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InvisiblePiptheGreAtest
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Constant Agar contamination? So Frustrating.
    #26473002 - 02/06/20 10:18 PM (3 years, 11 months ago)

I have done multiple runs of agar in patsywhtyes easy agar dishes, every time they have contaminated and failed. I’m inoculating with MS syringe in a SAB, I flame the needle with alcohol lamp. SAB is sprayed down with bleach cleaner and air sanitizer along with the room in a similar fashion. I choose to wear tyvek suit with mask and gloves all which get sprayed with disinfectant. The plates are prepared how pastsy describes and PC’d for 45 minutes. Why are these plates failing? Any advance cultivator if I could get tips on how to prevent this that would be great.


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InvisiblePiptheGreAtest
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Re: Constant Agar contamination? So Frustrating. [Re: PiptheGreAtest]
    #26473007 - 02/06/20 10:20 PM (3 years, 11 months ago)

Just recognized something if my agars wet will that increase the likelihood of contamination? Would you recommend a beginner to use pre poured plates?


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Offlinejbgtaa
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Re: Constant Agar contamination? So Frustrating. [Re: PiptheGreAtest] * 1
    #26473031 - 02/06/20 10:37 PM (3 years, 11 months ago)

Use Petri dishes and a media bottle for more consistent results. Bathroom may not be a good place to do work, although my mold infested basement worked for a long time.


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OfflineMH5109
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Re: Constant Agar contamination? So Frustrating. [Re: PiptheGreAtest]
    #26473040 - 02/06/20 10:40 PM (3 years, 11 months ago)

Are you hitting your arms/ the arms of the tyvek suit on your arm holes. The only reason I ask is cuz it looks like they are close together and I've had trouble with that in the past. If the syringe is fucked that could very well be the problem too, I speak from previous experience.


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OfflineRaccoon
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Re: Constant Agar contamination? So Frustrating. [Re: MH5109]
    #26473087 - 02/07/20 12:19 AM (3 years, 11 months ago)

Show pictures of the contaminated plates. Did you get cube myc to grow along with the contamination? Did you try transferring away from the contamination? Do the plates need GE holes? My petri dishes don't need them. Do you turn off the air in your house before doing sab work? If your technique is good, then the syringe could be the problem. Did you try not inoculating a test plate to see if your agar prep is okay?


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Invisiblegizmo1
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Re: Constant Agar contamination? So Frustrating. [Re: Raccoon]
    #26473103 - 02/07/20 12:32 AM (3 years, 11 months ago)

Buy a big ass can on corn in the bulk section, eat the corn then take the can and heat it up and use that shit to fix the arm holes in your SAB.
Also what is going on with your no pours it looks like the growth is on the lid? Is that polyfil? Not talking shit I honestly cant tell.


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OfflineFrankRhizo
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Re: Constant Agar contamination? So Frustrating. [Re: gizmo1]
    #26473325 - 02/07/20 06:24 AM (3 years, 11 months ago)

Control dish? One you never open or inoculate.
Have you tried a dry run, doing transfers with no spores or live myc?


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InvisibleSmartattack
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Re: Constant Agar contamination? So Frustrating. [Re: FrankRhizo]
    #26473327 - 02/07/20 06:28 AM (3 years, 11 months ago)

Dirty syringe. That's the whole point of agar with a syringe. As said, you need to transfer any myc away from the dirty plates.

And we need pics inside the plates not outside.


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OfflineSynKyd
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Re: Constant Agar contamination? So Frustrating. [Re: Smartattack]
    #26473576 - 02/07/20 09:44 AM (3 years, 11 months ago)

Increase PC time and make sure your lids aren’t your problem with a control plate as someone already mentioned would be my advice.  From there make sure your flaming your blade or instrument fully before you start cutting, that has dogged me before.

:cheers:


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InvisibleSmartattack
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Re: Constant Agar contamination? So Frustrating. [Re: SynKyd]
    #26473608 - 02/07/20 09:57 AM (3 years, 11 months ago)

Well he's already at 45 mins which is already more than necessary for agar. I do 2 liters at a time for 30 min no prob.


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OfflineBackbone
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Re: Constant Agar contamination? So Frustrating. [Re: MH5109]
    #26473697 - 02/07/20 10:42 AM (3 years, 11 months ago)

im thinking the syringe im not talking from experience


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OfflineA.k.aM
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Re: Constant Agar contamination? So Frustrating. [Re: Backbone]
    #26473758 - 02/07/20 11:25 AM (3 years, 11 months ago)

It is probably the syringe.

As long as plates you haven’t used yet are staying clean you should be able to transfer away from contams.

Also idk how big a deal it is but I was told to not use the lid as the bottom of the SAB, it can flex with the weight of moving things around and suck in/force out air stirring things up in the box.


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InvisibleInfiniteDreams
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Re: Constant Agar contamination? So Frustrating. [Re: A.k.a]
    #26473969 - 02/07/20 01:47 PM (3 years, 11 months ago)

Is that polyfill really packed in there tight?

And IDK, next to the toilet is about the last place I'd attempt sterile work.


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OfflineBackbone
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Re: Constant Agar contamination? So Frustrating. [Re: InfiniteDreams]
    #26474195 - 02/07/20 04:00 PM (3 years, 11 months ago)

you sure cus theres like zero air flow INSIDE the toilet...?


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InvisibleSmartattack
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Re: Constant Agar contamination? So Frustrating. [Re: Backbone]
    #26474280 - 02/07/20 05:10 PM (3 years, 11 months ago)

Toilet isn't the issue.


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Invisibleeatyualive
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Re: Constant Agar contamination? So Frustrating. [Re: Smartattack] * 1
    #26474291 - 02/07/20 05:18 PM (3 years, 11 months ago)

The syringe you are using could be contaminated. Have you tried swabbing a spore print?

I don’t think the plates are failing. It is one of 2 things:
1) bad technique
2) contaiminated syringe

Try testing plates with another source of spores. Preferably a print.

Another thing, you don’t need bleach or sanitizer for a sab. Simple 70 iso works great. Get a cookie rack and elevate your work area a bit off the bottom of the sab.
You can see one in this pic.



Changing to Petri dishes and media bottles will make no difference. It all works the same, technique is key. I use diy everything, I’ve used shmuvboxes, flowhood, SABs. My contam rate is literally the same through all methods. 0 to none.

I added magnetic vent covers to the holes in my sab. I cover my holes when not doing work inside to let the air settle. Do steady movements, not fast movements. Let the air settle 30 seconds to 1 minute before doing anything. If you move to fast, take your hands out, let the air settle again. Get into the habit of sanitizing with iso more often if you are having issues. Anytime my hands come out of my sab, they get iso before I put them back in. I also wear gloves but no sleeves. I simply wipe iso up to my elbows. I also keep my iso in a spray bottle.

Also pcing pasty plates more than 30 minutes isn’t necessary. I always do 30 at 15 psi and there is no difference in contamination rates. it’s the technique.

I use the lid of my sab as the bottom. A lot of what people tell you is horse shit.


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Edited by eatyualive (02/07/20 05:41 PM)


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OfflineTurtleEnvy
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Re: Constant Agar contamination? So Frustrating. [Re: PiptheGreAtest]
    #26474376 - 02/07/20 06:03 PM (3 years, 11 months ago)

Quote:

PiptheGreAtest said:
I choose to wear tyvek suit with mask and gloves all which get sprayed with disinfectant.




Do you tape your gloves to the sleeves of your suit?

Does your tyvek suit have a hood to cover your hair?

What type of mask are you using?


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OfflineA.k.aM
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Re: Constant Agar contamination? So Frustrating. [Re: TurtleEnvy] * 1
    #26474383 - 02/07/20 06:07 PM (3 years, 11 months ago)

Man that’s way overkill for a SAB.

As long as you have good technique and there’s no air currents you’ll be ok.

I’ve done a bunch of sessions with just a mask and gloves and a dog sleeping on my lap and been fine.

But it won’t hurt and I could definitely see constant contamination driving me to that point pretty fast lol.


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Invisiblegizmo1
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Re: Constant Agar contamination? So Frustrating. [Re: InfiniteDreams]
    #26474404 - 02/07/20 06:14 PM (3 years, 11 months ago)

Quote:

InfiniteDreams said:
Is that polyfill really packed in there tight?

And IDK, next to the toilet is about the last place I'd attempt sterile work.



I doubt it. There isnt much of it there. It doesn't even look like polyfil either the fibers look too short and close together. Almost looks like cotton???


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Invisiblegizmo1
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Re: Constant Agar contamination? So Frustrating. [Re: A.k.a]
    #26474414 - 02/07/20 06:19 PM (3 years, 11 months ago)

Quote:

A.k.a said:
Man that’s way overkill for a SAB.

As long as you have good technique and there’s no air currents you’ll be ok.

I’ve done a bunch of sessions with just a mask and gloves and a dog sleeping on my lap and been fine.

But it won’t hurt and I could definitely see constant contamination driving me to that point pretty fast lol.



Man that dog would be outside in the quickness. I dont mind my dog in my lap but I cant stand a clingy dog when im trying to work.


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Invisibleeatyualive
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Re: Constant Agar contamination? So Frustrating. [Re: gizmo1]
    #26474684 - 02/07/20 08:51 PM (3 years, 11 months ago)

You could try micropore tape over the holes instead of poly. But I don’t think that is the issue.


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InvisiblePiptheGreAtest
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Re: Constant Agar contamination? So Frustrating. [Re: eatyualive]
    #26479845 - 02/11/20 09:41 AM (3 years, 11 months ago)

I appreciate all the advice, I’m going to get a cookie sheet to raise my work space. I bought some pre sterilized plates just to see if it was my technique and work space or the peeping of my agar. The bathroom is never used so area shouldn’t be a problem. The poly fill in in there really tight and I guess that would be my GE for the plates.


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OfflineJarhead3521S
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Re: Constant Agar contamination? So Frustrating. [Re: PiptheGreAtest]
    #26479865 - 02/11/20 09:52 AM (3 years, 11 months ago)

I’m going to x2 on the smooth movement in the sab, and letting things settle.  Last round of petris I poured, was not smooth.  Used 10 out of the 20 the following day and left the unused stack in the box....been there for a couple weeks, started to do some work last night and noticed 2 of 10 had shit growing in them, and it most definitely came from my technique that night.


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InvisiblePiptheGreAtest
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Re: Constant Agar contamination? So Frustrating. [Re: Jarhead3521]
    #26479909 - 02/11/20 10:20 AM (3 years, 11 months ago)

Smooth movements and fix holes got it.


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OfflineSockadin
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Re: Constant Agar contamination? So Frustrating. [Re: PiptheGreAtest]
    #26479933 - 02/11/20 10:37 AM (3 years, 11 months ago)

Quote:

PiptheGreAtest said:
I have done multiple runs of agar in patsywhtyes easy agar dishes, every time they have contaminated and failed. I’m inoculating with MS syringe in a SAB, I flame the needle with alcohol lamp. SAB is sprayed down with bleach cleaner and air sanitizer along with the room in a similar fashion. I choose to wear tyvek suit with mask and gloves all which get sprayed with disinfectant. The plates are prepared how pastsy describes and PC’d for 45 minutes. Why are these plates failing? Any advance cultivator if I could get tips on how to prevent this that would be great.






Haven't we decided that the lids can be unmodified now days. Is the poly even needed?


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InvisiblePiptheGreAtest
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Re: Constant Agar contamination? So Frustrating. [Re: Sockadin]
    #26479945 - 02/11/20 10:45 AM (3 years, 11 months ago)

I’m stepping away from patsy plates. I need better visibility so I got some pre sanitize and patsy plates  dishes. I’m trying to isolate my problem but in further will either get glass Petrie dishes.


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InvisibleInfiniteDreams
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Re: Constant Agar contamination? So Frustrating. [Re: PiptheGreAtest]
    #26479966 - 02/11/20 10:59 AM (3 years, 11 months ago)

When you you inoculate your plates or whatever you are using, leave 1 or 2 with agar but don't inoculate them.  These can be your controls.  If they contaminate then you know you have an issue in your prep phase before you even get to inoculation.

If they don't contaminate but your inoculated plates do, then you know you either have issue with how you inoculate or it is just coming from the source.


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OfflineBackbone
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Re: Constant Agar contamination? So Frustrating. [Re: InfiniteDreams]
    #26480111 - 02/11/20 12:41 PM (3 years, 11 months ago)

i inoculated three plates in a sab pcd my agar beforehand, sanitized before and havent got any contams in 3 days, its probably his syringe, my methods werent perfect i touched a dish and had condensation in each plate...I got the idea to wait for the air to still in my sab, but i didnt use anything to elevate my plates i just made sure i wiped the entire area of the sab, this of course still pending. I heard only a couple days are needed before signs of contam show, its been three, ill keep watching.


Edited by Backbone (02/11/20 12:49 PM)


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OfflineA.k.aM
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Re: Constant Agar contamination? So Frustrating. [Re: Backbone]
    #26480149 - 02/11/20 01:11 PM (3 years, 11 months ago)

Well you’re supposed to wait for the air to settle before using it so if you haven’t been that could be a big reason.

I see some people work super high up in the box, my metal grate thing only keeps things maybe half an inch off the bottom but it’s been fine.


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