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OfflineNunyabiz
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Registered: 02/08/18
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Agar question for the experts
    #25155053 - 04/21/18 10:42 AM (5 years, 11 months ago)

I am struggling with agar after multiple attempts using pda, mea, sab, petris, mini rounds, lots of sterile supplies, etc. I have extensive practical experience in surgical asepsis. However, lots of contams in my efforts. Think it is the syringes. So, I ordered replacements from a different supplier. Before I inoculate another batch, I wanted to ask a couple of questions...

1. On average, how many contaminated plates should I expect out of a batch of 10?

2. What percentage of healthy plates do you go straight to grain with? How often do you have to do agar to agar?

3. Does using a flowhood significantly decrease agar contams in your experience?

Thanks in advance.

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Offlinenorthcrow
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Registered: 04/08/18
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Re: Agar question for the experts [Re: Nunyabiz] * 1
    #25155092 - 04/21/18 11:02 AM (5 years, 11 months ago)

Hey dude.  Thought I could answer these for sure.  I just started a new operation.  I bootstrapped everything so I built my own flow hood and what not.  Been working for about a month now.

1.  I wouldn't even expect 1 in 100 plates to be contam'd, let alone any out of 10.  I will, of course, attribute this to my flow hood.  I would estimate that I've probably done 80 or 90 plates in the past few weeks with no contam. 

2.  I actually do both with most of my plates.  Well, some of them.  So if you've got a healthy plate, feel free to chop that bad boy up and get it on grain.  I prefer to use isolates to colonize grain, so my first set of plates is usually intended for isolation purposes.  It's nigh impossible to get an isolate out of a syringe...but they're great for getting culture going.  For sure.  So I usually take a really nice specimen off of the leading edge of the hyphae, transfer it to it's own plate, and grow that out to colonize grain with.  I should also say that I use the leftovers from the syringe plates to colonize the grain as well, I don't just like...waste it. 

3.  Having a flow hood is so undeniably important.  I would never try to do any sort of culture work without one.  I know people have had luck without them, and kudos to them for sure, but to maintain my sanity I need a flow hood. 

So, my question is, then...What is your set up? 

I say this because I highly doubt that the syringes are the issue.  I would venture to say that the issue is either your environment or your sterile technique.  And if you say your technique is on par, then it must be the environment? 

So, yeah, what does the operation look like?

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OfflineBensi
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Re: Agar question for the experts [Re: Nunyabiz]
    #25155161 - 04/21/18 11:35 AM (5 years, 11 months ago)

Agar is a lot of fun and pretty cheap so if you do mess it all up you're not out that much. I use gulaman bar agar. Are you doing open air inoculations? if so I encourage you to build a SAB.

#1  I've been getting about 95-100% accuracy just using a still air box and exam gloves. I normally use glad minis. I did my first petri pour recently and had about 4 out of 20 contam but I was super clumsy with the petris and hot agar bottle...large learning curve for me so I had way less accuracy.

#2 All of them. Unless I'm trying to make an isolate or grow out a bunch of plates to get some masters going. If I see the myc spreading out quickly to the very edge of the agar like a big snowflake with no signs of bacteria or slowed growth I'll put it to grains on the first plate. I almost never get trich in my plates.

#3 I would say yes but I've never used one.  I have two bare bones still air boxes that I built. wipe it with iso and spray it with soapy water before doing myco work...easy peasy.

Edited by Bensi (04/21/18 11:44 AM)

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InvisiblebodhisattaMDiscordReddit
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Re: Agar question for the experts [Re: Nunyabiz]
    #25155195 - 04/21/18 11:50 AM (5 years, 11 months ago)

1. 0
2. ? Wut. How much water do you like to drink? What's your favorite color?
3. No

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InvisibleJHOVA
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Registered: 02/17/17
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Re: Agar question for the experts [Re: bodhisatta]
    #25155365 - 04/21/18 01:04 PM (5 years, 11 months ago)

Take your phone and record while laying on top of your sab. That will rule out bad sterile technique.


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