry" tests. A procedure in which you manipulate your plates as if you were to inoculate them but without actually putting anything in them. With this you can test if your blades, needles, petri dishes, etc. were properly sterilized. You can also test for contaminated spores and liquid culture but only after you determined that you have an adequate sterile technique, materials and agar.

3) As per the environment you already got plenty of advice above. You may also want to test if the procedure in another room gives you better results. Bathrooms often times get mold growth because of the high humidity after using the shower.

Good luck!

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InvisibleLadysKnight
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Re: High Contamination Rate despite best efforts [Re: Giermmo] * 1
    #28661793 - 02/16/24 08:24 AM (4 months, 6 days ago)

Get out of the bathroom

Sab and holes look too small, the larger the sab and armholes, the less turbulence

Try a no-pour agar tek to remove some variables

Try not cleaning or spraying anything into the still air box

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Offlinemea8
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Registered: 02/16/24
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Re: High Contamination Rate despite best efforts [Re: LadysKnight]
    #28662230 - 02/16/24 01:11 PM (4 months, 6 days ago)

You people are so nice!

@KingOfQuestions
To answer your questions:
Recently I have been doing only agar to agar transfers but before I was also doing spore germination directly from a spore print. I had similar levels of contamination. The window in the Bathroom seals well I'd say. I could try sealing the door.

Since a lot of people commented on the room choice: I have tried my Bedroom and my kitchen also. Both yielded similar results. The Bathroom in question is actually mostly disused. It has a washing machine in use but the shower is only used maybe once a year or so.

So my main takeaway is that I am going to do some "dry" runs with agar only and try some of your suggestions to see what the contamination rate is. I also had already suspected my SAB might be too small and will probably get a larger one (and make larger holes).

Many thanks! Your advice is much appreciated. I was really racking my brain over what the issue could be.

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OfflineGastronomicus
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Re: High Contamination Rate despite best efforts [Re: AspectOfTheCreator] * 1
    #28662239 - 02/16/24 01:18 PM (4 months, 6 days ago)

Quote:

AspectOfTheCreator said:
I found that spraying everything so much can be counterproductive. I just mist the walls of my SAB and go to work.




I don't even mist the SAB. I wipe down the surface with ISO and then get to work.

OP it reads like you're doing everything right. I do agree you should not do your work in the bathroom. You can light incense and watch the smoke to see if there are unwanted air movements happening.

Also maybe your technique is not good. You might be moving too aggressively or passing your hand/arm over the dishes when they're open. It took me a while to get good at working with agar


--------------------
Make my Funk the P Funk, I wants to get Funked up

LAGM2024

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OfflineMvlted
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Registered: 12/31/23
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Re: High Contamination Rate despite best efforts [Re: mea8] * 1
    #28662246 - 02/16/24 01:20 PM (4 months, 6 days ago)

so if drains dont get used in that bathroom, the drains that are supposed to hold water will evaporate over a length of time and essentially become vents from your sewer line into you room. some pretty nasty shit breeds im the air of your sewer drain so i really recommend trying another room as well!!


--------------------
New, please be patient! Open to advice!!

Edited by Mvlted (02/16/24 01:20 PM)

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Invisiblepacmanbreed
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Re: High Contamination Rate despite best efforts [Re: mea8]
    #28662347 - 02/16/24 02:20 PM (4 months, 6 days ago)

My first dishes are much worse, even if having a spare room for sab before, Just to add from mentioned.

I'd avoid bumping the sab - my arms touching the holes or using the top to lay tools while working as much.

toss the lid if you may, having the sab in a wider table is better.

as most folks do - work on a raised surface using a rack inside the sab, most of the particulates are in the sab floor, it made a great difference for me,

Edited by pacmanbreed (02/16/24 02:56 PM)

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InvisibleItsLinkNotZelda
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Registered: 01/20/24
Posts: 54
Re: High Contamination Rate despite best efforts [Re: pacmanbreed] * 2
    #28662444 - 02/16/24 03:30 PM (4 months, 6 days ago)

Please be careful when you are cleaning with bleach and alcohol. If you feel the need to use bleach, make sure you let it air out and dry before using any alcohol to clean anything, especially in a small enclosed space like a bathroom. Bleach mixing with alcohol could be very harmful to your health, even deadly. Clean agar plates aren't worth risking your health.

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InvisibleTriskelope
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Registered: 01/07/24
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Loc: In my head
Re: High Contamination Rate despite best efforts [Re: ItsLinkNotZelda]
    #28662605 - 02/16/24 04:49 PM (4 months, 6 days ago)

Just want to second 10k’s advice about running a true HEPA purifier, I have pets and has been extremely helpful to have this.

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OfflineSonomaFungi_707
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Last seen: 20 days, 8 hours
Re: High Contamination Rate despite best efforts [Re: ItsLinkNotZelda]
    #28662645 - 02/16/24 05:16 PM (4 months, 6 days ago)

I’m new at this, but I have been having good success working with “holy grail” style no-pour agar plates. I suggest giving them a try. In the meantime, the idea of doing “dry runs” to assess your sterile technique makes a lot of sense.

Once my SAB is cleaned and set up, I mist the interior lightly with soapy water. Then I spray each individual plate (donors and receivers) with ISO and rub it all around all the exposed surfaces with my gloved hands - one at a time, slowly/deliberately placing each agar plate into the SAB while it and my hands are still wet with ISO.

From there, I just try to work slowly/deliberately while keeping a constant awareness of where my hands are. They NEVER pass directly over an open plate, and they never move quickly while inside the SAB - just slow and steady.

Every time I remove my hands from the SAB, I spray and wipe my gloves and forearms with ISO before sticking them back in. I don’t wear long sleeves when I work in the SAB - bare skin is easier to sanitize with ISO than cloth.

As a general rule, everything that gets put into my SAB is wet with ISO when it passes through the arm hole.

Hope this helps!

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