u/GrumpyAlison

TLDR: I've been trying to sterilize begonias and they either die/melt or get contaminated (or both if I'm really lucky /s) and I'd love some suggestions about how to fix this because I thought begonias were supposed to be really easy beginner plants. More (very explicit) details of what I've tried below

Mostly I've been working in a SAB (made a DIY FFU recently). Control plates (i stuck my tools into the media for some of them) have been coming out clean long after the plants contaminate. I've had stored media for months with almost no contamination so it seems unlikely they're all being problematic right out of the pressure cooker. This leads me to believe it's a plant/sterilization issue most of the time?

So here's what I've tried (sometimes I try multiple sterilizations in a single batch / I'm not doing multiple bleach washes for each plant):

• Batch 1:
  • 5% bleach for 20 mins (wash 20 mins in tap water beforehand)
  • 10% bleach for 10 mins (based on protocol i found;(wash 20 mins in tap water beforehand))
  • contamination on all but 1 cup where the tissue died
  • bleach MAY have been bad/too old for this batch
• batch 2:
  • wash 20 mins in distilled water wish dish soap
  • 10% bleach for 10 mins (but i got new bleach)
  • cut plants into sterile water & put in cups
  • 4 days later: all contaminated and the rest dead/brown (probably should have washed before i cut?)
• batch 3:
  • orbital shaker wash for 20 mins
  • nadcc at ~225 ppm for 4 hrs (didn't rinse, just cut and put into TC)
  • bleach 10% for 20 mins then rinse
  • only 2 contaminated but most died before contamination; nadcc seemed to do maybe better than bleach?
• batch 4:
  • orbital shaker 20 mins
  • nadcc at ~225 and ~600 ppm (without tween because i forgot it like an idiot) for 1-3 hours on orbital shaker
  • 225 ppm nadcc had random contamination from 3-6 days after culture. visible tissue death from 3-6 days after.
  • 600 ppm nadcc had contamination starting at 4 days (improvement??) but tissue death starting at 2 days (so worse)
• batch 6:
  • run under tap water 20 mins
  • 1.11g nadcc to 500ml water (around 2000 ppm) + tween in mason jar for ~12-13 mins with manual agitation
  • wash 30 seconds > 2 mins > 2 mins in sterile distilled water
  • went 5 days before contamination showed up (record) but tissue started looking rough around 2-3 days
• batch 8: (still ongoing; I just did this like 2 days ago)
  • same as batch 6 but i used a diy FFU box fan instead of a SAB since someone suggested it was that. I felt very clumbsy in the FFU but my plates I did after the cultures seem clean so far
  • 2 days in : contamination in 1 cup
  • day 3 (today) : *most* leaf cuttings look not horrible except 2 which are totally dead/clear
• Bonus batch (from yesterday):
  • i tried resterilizing some bag plants (some of which were begonias; basically all of which came with bacterial infectinos) I got that had a clear bacterial infection with a 10 second dip in 70% IPA and then a 3-4 minute 3500ppm nadcc batch (realized this was higher than i intended and pulled them when the begonia leaves started activly looking like they were getting clear/raggy during the mix)
  • the next day 2 of the 5 things already have clear signs of contamination smh.

Notes

• I've been using nadcc over bleach because most people say it's gentler on the plant and I can't tell if liquid bleach has gone bad/lost potency or not.
• I'm using RODI water, not strictly speaking distilled (I have the filter for my reef tank, so I can make 0ppm water on command muahaha)
• usually I cut off a whole newish leaf, or the top few leaves from the stem, sterilize that and then cut it up

conclusions/observations;

• if i agitate begonias for longer periods, it tends to mangle the leaves, even if it's just water (magnetic stirrers are particularly bad about it)
• Adding higher concentrations of bleach/nadcc also does a number on begonia leaves (but the nepenthes I did seemed to take it better)
• Maybe I should get PPM? But that would just delay the bacteria as far as I can tell?
• I should perhaps try some other plants like philodendrons or anything less delicate just so I can see wtf is happening? (but all i really want to do is begonias)

And I'm wondering if maybe the begonias could have some bacterial issues or something in the plant itself that can't be cleaned off the surface, but most of the dots pop up on the surface of the plant and not around the edges. Do my begonias melt because they live in sealed boxes and maybe the leaves are super fragile or something? I feel like I'm losing my mind at this point because every sterilization protocol I've seen people use successfully doesn't get things clean at all or kills all the plant tissue?

I saw something about doing a wash in a fungicide after the bleach too - wondering if that would help or not.

Literally any suggestions are welcome at this point because I feel like I'm loosing my mind.

Thanks <3

TLDR: I've cleaned the fan and the laser and it's still significantly fumier than it used to be several weeks ago and I'm trying to figure out why, because I've tried all the common fixes to the problem that I've found and it doesn't seem to solve the issue.

I have the OneLaser XRF and a cloudline 6" inline fan for the exhaust that goes directly out the window in a mostly-straight 3-4' run of tube. I basically only cut 3-5mm wood. and I don't actually cut THAT much (maybe 75-150 hrs on the machine over a year), so I'd really doubt the fan is at the end of it's life span. The laser has a built-in PPM reader (which I also cleaned incase it was dirty) and jobs that used to max out at like 34ppm are now going up to 80 or 100+ (and the room will smell bad while engraving which didn't happen before other than a faint smell, so I don't think it's just that the meter suddenly went bad?) it's to the point where I wear a respirator for long cuts, which sucks because my office is hot).

I can't seem to smell specific areas of obvious leakage, but my sense of smell also isn't great, so who knows. It seems like it changes from piece to piece even if I'm not making any changes.

Things I've tried to fix the issue:

• clean the fan
• clean the laser
• clean ppm reader incase it was filthy
• replace tubing with custom hard tubing
• replace custom hard tubing with new ducting that isn't full of micro-holes
• turning off ambient room fan incase that was somehow sucking air out
• opening/closing door to office and other house windows
• using/not using external air assist
• Using/not using my DIY riser pegs & riser box (no apparent change)
• taping off the crevices by the lid (smelled fumy once; fix didn't help much)
• cracking the crumb tray to allow more air flow from the bottom of the laser
• measuring air flow as best as i could with one of those little wind speed measurer things (didn't yield specific results other than the small exhaust hole in the laser and the tube were choking flow a bit)

I'm really not sure what else to try short of buying a new or larger fan (which I'd rather not do if possible because they aren't cheap) or trying to deep clean the fan again, but I'm not sure if I can get it much better than it was last time I did that a week ago. Does using it as a laser fan just destroy the working life of the fan faster?

Any help/suggestions for troubleshooting or fixes is welcome, because this is starting to drive me nuts (and I refuse to cut anything super fumey like acrylic until this is fixed)

I have some ceramic media beads and a vibratory tumbler (different than a rock tumbler) lying around, I was just wondering if anyone had done this previously and had thoughts or suggestions on if it worked and the media y’all used.

For the record, this is for small stuff like beads etc, not whole giant mugs or whatever.

Thanks!

I clearly suck at registration but I feel like this couod have been way worse for a 6 color frameless print?

Clearly im bad at registration. Next time positioning my marks differently will help I think.

But not terrible considering this was my first attempt at multicolor and it was 6 screens I threw together this morning and they’re all frameless save the hangers i 3d printed at the top (for hanging to dry/storage but also to hold and keep the screen from warping during use. Seemed to work well). And the fact that the emulsion dropped a bit during drying (still messing with a good setup for my space for that…)

Are y’all doing more than one coat of emulsion on your frames? I haven’t found it’s made a huge difference on my prints so far between, but the more coats the more drippy it gets during drying (I tried 3 coats dried horizontally but that was nightmare sauce; this was 2 coats dried at a slant and it was better but still slightly wonky).

Not that this is relevant at all, but I have the rest of the emulsion sheet stored like an unholy fruit roll up in a cardboard box for later chopping and use lol. It

I was curious if there’s a way to make permanent screens (or super durable ones) for designs you just want to have on hand all the time. I assume washing them out will degrade them over time?

And if anyone has tried coating frameless screens. My end goal application (really small fabric prints and underglaze stencils) doesn’t need frames, so I was thinking maybe a temporary frame similar to a tufting frame or an embroidery hoop or window screen frames would work well enough to stretch the mesh and get the emulsion on and then just chop it into little pieces to expose? (I have tried ez screen with goo success, it’s just expensive in bigger sizes)

I know you can reuse frames but for recurring stuff it would’ve easier to not have to remake them all the time lol. I’ve also heard you can store frames before burning as long as they’re stored properly. Curious to hear peoples experience with that if they have any.

I was also considering using the ecotex pwr purple but im open to other suggestions. Using led to expose and primarily using water based inks atm.

Thanks!