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OfflineMycobum7
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Registered: 01/28/19
Posts: 180
Last seen: 11 months, 9 days
i keep getting bacteria in my agar dishes
    #26547200 - 03/20/20 05:30 PM (3 years, 10 months ago)

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.....


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


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Offlinerumfor69
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Registered: 08/05/11
Posts: 6,842
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
    #26547361 - 03/20/20 06:46 PM (3 years, 10 months ago)

Man that sucks, I feel for you cause I know how frustrating that is.
I don't have any insight into your problem but maybe consider trying
a no pour tek. I like using these flat wide mouth jars with a normal
4 hole lid, ring, sfd setup. I just toss in 1.17g pda and 30ml of water
Mix it a little, microwave till it bubbles, stir it a little again then
put on the lid, some tin foil and pc @15psi for 15mins


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Links For Beginners And Beyond:mspoil:


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OfflineSockadin
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
    #26547382 - 03/20/20 06:57 PM (3 years, 10 months ago)

Need more info, can you do a lighter picture in front of your FH? Also are you using a spore solution or swipe?


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OfflineMycobum7
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Registered: 01/28/19
Posts: 180
Last seen: 11 months, 9 days
Re: i keep getting bacteria in my agar dishes [Re: Sockadin]
    #26547504 - 03/20/20 07:56 PM (3 years, 10 months ago)

a lighter picture as in a photo of a lighter lit? i was doing that test earlier and flow seems good. to be clear, the contam seems to happen when I pour dishes, not when i get them from my trusted friend. This is my thought, i am new and have had issues with the mix fully dissolving, the water may not have been warm enough, so i would decant the solution to separate the sludge, therefore possibly making the solution too wet....?



something is definitely up and i gotta figrue this out.


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


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Offlinerumfor69
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
    #26547517 - 03/20/20 08:00 PM (3 years, 10 months ago)

Too wet or too dry would seem to have nothing to do with the sterility


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OfflineMycobum7
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Registered: 01/28/19
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Last seen: 11 months, 9 days
Re: i keep getting bacteria in my agar dishes [Re: rumfor69]
    #26547543 - 03/20/20 08:17 PM (3 years, 10 months ago)

hmm.....i was hoping too much moisture was the culprit because thats what i was gonna change. see, the flow hood has to be good because otherwise i would have seen bacteria on the many transfers ive done....right?

im doing an alcohol wash on gloves several times during the process, running filter for a while before, cleaning work station etc. basically following RR tek...



I need to come up w a plan before trying again but i dont know where to go.


--------------------
"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]
    #26547554 - 03/20/20 08:22 PM (3 years, 10 months ago)

Post a picture of a lite lighter 1 inch in front of your flowhood. We are going to look at the bend in the flame to check on the airflow and determine if you have to much or to little.

Could be flowhood, could be movements over dish, could be PC. Could be pouring plates method.

Do you get bacteria in your plates after you pour before you open again or only after you have used them?


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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: Sockadin]
    #26547563 - 03/20/20 08:25 PM (3 years, 10 months ago)

only after i pour. i was given dishes poured by a friend in front of the same hood and not only were they bac free but i did transfers with no issues, in front of the hood.

now im trying to make my own agar and with the same exact malt and agar i am getting LOTs of bacteria within 72 hours of pouring without opening them.

Now, im by no means a pro but ive studied the videos and understand the idea of whats going on.

i even perfected my taping technique ( was getting little rips in the film when i pulled it too tight)

and still i get blobby snowflake like growth of bacteria.


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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]
    #26547573 - 03/20/20 08:29 PM (3 years, 10 months ago)

Man that's weird. Pouring in front of flowhood.  Are they in the middle of the plate or around the edges?

Are you using Parafilm or syran wrap?

Unincoulated plates bacterial contaminatiom in front of a FH.. I'm thinking PC cycle maybe.


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OfflineMycobum7
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Re: i keep getting bacteria in my agar dishes [Re: Sockadin]
    #26547577 - 03/20/20 08:32 PM (3 years, 10 months ago)

it seems to come from the edges more often than not, and there seems to be a trend with the contaminated ones being a little more wet.
using parafilm.

What are you thinking about the PC that could be doing it? i did 30 minutes and a natural cool down for 500 ml

i said this once but ill say again, last time i didnt warm the water up much and got a lot of sediment, so i poured off what was dissolved and threw out some sediment, maybe i should just leave it next time?


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OfflineSockadin
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
    #26547585 - 03/20/20 08:34 PM (3 years, 10 months ago)

Maybe if it is at the edges your Parafilm is dirty?

Still want to check out that FH


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OfflineMycobum7
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Re: i keep getting bacteria in my agar dishes [Re: Sockadin]
    #26547593 - 03/20/20 08:42 PM (3 years, 10 months ago)

ok i messed with the hood and it appears it blows a little on the hard side so i doubled up the pre filter and can get about a 90 degree bend at maybe 4 inches. i will add pre filters to im prove on that

BUT

that doesnt address the fact that i have had lots of success transferring agar to agar and g2g near this hood without any of this bacteria nonsense.

SO... logic would indicate my tek contains fuckery....


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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: Mycobum7]
    #26547603 - 03/20/20 08:45 PM (3 years, 10 months ago)

because by now i would have certainly seen this happening on one of like 20 week old transfers im looking at here....


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OfflineSockadin
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
    #26547683 - 03/20/20 09:35 PM (3 years, 10 months ago)

Bacteria is hard to see when plates are fully colonized. I'm pretty sure I birthed a grain jar tonight that looked clean, but had a slightly sweet smell. Fucking oats.


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Online99.99
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Re: i keep getting bacteria in my agar dishes [Re: Sockadin]
    #26547701 - 03/20/20 09:43 PM (3 years, 10 months ago)

I also have a flow hood and bacteria was my biggest problem I could never seem to get rid of it until I started using a 5% bleach solution and now it’s been over a year and a half since I’ve had any bacterial contamination

Once in a wile I’ll get a mold on a dish but the bacteria never comes back


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OfflineSockadin
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Re: i keep getting bacteria in my agar dishes [Re: 99.99]
    #26547707 - 03/20/20 09:52 PM (3 years, 10 months ago)

Where are you putting the bleach? In your agar?


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Online99.99
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Re: i keep getting bacteria in my agar dishes [Re: Sockadin]
    #26547744 - 03/20/20 10:29 PM (3 years, 10 months ago)

Quote:

Sockadin said:
Where are you putting the bleach? In your agar?




Lol ... no I use the bleach solution to clean the inside of my flow hood spray down my rubber gloves and any tools that I might work with it’s a disinfectant

Just think about it the simple act of putting on rubber gloves to do work with does nothing to help sanitize things.. The gloves get all kinds of bacteria on them during the process of putting them on  you must spray down your gloves and pretend like you’re washing your hands for about 30 seconds ,the flow hood must be wiped down everything you put in it must be cleaned it’s a process


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Offlineorange771
Registered: 01/26/11
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Re: i keep getting bacteria in my agar dishes [Re: 99.99]
    #26547760 - 03/20/20 10:44 PM (3 years, 10 months ago)

That’s strange, I am rather new to pouring agar and I am pretty damn sloppy with my sterile technique and no issues. Are you loosening the lid on the agar container and wrapping the cap in foil? As soon as the pressure cooker drops all the pressure are you opening it and tightening caps back up ASAP and not opening until ready to pour? Are you spraying down the top inside of your still air box and water is dropping down? I stopped spraying down my box, don’t even put a wet paper towel down any more.


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Online99.99
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Re: i keep getting bacteria in my agar dishes [Re: orange771]
    #26547781 - 03/20/20 10:59 PM (3 years, 10 months ago)

Quote:






Thear is nothing wrong with my process now ,works fantastic but for your information I sterilize the agar in my pressure cooker for half an hour inside a beaker then I pour into the agar dishes while its still hot
let cool inoculate then wrap


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Offlineorange771
Registered: 01/26/11
Posts: 288
Last seen: 1 year, 8 months
Re: i keep getting bacteria in my agar dishes [Re: 99.99]
    #26547845 - 03/21/20 12:02 AM (3 years, 10 months ago)

I meant to reply to the op there, sorry about that.


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