Pressure cooker/autoclave: How to deal with excess condensation when sterilizing equipment?

[NeoBrew]

I'm just getting started on doing some yeast harvesting, storing, etc. I purchased some glass petri dishes that can I sterilize in my pressure cooker. I'm following the processes outlined in the Yeast Book by White and Zainisheff.

The plates I prepared came out of the pressure cooker covered in large droplets inside and out. I sterilized them inverted and separated in the pressure cooker. They were elevated on a platform above the water at the bottom of the pot. As I removed them from the cooker I shook them once or twice to shake some of the water off, but didn't want to leave them exposed for too long and risk contaminating them. I then mated the two halves to close them up. I followed the process for preparing the media with wort/agar. I've got them sitting on top of the refrigerator where it's warm now for a day to let them evaporate the condensation. They are in a plastic tote that is not air tight. I'm a little worried about them getting infected and/or not drying for a few more days. I'd like to tape them up to ensure they stay pure. (They are not inoculated yet.)

What do people do to help with the condensation? Should I be doing something different? Should I be sterilizing them closed? Wouldn't this just trap more water in them?

Help a noob out, would ya?

 

[Northern_Brewer]

Back in my lab days, we'd wrap anything like that in foil. If they're closed then it's harder for the water to get in in the first place.

 

[ESBrewer]

You can avoid condensation problems by sterilizing the (empty) plates with dry air in an owen. Wrap them in alu foil (either separately or by wrapping all the plates in a single foil package). Then heat the package in an owen at 170°C (338F) for one hour. Now the plates are sterile and dry and they won't get contaminated as long as the foil is kept closed. The wort agar can't be sterilized (completely) this way because without pressure water will only reach 100°C and there are some microbes and spores that can survive and cause contaminations later. Thus, you put the wort agar in pressure cooker (in an erlenmayer or some other suitable heat resistant glass vessel) for 20-30mins at full pressure. Close the erlenmayer by wrapping alu foil around the neck. This will not be airtight but it will prevent contaminants from dropping in to the flask post sterilization. After heating release the pressure !slowly! through the cooker's pressure release valve. Now the erlenmayer has some condensed water in it but it is still hot and if you open up the lid of the cooker just slighty to release vapour, the hot vessel it is going to dry soon from the outside. And the foil will prevent anything unwanted entering the wort-agar. While the agar is cooling down a bit, you take the pre-sterilized, dry plates from the foil package and put them on a (sanitized) table. Use sanitized latex/vinyl/nitrile gloves to prevent dirt from your hands entering the plates. Let wort-agar cool down until it is ~60°C. Agar will remain soluble until it is about 50°C, but even 50°C is difficult to reach (unless you put the flask in a water bath at 50-55°C). If you poor agar too soon (it is very hot) and you close the lid, a lot of condensation will take place inside the plates and they are too wet to be inoculated. So pour the plates at 55-60°C and then leave the lid just barely open for a short while so that the vapour can escape and the agar solidifies. A thin layer of agar will be enough and will minize the problems with condensation. This way you will be able to avoid any major problems. If some condensation takes place at the lid you can remove it briefly and heat it up in a flame (bunsen/gas burner) to dry it but be very careful because the glass is not resistant to the direct flame and may brake in parts if you leave it too long... It is a good idea to flame the erlenmayer opening between each pour as well. When the plates have cooled down, store them closed upside down, wrapped in alu foil again. The foil is very handy in preventing airborne microbes from landing in the cavity between the plate and the lid. When you are not growing anything on the plate, you can wrap the plates even with Parafilm(R) but this may not let air in at all which is considered bad if the yeast needs to multiply. In practise, at home conditions, from time to time you can see something unwanted growing on the plate (molds/bacteria). Usually they start from the side of the dish after entering through the cavity between the lid and the plate. This is not a huge problem as you can always inoculate two similar plates and then discard one if problems arise..

 

[bucketnative]

Back in my lab days, we'd wrap anything like that in foil. If they're closed then it's harder for the water to get in in the first place.

If the steam can't get in, then you aren't really performing steam sterilization. It's one of the biggest concerns when validating steam sterilization processes.

I like the dry heat idea presented by @ESBrewer. It's a simple solution and the foil provides a tortuous path barrier against contamination once you remove the plates from the oven.

 

[NeoBrew]

The oven does seem like a good idea. I hate to add more steps, but at least it would be a pretty hands free process.

 

[Northern_Brewer]

If the steam can't get in, then you aren't really performing steam sterilization. It's one of the biggest concerns when validating steam sterilization processes.

But we're not trying to validate a steam sterilisation process, we're just trying to do something that works (and is minimal extra work when one is autoclaving media at the same time). If one was autoclaving clinical waste, then one would be a bit more paranoid, but this is clean glassware. Even in the lab, which was rather more demanding than the average homebrew setup, we had no problems with stuff that was autoclaved in foil - it's not hermetically sealed, and the performance required is not as great as when you're dealing with waste.

I'm also a bit nervous about dry heat on ordinary glass, I have had things crack in the oven so it's not a panacea.

Just as a comment on ESB's post - we always put a foam bung in the mouth of a flask, then put foil over it - presents more of a barrier to fruit flies etc that can creep up under the edge of foil.

Best to wait until the pressure cooker is back below 100°C before releasing the pressure.

Condensation in the flask doesn't really matter - it's just distilled water.

I've found that the biggest single source of contamination at home was drying the plates - as ESB says, you want to leave the lids slightly ajar (say 1cm gap) until they're solid. It's not so bad in winter when there's fewer spores around and less convection, but for my typical 3-4 plates I sanitise a shoebox-sized Tupperware box with a Starsan spray, put the plates in and pour them (can be a bit awkward, but there's just about room), and then leave them a bit ajar but put the Tupperware lid on loosely. Once they're dry, you want to keep them inverted. I wouldn't sweat about oxygen access too much - I grow my yeast without any film on the plate but in the Tupperware, and then wrap them in foil or clingfilm if I'm storing them in the fridge. I do miss Parafilm though, I see you can get strips of it fairly cheaply on Ebay, it's good stuff.

Duplicating important plates/slopes is definitely a good idea.

 

[ESBrewer]

I'm also a bit nervous about dry heat on ordinary glass, I have had things crack in the oven so it's not a panacea.

With something like borosilicate 170°C is not a problem. You can minimize the heat shock by putting the plates in a cold oven before heating it up. A good reusable Petri dish should be made of heat resistant glass because it gets heated time after time either in autoclave, in pressure cooker or in the oven. But there are definitely dishes of ordinary glass that are not meant for microbiological use.

Condensation in the flask doesn't really matter - it's just distilled water.

Yep, inside won't be a problem but if the outside it is very wet and you touch it by hand or put it on the table, there may be a liquid bridge formed by (sterile) water that will carry the dirt from the fingers inside. This is exactly the problem with the plates when you remove em from the pressure cooker and there tends to be water all over and especially between the lid and the plate. Fortunately, hot erlenmeyer will dry almost instantaneously (from outside).

I've found that the biggest single source of contamination at home was drying the plates

This is true and can be examined bu leaving a plate open in the room for say 30mins and having a look at it the next day...

I sanitise a shoebox-sized Tupperware box with a Starsan spray, put the plates in and pour them (can be a bit awkward, but there's just about room), and then leave them a bit ajar but put the Tupperware lid on loosely.

A good idea! I wonder how many different containers a homebrewer needs...just bought a lot of food containers to store several specialty grains. I just put the foil-wrapped, inoculated plates inside a clean box and then put the box in a space where there is underfloor heating. It's pretty warm on the floor and this will speed up things slightly.

 

[gromitdj]

Help a noob out, would ya?

There is a really good YouTube channel from Sui Generis Brewing that covers some Yeast Lab basics. Including Agar plates, Plate streaking, etc.

Your Home Yeast Lab made easy Series

In the video below he covers dry sterilizing the plates.

 

[NeoBrew]

There is a really good YouTube channel from Sui Generis Brewing that covers some Yeast Lab basics. Including Agar plates, Plate streaking, etc.

That looks great, thanks for the resource. I'm going to find some time to run through these!

 

[Queequeg]

Your not going to reliably get sterile plates at home. Just buy pre poured irradiated plates it's so much easier (though more expensive)

 

[ESBrewer]

^One of those impossible things that we keep doing every day. At home and at work..

 

[NeoBrew]

I appreciate the advice. I spent $60 on glass plates, I'm going to make it work!

I did 2 hours in the oven at 150f last night. They are sealed in foil for now. I'll probably try to get them filled with wort/agar this weekend.

 

[Queequeg]

^One of those impossible things that we keep doing every day. At home and at work..

I never said it was impossible I said reliably. Also though you might think you have sterile plates because no contamination grow, it doesn't mean they are sterile. First anaerobes wont grow, low nutrient organisms won't grow and viable but non-cultivable organisms won't grow. Plus the conditions in a pressure cooker are far from ideal and if you open it without the dishes being bagged then they are instantly non-sterile

Even in the lab, pouring under a LFC they should not be considered sterile. That why in aseptic fill its normal to use aspect filled and terminally gamma irradiated plates, in triple wrapped plastic and not ones poured on site in the QC lab.

But once again it inevitably comes down to people using the term sterile wrongly when they actually mean aseptic.

 

[Queequeg]

I appreciate the advice. I spent $60 on glass plates, I'm going to make it work!

I did 2 hours in the oven at 150f last night. They are sealed in foil for now. I'll probably try to get them filled with wort/agar this weekend.

What agar are you using? powder or soild media in a glass jars you reheat to melt? If it powder unless its gamma irradiated ($$$$$) then it will likely be full of spores and vegetative cells.

If it is powder then you need to boil it, ideally run it through the pressure cooker. If you can get it at a good price get the solid re-heatable stuff in glass jars,

As for the plates at 150F, that is effectively low temp pasteurization (if wet). Dryness retards sterilization and sanitization. Dry heat sterilization is normally quoted as 662F.

My advice would be stick with the pressure cooker but get some self sealing autoclave bags like this:

https://www.ebay.co.uk/itm/PREMIER-...F-SEAL-AUTOCLAVABLE-BAGS-DENTAL-/391179823897

Keep the lid and base of the plate separate but in the same bag. Pressure cooker them for at least 20 mins (from when pressure is reached), ideally longer. You can then let them dry in the bag and assemble them again in the bag before opening the bag. As long as you keep them with the lid on top (the larger side) then by virtue of the swan neck inspired design the will remain sterile. You can tape them if you like.

When you come to pour use a heat proof rubber glove that you can sanitize and pour whilst the solution is hot, ideally above 160F. Its normal to let them cool with the lid off. But given you won't have somewhere clean to pour them i would cover them immediately.

You can then incubate them warm, at 90F for 3 days which will confirm if you have contamination. If you want to be really through, incubate them at 72F for 5 days first then 3 days at 90F. That way any contamination will grow on the plates prior use.

 

[ESBrewer]

I agree that it is always possible that the plates are not sterile. Even the €€€€ plates. If we need to split hairs at home brewing level, there is a term called sterility assurance level, just because probability that something is actually(reliably?) sterile after sterilization process can never reach 100%. Yet, this doesn't mean we need to buy the disposable plates to be succesful at culturing yeast for our brewing purposes.

 

[Queequeg]

Sterility assurance level is an artefact of not being able to prove a negative and only really applies to standardized process of sterilization like an autoclave, sterilizing oven or a gamma irradiation process and is generally referring to theoretical number of positive units based on abstraction of empirical data, because again you can't demonstrate sterility only absence of viability. This is achieved by demonstrating "kill" of a test strain at a known concentration and then extrapolating that information, with a 6 log reduction in population equating to 1 positive unit in 1,000,000 subject to the sterilization program in question. So for those pre-sterile plates 1 in 1,000,000 is theoretically sterile if it has a typical SA of 10-E6.

The issue is with homebrewers is that sterility is considered a desirable despite it being something largely beyond most people capabilities and largely completely unnecessary.

Instead what homebrewers need is a suitable level of asepsis in their processes and in most part boiling is sufficient and trying to emulate autoclaving by using a pressure cooker isn't really necessary imo unless you have a media containing a know bioburden of thermally aerobic resistant spores, which honestly is unlikely.

So people get contamination free results, without sterilization which is all they need. Reality is these procesess are likely nowhere near sterile (in the sense of how sterility is understood scientifically) nor do they need to be.

If people are asking how they sterilize things at home, I can either tell them to buy an autoclave and a LFC or say its not possible. If people want to know best practices for minimizing contamination and maximizing aspetic state then that's a different thing entirely, the easiest path is buy pre-poured plates.

The above method I have suggested to Neobrewer will probably get him the most aspetic self pored plates at home, which is way beyond what he probably needs.

He could equally submerse his plates in a 1% solution of peracetic acid, drain them and get the same results. Or boil the plates, or put them in the oven at 392F for an hour. Ultimately the weakest point in the process is using the plates, as you are exposing them to the room when you subculture.

I recommended pre -poured plates because it is the most elegant solution, the only compromise is price. But that said glass petri dishes are not cheap and neither is media.