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InvisiblePiptheGreAtest
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Registered: 07/18/16
Posts: 464
Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
    #26547882 - 03/21/20 12:28 AM (3 years, 10 months ago)

Quote:

Mycobum7 said:
on two separate attempts now i have had a significant amount of dishes grow bacteria on them post pour and before a transfer. I have watched several videos and read several teks. i noticed my mix came out a little watery, could that be the culprit? i am quite confident on the PC and am using a flow hood so not sure why Im having so many issues. Plus, when i transferred to plates given to me that were prepped in front of the same hood by someone else (more experience) i dont have  a issue.

Im almost sure it has to do with my tek/ too watery agar mix, but am pretty confused. I want to have a change in tek or recipe before i break open yet another bag of 20 sterile dishes.....





I had these same questions when my agar kept failing using pour plates, mine were also on watery side. I decide test my sterile technique by order pre pour sterile plates off the internet. I had successful growth on all the plates. I now follow C100 recipe to the t no more watery plates and have over 100 successful transfers. If you get dish off the internet and it works you know it’s your recipe or prep methods figuring you do everything in front the hood the same.

** Recent successful transfers**



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OfflineMycobolical
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Registered: 06/24/17
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Re: i keep getting bacteria in my agar dishes [Re: PiptheGreAtest]
    #26547915 - 03/21/20 12:50 AM (3 years, 10 months ago)

When you pour your plates are you getting condensation inside? Even with good sterile techniques condensation can cause contamination on fresh plates.


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OfflineSockadin
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Re: i keep getting bacteria in my agar dishes [Re: Mycobolical]
    #26548135 - 03/21/20 05:38 AM (3 years, 10 months ago)

How can condensation cause contamination? Please enlighten me on how this process works?


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OfflineMycobum7
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Registered: 01/28/19
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Re: i keep getting bacteria in my agar dishes [Re: 99.99]
    #26548393 - 03/21/20 08:53 AM (3 years, 10 months ago)

5% bleach to clear your work area? i also do this... what is your cleaning routine tho?


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"This isn't my first rodeo ya know.......its like my third."


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OfflineMycobum7
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Re: i keep getting bacteria in my agar dishes [Re: orange771]
    #26548401 - 03/21/20 08:55 AM (3 years, 10 months ago)

agar has poly filter and is wrapped until time to use.


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"This isn't my first rodeo ya know.......its like my third."


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OfflineMycobum7
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Re: i keep getting bacteria in my agar dishes [Re: Sockadin]
    #26548404 - 03/21/20 08:57 AM (3 years, 10 months ago)

it is stated in many teks that excess precip may be associated with contam, its why in some professional labs they store the dishes upside down


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"This isn't my first rodeo ya know.......its like my third."


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OfflineAtmozFear
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Registered: 01/25/19
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
    #26548405 - 03/21/20 08:58 AM (3 years, 10 months ago)

I had the same problem, and I think it was because I was not doing the parafilm efficiently or fast enough so I always just do no pour now.


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OfflineMycobum7
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
    #26548409 - 03/21/20 08:58 AM (3 years, 10 months ago)

i know this thread is getting lengthy but im gonna keep dropping this because i think its important.....

I have dont A LOT of transfers in front of this hood  onto agar that someone else prepped using this hood and have never seen any bacteria at at all

HOWEVER... when I do the the pouring....well.....


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"This isn't my first rodeo ya know.......its like my third."


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OfflineAtmozFear
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
    #26548412 - 03/21/20 09:01 AM (3 years, 10 months ago)

yeah someone on here said "no pour is for pussies!"

He was right but I don't give a shit, pouring is just one more variable.


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OfflineMycobum7
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Re: i keep getting bacteria in my agar dishes [Re: AtmozFear]
    #26548419 - 03/21/20 09:04 AM (3 years, 10 months ago)

im not ready to try a new tek yet but im sitting here like WTF am i doing wrong? this sucks but gives me motivation to try new things and succeed at this...


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"This isn't my first rodeo ya know.......its like my third."


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OfflineSockadin
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
    #26548423 - 03/21/20 09:05 AM (3 years, 10 months ago)

You seem convinced it isn't your flowhood. Did you say you poured in SAB with the same problem?

I don't wipe anything down other than dish with ISO and jars. I had a lot of issues anout 4 years ago with bacterial jars and overtime learned to prefect my grain spawn and PC times. But it was all from dirty cultures and not being patient.


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OfflineMycobum7
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Re: i keep getting bacteria in my agar dishes [Re: Sockadin]
    #26548437 - 03/21/20 09:09 AM (3 years, 10 months ago)

i mean if it was the hood i would have certainly seen bacteria within 3 days of doing transfers, it wouldnt be able to hide because there would be plenty of uncolonized agar for it to show up in. Hasnt showed in one transfer...so thats where i get that logic

i have not used the SAB to do pours i use that mainly for making prints...

im getting new parafilm, try to make a less "watery" mix. other than getting new sterilized dishes then im kinda not sure what to do other than those above mentioned things.

I mean if the hood was failing, then i would be seeing bacteria on at least some of my transfers....right?


--------------------
"This isn't my first rodeo ya know.......its like my third."


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InvisibleLadysKnight
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Registered: 10/09/15
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
    #26548458 - 03/21/20 09:24 AM (3 years, 10 months ago)

This another thing I don't like about "pours". You have to take someone else's word for it that your dishes are sterile. Fuck that. When it comes to sterility, trust no one.


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OfflineMycobum7
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Re: i keep getting bacteria in my agar dishes [Re: LadysKnight]
    #26548474 - 03/21/20 09:30 AM (3 years, 10 months ago)

well i mean theyre in a sleeve from a lab supply site...but yes there is a faith element when using stuff form anywhere. can you drop a link to your preferred no pour tek?


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"This isn't my first rodeo ya know.......its like my third."


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InvisibleLadysKnight
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
    #26548483 - 03/21/20 09:36 AM (3 years, 10 months ago)

I use glass petris dishes.

Pour from pot to plates on kitchen counter (at any temp)
Let plates cool, lids off.
Add lids, stack in groups of 5, wrap in foil.
PC for 20, let cool completely before moving off burner
I use jar rings and trivet, add just enough water to cover trivet.


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Offlinehallmaker
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Re: i keep getting bacteria in my agar dishes [Re: LadysKnight]
    #26548606 - 03/21/20 10:42 AM (3 years, 10 months ago)

I think you should stick with the pour tek. I buy my petri dishes from amazon at this link
https://www.amazon.com/gp/aw/d/B07S8ZCFYZ/ref=ppx_yo_mob_b_track_package_o0_img?ie=UTF8&psc=1

It could be your dishes. I use a my own SAB for pouring.



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InvisibleInfiniteDreams
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Registered: 10/25/19
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Re: i keep getting bacteria in my agar dishes [Re: hallmaker]
    #26548696 - 03/21/20 11:18 AM (3 years, 10 months ago)

I use pour plates. 
They come sterile in a sleeve. 
I sterilize my agar in the PC before I pour. 
I pour in a SAB. 
I do not use ISO. 
I do not use gloves. 
I do wash my hands.
I do not wrap the plates.
After they have set, I do put them in ziplock bags to store on a shelf until time for use.

I have not yet found a single case of contamination when I go to use them, sometimes weeks later.


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OfflineMycobum7
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Re: i keep getting bacteria in my agar dishes [Re: InfiniteDreams]
    #26548832 - 03/21/20 12:30 PM (3 years, 10 months ago)

statements like yours really make me question my dishes


--------------------
"This isn't my first rodeo ya know.......its like my third."


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OfflineMycobolical
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Registered: 06/24/17
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Re: i keep getting bacteria in my agar dishes [Re: Sockadin]
    #26548842 - 03/21/20 12:38 PM (3 years, 10 months ago)

Quote:

Sockadin said:
How can condensation cause contamination? Please enlighten me on how this process works?




When you have condensation in your dishes it eventually falls off the lid onto the surface of the agar. It can also fall between the lid and the outside. From what I understand and have experienced myself this standing water can act like a wick for bacteria to get I to your otherwise clean dishes. It's the same reason we cover our jar lids with foil to keep the filters dry. Wet filters allow bacteria to work it's way in. There are a few things you can do to hell prevent this.

1. Wait until your agar cools a bit before pouring your dishes.

2. Make extra agar in a jar with no filter on the lid. Set that jar on top of your dishes as they cool to help equalize the temperature across the dishes as they cool.

3. If you the room your pouring in wont be disturbed,cleave them closed but unsealed in the flow of your hood for 12-24 hours. It is sterile and will give the dishes condescension time to evaporate. (I do this every time.)

4. Store your dishes upside down both before and after transferring. This will help prevent any standing water on top of the lid from falling onto the substrate.


Also how are you wrapping your dishes? Pics would be helpful.


Edited by Mycobolical (03/21/20 12:40 PM)


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InvisibleLadysKnight
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Registered: 10/09/15
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Re: i keep getting bacteria in my agar dishes [Re: InfiniteDreams]
    #26548849 - 03/21/20 12:41 PM (3 years, 10 months ago)

Quote:

InfiniteDreams said:
I use pour plates. 
They come sterile in a sleeve. 
I sterilize my agar in the PC before I pour. 
I pour in a SAB. 
I do not use ISO. 
I do not use gloves. 
I do wash my hands.
I do not wrap the plates.
After they have set, I do put them in ziplock bags to store on a shelf until time for use.

I have not yet found a single case of contamination when I go to use them, sometimes weeks later.




Hey InfiniteDreams, I'm also on team Ziploc, no wrap, and store in the open.
When using the plate, I wipe the baggie with iso outside the sab, open baggie in sab, toss baggie.
So you don't wipe the baggie at all?
Do you open it inside or outside sab?


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