Seed flasking: lab having difficulty with disinfection - Orchid Board

Orchid Whisperer · #1 05-28-2020, 01:43 PM

Summer of 2019 I made a Cattleya alliance cross that I had high hopes for. Seed pod dehisced in April, I sent seed to a flasking lab in April, but also saved some seed (placed in jar with dessicant, refrigerated, changed out dessicant for new, back in jar, placed in freezer, per recommendations found online).

I checked with the lab this week to see if there were any signs of germination. Good news bad news, germination yes, but also bacterial growth in 2 mother flasks. Lab still had some seed, disinfected per usual procedure, same result.

The lab believes that the bacteria came in on the seed, and is resistant to disinfection. He suggested that sometimes bacteria can be benign, suggested waiting and seeing what happens before giving up.

Just wondering if anyone has had a similar problem with resistance to seed disinfection? Any suggestions as to steps to try to overcome the problem? I have a few ideas for things to try but wanted to get other thoughts and opinions first.

Anyone work with a lab that has a really good track record on successfully disinfecting (difficult) seed without killing it? I don't mind losing some of the seed, if some also germinates successfully.

Thanks!

Dorchid · #2 05-28-2020, 02:53 PM

I would think the addition of gentamycin or some other antibiotic to the media would be helpful (if it isn’t already a standard additive). There could possibly be some adverse effects on the plants but I just pulled up a study showing no issues with catts. Here’s a paper on it: https://escholarship.org/content/qt9...qt9f26x5vr.pdf

Oyarzabal · #3 05-28-2020, 03:40 PM

In my experience. Infections the seed had, or you introduce will be showing in the first three days. The only remedy for that is a better technique and some common sense.
Wear long sleeves and a mask when flasking. Your skin sheds and you have bacteria in your mouth that can fall in the flasks. I used to add PPM, but now I think it is a waste of money.
A more insidious contamination comes from the flask "breathing" and bacterium or fungus showing up three month into the process.
If you have the seed pot. The best thing to do is to seed 6 or 8 mother flasks. If 99% of them get contaminated the one that still intact will produce more orchids that you need.

---------- Post added at 01:40 PM ---------- Previous post was at 01:28 PM ----------

Quote:

Originally Posted by Dorchid

I would think the addition of gentamycin or some other antibiotic to the media would be helpful (if it isn’t already a standard additive). There could possibly be some adverse effects on the plants but I just pulled up a study showing no issues with catts. Here’s a paper on it: https://escholarship.org/content/qt9...qt9f26x5vr.pdf

Most of those antibiotics are specific to a group of bacterium not the whole domain. And them you have fungus and other more exotic life forms that are not killed by antibiotics.
There is a chemical compound called PPM. I think is Plant Preservative Mixture that is a broad spectrum abiotic liquid. It is expensive, but 2ml will do for liquid of media. There is no substitute for being careful when you flask, developing a method and sticking to it to avoid contamination.

Plant Preservative Mixture (PPM) 250 ml

Dorchid · #4 05-28-2020, 03:44 PM

Aseptic technique is definitely the key. I’m curious if his flasker is actually a lab working in front of a hood or just someone working in a sab at home. I’ve yet to germ any seeds but I grow fungi and would expect the procedures would be the same. I would also think a reputable flasker would have good technique and proper equipment.

@ Oyarzabal - I just brought up gentamicin as it’s a broad spectrum antibiotic and was also referenced in the study I linked to. Personally, I’ve never used it but wasn’t sure if it was commonly used in flasking. On top of aseptic technique, I would think the “green pod” technique would also improve results. Sounds like PPM would be the better option if indeed one were having issues with contams.

Oyarzabal · #5 05-28-2020, 05:44 PM

@Dorchid
Indeed. Green pod is the way to go and even newly crack pods will do regarding infection. The advantage of green pod is that they germinate way faster. Withing 9 days you will see green micro protocorns if you use a flashlight and point towards you.
This is what I do. I have read all the common literature, some peer review papers, and I am aware of what most people say to do; however, I do have a mind of my own and this work for me.
I do use (18X36) a laminar flow cabinet that I built and I only do Cattleyas, so I would not have a clue about other species. My place is far from aseptic since I have bunch racks with seedling in the same room where I do my flasking. I collect the seed pods after 7 months, sometimes later and sometimes when I realize they are cracking. I cut the top and bottom of the seed pot without opening the pot.
Then I cleaned with some towel paper and alcohol. Not crazy just a quick cleaning to remove dust or anything attached to the pot that can fly away and contaminate the flask.
Before hand, I autoclave some water, a set of small spatulas, and some petri dishes made of glass. Everything is individually wrapped in aluminum foil and place in the laminar flow cabinet before I get the seed pot. I get gloves, mask, drench everything in alcohol and I place the seed pot in a petri dish
Next, I flame a blade, this can be a shaving blade, exacto knife, or a scalpel and make three longitudinal cuts in the seed pot if it is really green. If not I use the crack lines and open the seed pot into two or three pieces. Be carefully not to spill, or disturb the seed. You want them to be contained withing the pot that is sterile or partially sterile.
From there I take my baby spatula our its foil wrapping and pick a tiny bit of the most white visible seeds. This work even for pots that have been open for a three or four days. Usually, the top of the pot is still sterile. (Pots usually open from the bottom up) You can seed the difference in color of the seeds. I will change the spatula if I touch anything that is not seed, and that will include the flask, or any green part of the seed pot.
I just did nearly 44 flask a week ago and had one pot that was totally contaminated that ruined 6 flask and a single one with a different kind of bug from the other pots.
Once you have the pot seeds are cheap and it is a number's game. I usually do between six and eight flask for a pot and when I cross the orchids I cross at leas two flowers, so I can use two pots. If one is contaminate I do not care. I have a good one left.

Dorchid · #6 05-28-2020, 05:54 PM

Ray · #7 05-28-2020, 11:46 PM

The discussion of the benefit of greenpod sowing is nice, but doesn't help Orchid Whisperer at all...

Dorchid · #8 05-29-2020, 12:00 AM

Sorry, I don’t know of any lab that has a really good track record on successfully disinfecting (difficult) seed without killing it. Our discussion also included topics such as aseptic technique, antibiotics and biocides, and a run down of procedure, which might be of use to op. So, do you have anything pertinent to contribute? Or, I could simply say, “neither does your snarky comment.”

Oyarzabal · #9 05-29-2020, 12:25 AM

I will purposely ignore your snarky comment, and I would quote Dorchid:

" So, do you have anything pertinent to contribute?"

Ray · #10 05-29-2020, 08:45 AM

Sorry, it was not meant to be snarky. OW said his lab was having an issue and discussing how it should have been done was off-topic and seemed to be a bit of a disservice to him, so it was an apparently poor attempt to get the discussion back on track.

Had I ever done other than green pod culturing, I might have contributed more.