Methods for preparing agar without a flow-hood?

[Merlyn]

Good day everyone,

I am at that point where i’ve been deeply considering isolating pure cultures.
Right now, my approach has been rather low-tek, to get me by with just a still air box and a pressure canner.

However, pouring agar in a still air box seems highly impractical and was wondering if there are any alternatives; that don’t require a flow hood for pouring agar efficiently?

Considering turning 50ml glass jars into big agar plates, with a injection and air-port. That may do the trick; just pre-pour the agar and pressure sterilize it. Then inoculate…

[moss_crime]

hey merlyn,

what issues have you been running into with pouring agar in the SAB? my general process has been to mix and pre-sterilize agar in lab media jars in a pressure cooker, then pour a few stacks of plates in a SAB.

i’d say after some practice that contamination rates are maybe 5% or less. often batches are poured with no contaminated plates.

[Lando]

I think you are onto the right answer. In basic terms:

• Prepare the medium
• Pour in sterializable containers (but not in sterial conditions)
• Then sterialize the filled containers.

I’ve seen lots of people get by with wide but short jam jars for this. Another cool thing I saw was small liquor bottles (like the 2€ ones you see at the checkout) laid on their side to cool.

I haven’t done it this way but the main drawback I see is the number of “plates” you can make out of one run of the autoclave/pressure cooker is lower. But in terms of effectiveness I think it is great.

[glyph]

@moss_crime

what issues have you been running into with pouring agar in the SAB?

+1 to this question. My technique for pouring plates is identical whether using a flowhood or SAB. The only real difference I experience is the slight restriction of movement of my arms and reduced visibility when using the SAB.

I’d say the practice of pouring plates in a SAB also improves overall comfort and success with other SAB work, such as doing transfers.

@moss_crime

i’d say after some practice that contamination rates are maybe 5% or less. often batches are poured with no contaminated plates.

+1

The pre-pour technique outlined by @Lando is a solid alternative. I’ve done this with glass (soda lime) petri dishes and it works well. The main consideration is ensuring the stack is kept upright throughout the sterilization run and especially during the cooling phase (assuming you let the agar cool and solidify in the PC), otherwise you’re going to have a mess.

Using small glass jars instead of petri dishes could save you a lot of money. I wouldn’t bother with the air-port; not necessary for agar. I wouldn’t bother with the injection port either but I’m assuming you want a way to do agar → LC and LC → agar transfers in the open air.

[Merlyn]

Hey Moss,

Haven’t tried pouring agar yet. Been mentally preparing myself and just heard from other friends in the field that pouring agar in a SAB is very difficult. Almost getting the impression that its undoable.

However, always; where there is a will, there is a way and i trust that.

@Lando and @glyph, thank you for those awesome options. I am going to try a pour and no-pour technique and see which one works easier for me in the long run.
@glyph interesting, the intention was to use an injection port on a few just for the initial A1 inoculation. Afterwards I will just sub-culture from the master plate/jar.
I got a whole bunch of plastic (yes diabolical plastic!) petri dishes that i dont want to waste so I’ll definitely try that in the SAB with regards to what you outlined.

Got a whole bunch of 50ml glass jars leftover from a past business adventure that stopped, so will be lovely to recycle and use those. In the steps outlined by @Lando

Thanks everyone, ill keep you all posted with pictures of your suggestions in implementation.

[glyph]

@Merlyn

Been mentally preparing myself and just heard from other friends in the field that pouring agar in a SAB is very difficult. Almost getting the impression that its undoable.

This hurts my heart Just kidding But seriously, I hope you’ll give the SAB a shot before deciding it’s undoable. I’ve poured literally hundreds of plates in a SAB with super minimal contam rates. I wanted to link to some Shroomery threads on this subject but it appears to be down at the moment. So much of this hobby is about experience - take what you hear from hobbyists and try it for yourself.

One thing you could do is use those plastic petris you have to practice your technique. Even if you don’t intend to do transfers, you can pour the plates in the SAB, seal them, and then set them aside to incubate. If your plates are clean after a couple of weeks, then your technique was solid.

When doing transfers, it’s good practice to leave one blank plate aside from each batch. If you end up with contams in your transfer plates, the blank plate will help you to identify when the contams entered the picture (ie. If the blank is still clean, then it’s likely that the contams were introduced during your transfers. If the blank is contaminated, then they were likely introduced during the initial pouring).

@Merlyn

Got a whole bunch of 50ml glass jars leftover from a past business adventure that stopped, so will be lovely to recycle and use those. In the steps outlined by @Lando

Nice! That sounds great. I also thought I should mention that PP5 plastic containers can be sterilised. My local sushi place uses small ones for soy sauce and they’re perfect for agar (and can be reused). You could pre-pour those (they come with snap-on lids). Bigger (PP5) plastic containers work well for grain jars etc.

[moss_crime]

@glyph

This hurts my heart Just kidding But seriously, I hope you’ll give the SAB a shot before deciding it’s undoable. I’ve poured literally hundreds of plates in a SAB with super minimal contam rates. I wanted to link to some Shroomery threads on this subject but it appears to be down at the moment. So much of this hobby is about experience - take what you hear from hobbyists and try it for yourself.

+1 on this. I’m far newer than glyph on this stuff but i’ve come to really enjoy the SAB work and and pretty pleased with the bone simple mechanics of how it works. i was also reluctant/nervous about lack of lab skills just starting off and needlessly intimidated. first batch i did lost a few but even the contam rates for that weren’t awful. muscle memory and technique improve pretty quickly. now SAB work is a pretty relaxing way for me to slow down and sync with the pace of air perturbation dynamics.

end of day for me i think it’s pretty pleasing knowing i can drop a few grand on a flow hood, or build one for like 600 bucks orrrrrr just drop like 15 bucks on a clear plastic tub and get nearly the same results.

keep us updated @Merlyn i’d love to hear how either the pour or no-pour trials go.

[Merlyn]

Just for you @glyph
I did this in a SAB. Its a black carbon MEA premix. Added 1 liter of distilled water to about 50 grams of mix, pressure cooked it for 25 mins at 10psi.

I sterilized the box and poured my plates. I think the handling of the bottle was the most important lesson in terms of getting into an efficient rhythm.

Wrapping the micro-pore tape was definitely the longest part of the process haha.

Cant wait to inoculate!

[Merlyn]

Excuse me, I was busy replying further and my device ran out of battery.

@glyph ill keep some blanks out, thats a great suggestion. Thank you.

I am quite confident that whatever happens will be a great learning experience which it has been already.

Those PP5 sushi plastic containers you speak of - when you say sterilize - you mean in a pressure cooker? Thats very interesting…

@moss_crime it’s funny you say that cause I was starting to feel very relaxed after awhile doing the process in a SAB. After the initial sweat that is…I could see how after a while it will become more like riding a bicycle. Second nature that is.

I haven’t got around to trying a no-pour technique just yet. Very excited to give that a go as well

+1 to SAB’s and “low” tech mycology!