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Mycobum7
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i keep getting bacteria in my agar dishes
#26547200 - 03/20/20 05:30 PM (3 years, 10 months ago) |
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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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rumfor69
Bodhicitta Cultivator



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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) |
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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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Sockadin



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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) |
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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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Mycobum7
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Re: i keep getting bacteria in my agar dishes [Re: Sockadin]
#26547504 - 03/20/20 07:56 PM (3 years, 10 months ago) |
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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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rumfor69
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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) |
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Too wet or too dry would seem to have nothing to do with the sterility
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Mycobum7
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Re: i keep getting bacteria in my agar dishes [Re: rumfor69]
#26547543 - 03/20/20 08:17 PM (3 years, 10 months ago) |
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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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Sockadin



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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) |
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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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Mycobum7
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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) |
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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.
-------------------- "This isn't my first rodeo ya know.......its like my third."
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Sockadin



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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) |
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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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Mycobum7
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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) |
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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?
-------------------- "This isn't my first rodeo ya know.......its like my third."
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Sockadin



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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) |
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Maybe if it is at the edges your Parafilm is dirty?
Still want to check out that FH
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Mycobum7
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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) |
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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....
-------------------- "This isn't my first rodeo ya know.......its like my third."
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Mycobum7
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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) |
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because by now i would have certainly seen this happening on one of like 20 week old transfers im looking at here....
-------------------- "This isn't my first rodeo ya know.......its like my third."
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Sockadin



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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) |
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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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99.99
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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) |
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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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Sockadin



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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) |
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Where are you putting the bleach? In your agar?
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99.99
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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) |
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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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orange771
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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) |
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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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99.99
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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) |
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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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orange771
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Re: i keep getting bacteria in my agar dishes [Re: 99.99]
#26547845 - 03/21/20 12:02 AM (3 years, 10 months ago) |
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I meant to reply to the op there, sorry about that.
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PiptheGreAtest
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
#26547882 - 03/21/20 12:28 AM (3 years, 10 months ago) |
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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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Mycobolical
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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) |
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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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Sockadin



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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) |
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How can condensation cause contamination? Please enlighten me on how this process works?
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Mycobum7
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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) |
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5% bleach to clear your work area? i also do this... what is your cleaning routine tho?
-------------------- "This isn't my first rodeo ya know.......its like my third."
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Mycobum7
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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) |
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agar has poly filter and is wrapped until time to use.
-------------------- "This isn't my first rodeo ya know.......its like my third."
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Mycobum7
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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) |
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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
-------------------- "This isn't my first rodeo ya know.......its like my third."
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AtmozFear
just a shade of myself


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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) |
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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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Mycobum7
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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) |
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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.....
-------------------- "This isn't my first rodeo ya know.......its like my third."
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AtmozFear
just a shade of myself


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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) |
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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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Mycobum7
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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) |
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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...
-------------------- "This isn't my first rodeo ya know.......its like my third."
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Sockadin



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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) |
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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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Mycobum7
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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) |
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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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LadysKnight
Hello Ladies


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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) |
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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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Mycobum7
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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) |
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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?
-------------------- "This isn't my first rodeo ya know.......its like my third."
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LadysKnight
Hello Ladies


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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) |
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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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hallmaker
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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) |
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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.
-------------------- Hello.
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InfiniteDreams


Registered: 10/25/19
Posts: 1,224
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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) |
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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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Mycobum7
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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) |
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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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Mycobolical
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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) |
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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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LadysKnight
Hello Ladies


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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) |
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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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hallmaker
Busy



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Re: i keep getting bacteria in my agar dishes [Re: InfiniteDreams]
#26548926 - 03/21/20 01:21 PM (3 years, 10 months ago) |
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What is ISO?
-------------------- Hello.
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BooShow
Spooky



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Re: i keep getting bacteria in my agar dishes [Re: hallmaker]
#26548929 - 03/21/20 01:23 PM (3 years, 10 months ago) |
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Quote:
hallmaker said: What is ISO?
Isopropyl Alcohol
-------------------- You are what is. That's all.
  
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LittleBoard
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
#26549082 - 03/21/20 03:02 PM (3 years, 10 months ago) |
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Quote:
Mycobum7 said: 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?
At which step did you do this and why? I assume before PCing. I imagine that this can throw of your ratios and you get "watery" agar. What is your friend's procedure where there is no contam with apparently all factors the same as yours?
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Sockadin



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Re: i keep getting bacteria in my agar dishes [Re: LittleBoard]
#26549188 - 03/21/20 03:53 PM (3 years, 10 months ago) |
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So he is ordering pre poured plates and getting bacteria? I didn't realize this. I have no experience in the matter, but if you let someone else make your plates why not let them grow your shrooms?
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Kizzle
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
#26549281 - 03/21/20 04:54 PM (3 years, 10 months ago) |
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Quote:
Mycobum7 said: statements like yours really make me question my dishes
That's the first thing I'd suspect, all things considered. Pouring is relatively easy so if you're not having problems when doing transfers I would be surprised if it was your sterile technique or flowhood that's the problem. You could do a test and try pouring some into a sterile mason jar or sterile PP container and see if it still happens with that. I've never heard of anyone getting presterilized dishes that were compromised so I don't know how likely it is.
Something else to consider though is if the plates your friend made were made with sabouraud agar or antibiotic agar it might prevented the bacteria from showing up.
--------------------
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InfiniteDreams


Registered: 10/25/19
Posts: 1,224
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Re: i keep getting bacteria in my agar dishes [Re: LadysKnight]
#26549335 - 03/21/20 05:27 PM (3 years, 10 months ago) |
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Quote:
LadysKnight said:
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?
At first I did that also. Now I don't use iso. I also am reusing the same ziplocks. The ones I"m using have a slide close and can fit about 20 plates in a single bag. I've been opening the bag outside the SAB. But I am right in front of it and pass the stack of plates through the hole and set them on the rack inside my SAB. The plates themselves remain closed the entire time. After they are inoculated I will wrap them with parafilm.
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LadysKnight
Hello Ladies


Registered: 10/09/15
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Re: i keep getting bacteria in my agar dishes [Re: InfiniteDreams]
#26549387 - 03/21/20 05:54 PM (3 years, 10 months ago) |
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Ok, I see, not too similar, mine go from PC to sab into individual ziplocks after inoc. I may still try taking the plate out before sab.
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Mycobum7
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Re: i keep getting bacteria in my agar dishes [Re: Mycobolical]
#26549500 - 03/21/20 06:45 PM (3 years, 10 months ago) |
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this is great information, thank you. i am wrapping my dishes with parafilm, i use two squares, fold once hot dog way so double layered, and wrap. nicely so it seals the overlap with no tears, this is done in front of the hood.
-------------------- "This isn't my first rodeo ya know.......its like my third."
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Mycobum7
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
#26549504 - 03/21/20 06:48 PM (3 years, 10 months ago) |
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no these are not pre poured plates, i am pouring. i think i will do another pour tonight with the best tek i have yet done, keeping in mind things like moisture content / condensation and see what happens.
-------------------- "This isn't my first rodeo ya know.......its like my third."
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Mycobum7
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Re: i keep getting bacteria in my agar dishes [Re: PiptheGreAtest]
#26549514 - 03/21/20 06:55 PM (3 years, 10 months ago) |
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can you drop a link to the c100
-------------------- "This isn't my first rodeo ya know.......its like my third."
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Sockadin



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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
#26549561 - 03/21/20 07:22 PM (3 years, 10 months ago) |
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What is your recipe? Are you venting the PC for long enough? I'm grasping at straws here man you got a good puzzle on this one.
Thought I just had. Make a batch of agar and don't pour it. PC it and let it cool in the bottle till it gets all solid infront of the FH. Wait a few days. If you get bacteria in the bottle you can start there and work backwards. If it doesn't just rePC and work forwards
Edited by Sockadin (03/21/20 07:24 PM)
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Mycobum7
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Re: i keep getting bacteria in my agar dishes [Re: Sockadin]
#26549859 - 03/21/20 09:55 PM (3 years, 10 months ago) |
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i was thinking something like that, i was thinking maybe doing a sterilized jar and pouring into that to control jar vs. dishes.
When you say venting the pc what exactly do you mean and what is its importance?
recipe is 10 g agar 10 g malt per 500 ml, seems to be a relatively standard recipe. doing a cook right now and gonna do some cleaning and do a pour,
im thinking ill try to leave a small amount of agar in the container and see if any bacteria grown in there, the idea being having a control between a PC container and a sterile dish, although its not perfect its a place to start.
-------------------- "This isn't my first rodeo ya know.......its like my third."
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Mycobum7
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
#26549893 - 03/21/20 10:16 PM (3 years, 10 months ago) |
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been reading lots on altitude, i live at about 5 k and have been doing standard pc times
anyone with info on this?
should i mod the pc or change times to account for this?
-------------------- "This isn't my first rodeo ya know.......its like my third."
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Sockadin



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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7]
#26549908 - 03/21/20 10:26 PM (3 years, 10 months ago) |
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Don't mod the PC.
When you are up to temperature you want to leave the weight off for 5 or so minutes until it stops spitting bursts or air and gives a full set of steam blowing out of the weight vent for 5 minuets. Displaced air can make your PC run worthless.
Also would be good to check on the flowhood pressure via a lighter. You want to see what the bend is because to much and you create turbulant air, to little and you don't create laminar flow.
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Kizzle
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Re: i keep getting bacteria in my agar dishes [Re: Mycobum7] 1
#26549922 - 03/21/20 10:38 PM (3 years, 10 months ago) |
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Just run though the possibilities and check for whatever you haven't ruled out yet.
1. The agar is not being completely sterilized 2. The petri dishes are not sterile 3. Your hand is dropping contaminants onto the plate when you pour 4. The outside of the bottle is dropping contaminants onto the plate when you pour 5. The inside of the agar bottle is being contaminated as you open it 6. Your hand is touching the inside of lid while opening the petri dish 7. The flowhood is producing turbulence instead of laminar flow 8. A contaminated surface is coming between the front of the flowhood and the petri dish while the lid is open 9. A contaminated surface is coming between the front of the flowhood and the bottle while the bottle is open 10. Mites are getting into the culture
Like InfiniteDreams said, I can't recall a single time a plate contaminated before it was inoculated with exception of the period when I had a mite problem. If that's the case you'll see little trails on the agar like this.

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Sockadin



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Re: i keep getting bacteria in my agar dishes [Re: Kizzle]
#26549932 - 03/21/20 10:44 PM (3 years, 10 months ago) |
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Wow that is amazing. As always if you got contams ask kizzle.
Also how soon are you opening your PC after it cools? You can open to soon and cause issues there.
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Mycobum7
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Re: i keep getting bacteria in my agar dishes [Re: Sockadin]
#26550100 - 03/22/20 02:09 AM (3 years, 10 months ago) |
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ok i poured 20 new dishes and i feel this is my best pour session yet. what a cluster fuck this has been.
the first time the mix was likely off and was super hot when i poured it which just sucked. lots of excess moisture
second time, i let it cool too long and it was solidifying as i did the plates, was messy, tried to re heat it, container is too tall for microwave....i digress..
THHIRd time.. they say third is a charm .. i fucking hope. i poured 20 plates with what i consider to be ideal technique, nice and even pours and less moisture.
i will update in 72 hours either stoked about no bacteria or super frustrated and confused about the bacteria...
-------------------- "This isn't my first rodeo ya know.......its like my third."
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tramalot
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Re: i keep getting bacteria in my agar dishes [Re: 99.99]
#26550255 - 03/22/20 06:03 AM (3 years, 10 months ago) |
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Quote:
99.99 said:
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
Shouldn't you use a media bottle with a cap? An open beaker is not a common method!
-------------------- It's the Journey I enjoy!
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Sockadin



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Re: i keep getting bacteria in my agar dishes [Re: tramalot]
#26550420 - 03/22/20 08:30 AM (3 years, 10 months ago) |
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I use to use old whisky or wine bottles with an aluminum foil homemade lid with no issues. As long as you are keeping the lid on until in the SAB or FH it will be fine. Same thing for the breaker, if you are using a cover of some kind it should be fine.
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Mycobum7
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Re: i keep getting bacteria in my agar dishes [Re: Sockadin]
#26556630 - 03/25/20 09:00 AM (3 years, 9 months ago) |
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update....this pour appears to have no bacterial growth yet. its been about 3 days or so. i think this is what i learned..
too much moisture may possbly wick bacteria into the dishes in the end i think the bacteria may have actually come from old parafilm
i had been given some parafilm from a friend and it didnt seem to adhere and peel off the backing as nicely as new parafilm, and after considering all things i believe the excess moisture combined with shit parafilm was an issue...
just saw that quite a few people looked at this post so thought i would share what i learned from it.
-------------------- "This isn't my first rodeo ya know.......its like my third."
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