Tissue Culture: Converting a homemade sterile glove box into a filtered postive pressure system

[JustSumTomatoes]

Excellent info man, thanks for sharing that. I guess sterile air/glove box is a bit of a misnomer then. I figured making a space completely sterile 100% of the time was next to impossible but wasn’t aware there was mathematical theory behind it. That’s really interesting about the log reduction. No one ever explained that to me in any of my biology or microbio classes. That, or they did and it went in one ear and out the other. That also makes sense about the gloves in the box acting as pistons. That’s what I was observing with the leaking.

I imagine you’d have to have your SAB in a draft-free environment or it would be defeated. What I’m hoping to have with what I’m building is not having to worry about that at all. Don’t have to be concered about anything floating in or up cause it’s all (at least in the 99% range) being pushed out. What I’m having issues with is mold spores, fairly large in size (compared to bacteria) and should be easy to filter out.

I definitely will keep everything you shared in mind and may even make one of those just to compare. A still air box could very easily be made, in fact I had one before I added all the bells and whistles lol. Thanks again for the advice!

[JustSumTomatoes]

Yes I have. I use a quick dip in 70% rubbing alcohol, 2-3 minutes in 3% hydrogen peroxide, and 10 minutes in a 10% bleach solution (way too strong actually.) I then do a wash for 3 minutes in pure H2O that’s been autoclaved in a Culturejar G9 w/ a non-vented Phytocap.

[JoeCrowe]

I’m lovin’ it!

[JustSumTomatoes]

The paper towels on the bottom with hydrogen peroxide is a good idea. I don’t know about fumigating the room with lysol, might need to save the gas mask for myself then haha. I like the whole furnace filter on the fan idea, closest thing to an air purifier I can afford lol.

[anon93244739]

Clearance at kroger, 2 for $5

[JustSumTomatoes]

Thanks for the compliment! I use a 10% bleach solution to sterilize my instruments. It was common protocol when doing surface sanitization with all the nasty stuff in my microbiology courses. I would much rather be flaming my forceps and scalpel though. Need to get one of those little propane canisters for camping and throw a burner on it.

[JustSumTomatoes]

@cannabissequoia Lmao, been there done that.

[MidwestMover]

@JustSumTomatoes

SAB in a draft-free environment

I try. I’ve forgot to turn off the A/C or heat a few times and felt it blowing on me and thinking I was screwed. I really didn’t have much contamination in my efforts though. I’m waiting on some Petri dishes and I’ll start up again.

[MidwestMover]

Pass it over lol. I remember smoking out of a oxygen mask with a blunt in it. Guy told me to inhale thru my nose, that tore me up.

[curiouscat]

Just KISS approach… remember that the more factors you add higher are the chances of getting something wrong. It is important to clean everything but if your last steps aren’t “clean” then your done… all that care down the drain

If you have a pressure cooker, just use that. Put all your stuff above the water surface so it gets steamed. Once the temperature is right, 120° Celsius (if you splash some water on the lid and it evaporates quickly your good to go) count 30 min and the content is sterile.

However once you open the lid of the pressure cooker (unless you are inside a lab with controlled environment which is not the case ) all the nasty stuff will want to cling.

A trick for jars and lids for example is have them well washed with soap, then you put the lids on slightly screwed but not tightly. So that when the “cooking” has ended the interior of the jars is untouched. When you need to use the jar, you wipe it down with alcohol 75 proof before putting it inside the glove box. Close the glovebox and wipe everything down with alcohol again.

Soap will suffice to get rid of many bacteria and viruses by distroying their phospholipid layers in other words it kills most stuff, the problem is that it won’t kill those that sporulate or fungi, hence the Pressure Cooker method.

For tweezers, scalpels and all other materials you should do a flame sterilisation. Or high heat sterility by placing them inside craft paper and them placing in a hot oven for 2 hours at 170° Celsius, this last will ensure it is sterile until you compromise the packaging/craft paper.

It is the handling of the materials and your movements that will dictate the end results.

You mentioned that you rinse the jars with H2O but is it Pressure Cooked?

I’ll give you an example, in mycology there are many techniques used to make media viable for growing a certain mushroom or fungus. There are complex ways of doing so, there are also very simple ways.

Take this one for example. Sawdust pellets hot water and H202 and that’s it. It is very successful and simple way but there is a catch, the species is a very prolific one so it does not give time to others to take over.

I guess, and I don’t have any experience here, that tissue culture takes some time to develop. Maybe you should consider also buying media with some sort of antipathogen? An antibiotic? So in this case a selective media.

Hope this helps and is useful.

Have a look here just to get an idea of what I was rambling about

Let's Grow Mushrooms! 1 1 Getting Started with BRF Cakes

PS: I know your not growing mushrooms, but the principals aply

[JustSumTomatoes]

Wow, thanks for all of that useful information and the video for reference! I try asking questions in the carnivorous plant community and everyone is tight lipped and afraid to reveal their secrets. Come here and the knowledge flows! OG is legit as it gets.

You’re right, I definitely need to invest in a bunsen burner. The craft paper is a good idea. I could even create a sealed pouch or something of that sorts and sterilize when I’m done so the next time I want to work I can just put the pouch in the box, clean it, tear it open and start.

I’m very familiar with pressure cookers (poor man’s autoclave.) I have a process for preparing and sterilizing my nutrient and agar media in the cooker. Yes, the water I do the wash with is sterilized in a Culturejar G9 and then opened once inside the box. Believe me, I’m very OCD and meticulous about making sure everything is completely wiped down and clean before even starting and have a bit of a system worked out.

I add an an antibiotic during my media preparation called PPM-- Preservative for Plant Tissue Culture. It does wonders for bacteria, but if the mold attaches itself to the explants it can take over.

Interestingly, this doesn’t happen to a jar just containing media. I tried to make it happen. One of my jars was damaged in the cooker so for fun I pulled the cap off and left it open overnight, then recapped it. This was back in October and still nothing has grown. That PPM is good stuff…

I even tried a final dip of diluted PPM for my explants after the H2O wash and this really helped at first, but a month in the mold started to pop up on some explants. Back to the drawing board.

[anon93244739]

Where did you get this? Link?

[JustSumTomatoes]

Continued on the box today. First off, always use the proper safety gear when doing projects. It’s cheap and easily replaced if damaged, your organs aren’t…

Let’s build a flange out of a surplus Russian filter…

Time to break out the Dremel tool…

Another gadget of mine, an old furnace motor bolted to a bench, set up for 120 volts with an on/off switch installed. Perfect to attach grind stones and wire brushes too…

Now for some holes for the screws…

Not the best tool for the job but I’m willing to improvise…

More holes for the lid…

Now for some adhesive to seal the hole and to put the screws and nuts in…

Done for today. Going to let that adhesive dry and pick back up over the weekend.

[JustSumTomatoes]

Plant Preservative Mixture (PPM) 30 Milliliters.

[JustSumTomatoes]

Also swapped out the germicidal UVC light (left) for a 6-watt flourescent that won’t cook my eyes out when working…

[MBVapester]

I sure agree with that from my mushroom growing days. Pressure cooker and rye then in the box.

[JustSumTomatoes]

Installed the screen onto the flange. This should help widen the flow of air. Wish I could have used something nicer than electrical tape to secure it, but it’s very practical. If I need to get back in there I can just remove it…

[Guitarzan]

@JustSumTomatoes

I add an an antibiotic during my media preparation called PPM

If you’re using PPM, and you’re still getting mold, you aren’t sterilizing your explants enough prior to plating them up. The sterilization of your explants is critical. We bought a starter kit for TC, and it came with a booklet of procedures.

Here are the relevant pages that deal with media prep, explant sterilization and plating, transfers, the whole she-bang. Media Prep-Explant Sterilization-Plating.pdf (3.3 MB)

It sounds to me like once you get your sterilization procedures down, you should start seeing some plantlets. It’s a very cool thing to watch a plant grow from a hunk of stem.

Good luck with it!!

[JustSumTomatoes]

Thanks for all that good info! Phytotech is where I got a good amount of my supplies. I printed that whole .pdf file out and put it in my protocol binder. Tissue culture obviously has a bit of a learning curve, but I’ll get this figured out. Thanks again for your help!

[JustSumTomatoes]

Got the filter, hose, and fan assembly put together. Electrical tape worked great for the filter to fan, but the hose to fan could use a piece of pvc pipe with electrical tape to hold them together. Both are airtight though. Cleaned the ends with rubbing alcohol first.

These are the hoses I connect together. They are from the same era as the Russian gas mask I showed earlier. This why I selected the Russian filter to build the flange out of. The threads on the hose match the old filter…

I plugged it in for a spin and it’s very close to being completed…