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OfflineCenterfinger
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agar contam, please ID
    #21792477 - 06/11/15 10:32 AM (8 years, 7 months ago)

I've looked through thousands of pictures and just cannot figure out what happened with my agar.  I thought things may be ok until I noticed this same type of growth on two of my control plates.

I made 8 jars of agar (potato boil water 100g, 5g agar and a teaspoon of karo), Pc'd twice for 20 minutes.  I used a GT multispore on 2 of them.  This was 4 days ago.  2 of my control jars have similar growth and 4 of them are 100% clean.

I saw one picture that looked like mine, but the contam was not identified.  I'm sure it's gotta be a common contam. I have circled the locations where 1 drop of spore solution was dropped.  one jar had 3 drops and one had 1 drop.

any assistance is appreciated. Sorry for the crappy pics, I just snapped them quickly before heading to work.

Also, the pics don't show it, but this is not fluffy stuff more of a film and kinda looks like the surface of the moon.

Jar 1 3 drops multi spore

Jar 2 one drop MS

Control jar 1

Close up of the edge on jar with 3 drops


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Offlineinvitro

Registered: 05/03/13
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Re: agar contam, please ID [Re: Centerfinger]
    #21795699 - 06/12/15 01:21 AM (8 years, 7 months ago)

what do u mean by control plate?  Can you get me a pic of your lid?


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OfflineCenterfinger
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Re: agar contam, please ID [Re: invitro]
    #21796531 - 06/12/15 09:13 AM (8 years, 7 months ago)

I'll grab a pick of the lid, but it's standard Mason jar lid with a quarter inch hole in the center with two layers of micropore tape.

By control plate I meant that i had 5 agar jars left over with nothing in then but agar. I left them just sit there to ensure there was not a problem with the agar prep. The jars with the spore drops got the infection right away, first signs were after 24 hours. Two of the unused agar jars didn't show signs of contam until day 4.

I just assumed its some sort of bacterial infection, but maybe its something I can correct in my procedure. I thought it had to be a common contam, but I guess its not that simple.

I made 8 more agar jars last night and will let them just sit around a few days before trying again with spores.

The spore solution was in my fridge for 2 years, but looked like I made it yesterday. Most of my supplies are about 2 years old, I'm just trying to some clean growth going.

With my 3 agar jars that were still clean from the first round, I tried one with the ms syringe again, the other two got spores from 2 different prints.

Thanks for any assistance.


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Offlineinvitro

Registered: 05/03/13
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Re: agar contam, please ID [Re: Centerfinger]
    #21796697 - 06/12/15 10:00 AM (8 years, 7 months ago)

Well if your control is getting bacteria then something is up with your procedure right? 

You might want to look at:
how long you sterilize
How you pour (is it a no-pour plate)?
and lastly, maybe something wrong with your lid?


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OfflineCenterfinger
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Re: agar contam, please ID [Re: invitro]
    #21796766 - 06/12/15 10:16 AM (8 years, 7 months ago)

I agree there is something wrong with my procedure. So with this next round of agar, I have tried 2 jars with solid lids and increased PC time to 30 minutes.  I can't recall exactly, but with the first round of contamed agar I'm pretty sure I left the foil on a couple of jars for a couple of days and if those were to two the got the contam, I would then know to remove the foil immediately after they come out of the PC.

It's also my first time using micropore tape and I don't think I like or need it.  Since I'm just trying to get a bit of growth going in order to clean it up and make some transfers I really dont think that I need GE.


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Offlineinvitro

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Re: agar contam, please ID [Re: Centerfinger]
    #21799916 - 06/12/15 10:23 PM (8 years, 7 months ago)

Are these no pour plates?  Are you taking them out of the pc before they are cool?


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OfflineCenterfinger
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Re: agar contam, please ID [Re: invitro]
    #21813901 - 06/16/15 09:32 AM (8 years, 7 months ago)

yes, these are no pour agar.  They were cool enough to handle and put in my SAB.

With my 3 jars that were clean from the first round, I went print to agar with two of them and one got some MS syringe.  One of them from a print got the same contam, but the other two are looking good and clean.  I did this last Thursday so I hope to see germination by the end of this week.

I did another 8 jars of agar on Thursday as well and all of them look good as of right now.  So if and when I start to see some germination, I'm good to go with some clean agar for transfers.

I'm running a 25% contam rate with agar at this point, which I think is pretty high, but I'll just have to get my proceedure cleaned up and maybe build a new SAB.


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Offlineinvitro

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Re: agar contam, please ID [Re: Centerfinger]
    #21813989 - 06/16/15 09:57 AM (8 years, 7 months ago)

I'm curious about your technique.  How are you doing it with the print, an inoculation loop?  With the ms syringe are you cracking the lid or going through a ship?  If you get your technique down I'd like to hear how you changed it.


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OfflineCenterfinger
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Re: agar contam, please ID [Re: invitro]
    #21822831 - 06/18/15 09:14 AM (8 years, 7 months ago)

I used a scalpel like apparatus for the print, heated to red hot placed in SAB then wiped down with alcohol paper towel.  I dipped the scalpel in the agar, then to the print then back to agar.  I don't have any ships in these lids so I do open the lid to complete the transfer.

Things I did differently on the second round of agar that all came out clean (7 days, absolutely nothing growing in them), was I PC'd them 2 times vs one.  I took the tin foil off a few of them right away to see if that may have been the issue, but that did not make any difference.

It's been 7 days and I can finally see some growth on both the spore and MS agars, so I should be able to make some transfers in a couple of days.  I'm going to transfer each one into 2 separate jars just in case I get the same contam. 

At least my particular contam starts and grows really fast so I can see if I have issues in a day or two.


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OfflineCenterfinger
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Re: agar contam, please ID [Re: Centerfinger]
    #21827896 - 06/19/15 11:58 AM (8 years, 7 months ago)

I'm getting really pissed off at working with agar.  of the two jars with what I thought was growth, one definitely has a penny sized growth of the same contam and the other has a pin head sized growth of bright white myc, but has not spread or grown in two days.

On the flip side I knocked up a pint size jar of prepped oats at the same time as the two of these plate and it's moving along quite nicely.

I think I'm going to run down to the lab supply store and get some proper agar and petri's.


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InvisibleTheEaglesGift
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Re: agar contam, please ID [Re: Centerfinger]
    #21830644 - 06/19/15 11:36 PM (8 years, 7 months ago)

Quote:

I don't have any ships in these lids so I do open the lid to complete the transfer.





Even if you has ships, how would you use them with a scalpal?

Are you working in a still air box?


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InvisibleMad Season
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Re: agar contam, please ID [Re: Centerfinger]
    #21831623 - 06/20/15 08:16 AM (8 years, 7 months ago)

Quote:

Centerfinger said:
I used a scalpel like apparatus for the print, heated to red hot placed in SAB then wiped down with alcohol paper towel.  I dipped the scalpel in the agar, then to the print then back to agar.  I don't have any ships in these lids so I do open the lid to complete the transfer.



Next time don't use alcohol. Flame sterilizes things. Alcohol sanitizes. You should see what a surface wiped with alcohol does under a microscope. Just moves the contaminations around. You basically cleaned the blade then wiped it with shit and made it unsterile again. You don't even need to dip in the agar. Also knives can damage spores. Use a loop or a blunt object that can scrape. Flame til red hot, and wait a few seconds for it to cool down. No agar dips or alcohol wipes.

Famous saying is its like taking a shower then wiping your face with shit afterwards.

Also is it in a still air box? Never let media stay open for long and don't put your arm/gloved hand over the media. If you do consider yourself fucked.


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Edited by Mad Season (06/20/15 08:20 AM)


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OfflineKizzle
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Re: agar contam, please ID [Re: Mad Season]
    #21839383 - 06/21/15 09:26 PM (8 years, 7 months ago)

Quote:

Mad Season said:
Quote:

Centerfinger said:
I used a scalpel like apparatus for the print, heated to red hot placed in SAB then wiped down with alcohol paper towel.  I dipped the scalpel in the agar, then to the print then back to agar.  I don't have any ships in these lids so I do open the lid to complete the transfer.



Next time don't use alcohol. Flame sterilizes things. Alcohol sanitizes. You should see what a surface wiped with alcohol does under a microscope. Just moves the contaminations around. You basically cleaned the blade then wiped it with shit and made it unsterile again. You don't even need to dip in the agar. Also knives can damage spores. Use a loop or a blunt object that can scrape. Flame til red hot, and wait a few seconds for it to cool down. No agar dips or alcohol wipes.

Famous saying is its like taking a shower then wiping your face with shit afterwards.

Also is it in a still air box? Never let media stay open for long and don't put your arm/gloved hand over the media. If you do consider yourself fucked.



If you dip it in the agar to cool it'll help the spores stick to it a little. Might not be good idea if you plan to reuse the print at a later time.


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OfflineCenterfinger
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Re: agar contam, please ID [Re: Kizzle]
    #21841275 - 06/22/15 10:57 AM (8 years, 7 months ago)

Thanks for the additional tips.  Next round I will ditch the alcohol wipe after the flame. I also made an innoc loop and tested it out with a new print that arrived last week.

I did get some malt extract and some new agar agar on friday that I will use for my next attempt.

I use a sterlite SAB, but I think I may make a better alternative because it's kinda hard to see through the milky sterlite.  I was thinking of laying a fish tank on its side and build some sort of  lexan extension for the hand holes and angle the view window.

My MS pin sized growth is about the size of a nickel now, but I think it's still to small to make a transfer. The 8 jars of agar from round 2 are still very clean.


Edited by Centerfinger (06/22/15 11:21 AM)


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