Just an idea...

[Dane]

I've read up about a Laminar flow cabinet and they are so expensive, especially for me while i'm still a scholar. So I've come up with and idea to make a cabinet without HEPA filters. When I was looking at one of my flasks that I bought I noticed the non-absorbent cotton wool in the top which allows air flow but doesn't allow fungal spores through similar to a HEPA filter. So I've came up with the idea of making a filter with what we call 'batting' which is closely stacked non-absorbent wool which is produced in sheets. I've decided to make a filter by closely stacking about 10-15 sheets together and allowing a fan to blow through the one side of the filter similar to a laminar flow cabinet. And for extra protection I could spray sterilizing agent on the filter before turning on the fan to start flasking. It might be a cheaper alternative?

Any comments on my thought? Does anyone think it will work or has tried it?

 

[ALToronto]

How sterile is the wool? One thing about HEPA filters is they're synthetic, made in clean room conditions. Can't say the same about the wool. Why would you not want to use synthetic batting?

 

[gonewild]

How many flasks do you plan to do?
How many seed pods are you prepared to loose?

Remember you are basically reinventing the wheel.
Before laminar flow hoods were available orchid growers tried just about every possible method to solve the problem.

I started out in a bathroom full of steam then tried an adapted aquarium cabinet with fixed rubber gloves and a bunch of other ideas. Most worked to a degree but there was always losses due to contamination. Then I gave up and sent my seed to G&S labs and never had any more losses.

Don't expect anyone to tell you the layered batting will work as good as a LF hood because it won't.....but it can work if you take enough care.

 

[Dane]

It is a synthetic wool produced in sheets

I just need a more reliable way to insure that the % of contamination is a lot lower, including the risk.

 

[Dane]

I just want to do some flasks from start to finish, most of the plants die at replating.... I've had to teach myself over the years from reading the little said about flasking on the web and by watching youtube videos. I'm also 18 years old and I really cannot afford expensive laminar flow cabinets. I enjoy doing flasking as a hobby and watching the seeds germinate I would like to grow at least one flask from start to finish... I never stop learning and im learning in stages from finally getting them to grow from seed with minimal contamination. But when I replate I often have to open the flasks and almost all the flasks become contaminated in the glove box and i'm unsure what size the plants have to be before deflasking

 

[TyroneGenade]

Dane, the batting is not going to work. Batting is used in the laminar flows as a prefilter before the air passes through the HEPA filter. The batting (cotton wool etc...) will still allow bacteria and fungal spores to pass through and get into your flasks. Your glove-box is perfect for what you are doing. Don't fix what isn't broke.

 

[Dane]

The only problem that i'm experiencing with my glovebox is that its so difficult to work in and so difficult to do replating in as the plants crush so easily when working with thick gloves, and contamination often occurs because it is so difficult to work in... opening flask is difficult because the gloves will slip.

 

[TyroneGenade]

See http://www.ukorchidforum.com/viewtopic.php?f=26&t=1292&start=40#p14808 . There are alternatives to big thick gloves. A local vet should be able to help you find a source for the shoulder-length gloves, else try ebay. I think you can find them in floppy plastic as shown in the thread or more skin-tight latex which would give you better dexterity. I am toying with setting up a system like what Terry has for home flasking and doing some research... so, some caution for sensitive viewers (like myself), make sure "safe search" is on if you Google shoulder length latex gloves else you may get a nasty shock.

To avoid the problem of opening up flasks, use plastic tubs. You can see the type I mean at http://www.saorchids.co.za/laboratory.html . You can get them at most plastic/food packaging shops. You don't need such a tough plastic tub, all it must be able to do is take temperatures above 100 oC and snap fast tightly. I put some cling-film over the top to be more sure than nothing can get through. These tubs breath well and don't need to be vented. The biggest problem is the lack of water due to evaporation but frequent replating is better for growth in any case.

 

[Dane]

See http://www.ukorchidforum.com/viewtopic.php?f=26&t=1292&start=40#p14808 . There are alternatives to big thick gloves. A local vet should be able to help you find a source for the shoulder-length gloves, else try ebay. I think you can find them in floppy plastic as shown in the thread or more skin-tight latex which would give you better dexterity. I am toying with setting up a system like what Terry has for home flasking and doing some research... so, some caution for sensitive viewers (like myself), make sure "safe search" is on if you Google shoulder length latex gloves else you may get a nasty shock.

To avoid the problem of opening up flasks, use plastic tubs. You can see the type I mean at http://www.saorchids.co.za/laboratory.html . You can get them at most plastic/food packaging shops. You don't need such a tough plastic tub, all it must be able to do is take temperatures above 100 oC and snap fast tightly. I put some cling-film over the top to be more sure than nothing can get through. These tubs breath well and don't need to be vented. The biggest problem is the lack of water due to evaporation but frequent replating is better for growth in any case.

Thanks so much I remember getting shoulder length gloves a while back, I've also just started with plastic flasks and I've also noticed that the medium evaporated extremely quickly and then the protocorms shrivel up with the medium. :clap: I must change my set-up

 

[orcoholic]

I assume since you are "still a Scholar" you can check with your science department to see if they are getting rid of any older hoods.

Also, there are cheap HEPA fan/filters on AMazon and maybe you could build something around them. I don't know if they are as good as the filters in a hood, but if you put one on the top of an aquarium, remove the front glass, and seal the openings real well, maybe it will help reduce your losses.

 

[Dane]

I assume since you are "still a Scholar" you can check with your science department to see if they are getting rid of any older hoods.

Also, there are cheap HEPA fan/filters on AMazon and maybe you could build something around them. I don't know if they are as good as the filters in a hood, but if you put one on the top of an aquarium, remove the front glass, and seal the openings real well, maybe it will help reduce your losses.

Thanks i'll give it a look

 

[Dane]

See http://www.ukorchidforum.com/viewtopic.php?f=26&t=1292&start=40#p14808 . There are alternatives to big thick gloves. A local vet should be able to help you find a source for the shoulder-length gloves, else try ebay. I think you can find them in floppy plastic as shown in the thread or more skin-tight latex which would give you better dexterity. I am toying with setting up a system like what Terry has for home flasking and doing some research... so, some caution for sensitive viewers (like myself), make sure "safe search" is on if you Google shoulder length latex gloves else you may get a nasty shock.

To avoid the problem of opening up flasks, use plastic tubs. You can see the type I mean at http://www.saorchids.co.za/laboratory.html . You can get them at most plastic/food packaging shops. You don't need such a tough plastic tub, all it must be able to do is take temperatures above 100 oC and snap fast tightly. I put some cling-film over the top to be more sure than nothing can get through. These tubs breath well and don't need to be vented. The biggest problem is the lack of water due to evaporation but frequent replating is better for growth in any case.

How do you then sterilize the inside of the cabinet, can I use a spray bottle with disinfectant?

 

[TyroneGenade]

Hello Dane,

I spray everything down with hydrogen peroxide. I switched from ethanol to hydrogen peroxide as I'm a bit messy and get ethanol in the flasks which is death to the contents. I do this for the plastic tubs as well.

The plastic tubs should not dry out as fast as you describe. Mine didn't, but I had wrapped clingwrap over the top.

In South Africa, if a lab is getting rid of a laminar flow it is because it can't be fixed any more so don't get too excited about a hood being offered. Make sure the thing works or can be fixed. Fixing a badly broken hood will cost more than the hood...

If you can get a cheap air source and HEPA filter then it isn't very difficult building a hood. There are plans online... but I would stick to what works before trying something new. The airflow rate has to be just right, and the flow totally laminar (no cavitation!) else all you have is a glorified spore distribution system.

 

[Trithor]

Hi Dane,
A laminar flow only supplies a slow movement of clean air as a form of positive pressure. If your sterile technique and items that you are working with are not sterile, you will still get contamination. The benefit of a laminar flow is ease of access, as the frontal opening is large.
If you are getting contamination at the replating stage, then the contaminants are not the result of the glove box, but due to something that you are introducing at that time. You need to make sure that the external surfaces of the mother flask are well sterilized. The most difficult point of contamination to correct is the junction of lid to bottle/tub. This can be kept clean by applying clingfilm to the lid/neck at first flasking.
If you managed to produce clean mother flasks in your glovebox, then you should be able to replate. Personally I would loose the gloves. Air born contaminants move on air currents and by settling. If you move slowly and wash your hands and forearms well before you start, you should have no problems. Spray your hands and forearms with 70% alcohol each time you introduce them into your clean box, and remember, move slowly and keep your movements smooth (reduce air currents).
I use 70% alcohol to surface sterilize, (Dischem sell a good surface spray in a red bottle). Your flasks should not be in the box and certainly not open when you spray the inside of the box or sterilize the surfaces (Tyrone getting alcohol by being messy is confusing)
The easiest way to do your first replate is to squirt 1 or 2 ml of sterilized distilled water into the mother flask, loosen the protocorms with a spoonula, swirl and pour a bit into each replate. Be very sure that the neck of the motherflask is sterile before you do this. The thinned mother flask can be kept and recapped again. This works well, is supa fast and because of the speed, the replates are open only for the briefest moment. Also, you are not repeatedly introducing an object (spoonula or other) into your replate flask. Remember contamination is more likely the more times you interfere with the flask as well as the length of time that it remains open.
Obviously the 'pour' method does not work with subsequent replates as soon as there are roots and leaves involved.

 

[Dane]

Hi Dane,
A laminar flow only supplies a slow movement of clean air as a form of positive pressure. If your sterile technique and items that you are working with are not sterile, you will still get contamination. The benefit of a laminar flow is ease of access, as the frontal opening is large.
If you are getting contamination at the replating stage, then the contaminants are not the result of the glove box, but due to something that you are introducing at that time. You need to make sure that the external surfaces of the mother flask are well sterilized. The most difficult point of contamination to correct is the junction of lid to bottle/tub. This can be kept clean by applying clingfilm to the lid/neck at first flasking.
If you managed to produce clean mother flasks in your glovebox, then you should be able to replate. Personally I would loose the gloves. Air born contaminants move on air currents and by settling. If you move slowly and wash your hands and forearms well before you start, you should have no problems. Spray your hands and forearms with 70% alcohol each time you introduce them into your clean box, and remember, move slowly and keep your movements smooth (reduce air currents).
I use 70% alcohol to surface sterilize, (Dischem sell a good surface spray in a red bottle). Your flasks should not be in the box and certainly not open when you spray the inside of the box or sterilize the surfaces (Tyrone getting alcohol by being messy is confusing)
The easiest way to do your first replate is to squirt 1 or 2 ml of sterilized distilled water into the mother flask, loosen the protocorms with a spoonula, swirl and pour a bit into each replate. Be very sure that the neck of the motherflask is sterile before you do this. The thinned mother flask can be kept and recapped again. This works well, is supa fast and because of the speed, the replates are open only for the briefest moment. Also, you are not repeatedly introducing an object (spoonula or other) into your replate flask. Remember contamination is more likely the more times you interfere with the flask as well as the length of time that it remains open.
Obviously the 'pour' method does not work with subsequent replates as soon as there are roots and leaves involved.

Thanks, i'll give it a try with some of my flasks where the medium is starting to shrink and the protocorms are growing

 

[TyroneGenade]

Trithor, I'm a little more paranoid than you and work with gloves on at all times and I keep these sodden with disinfectant. I also surface sterilize instruments as I move form flask to flask. We didn't work with flames in our hoods (too many students setting themselves on fire) so some alcohol inevitably dripped into flasks... So I switched to H2O2. I add a little detergent to the H2O2 solution (1 part 10 volume H2O2 to 1-2 part water). Sunlight soap works well but just a tiny bit. This breaks down the surface tension on tubs, glassware etc... and allows for efficient surface sterilization. I used tween, not sunlight soap but the sunlight soap shouldn't cause issues.

The constant extension and retraction of limbs in a glove box will cause large air currents that will contaminate everything that isn't sealed. I do agree that the major site of contamination is at the junction between the flask and lid. Clingfilm helps a lot here. I always spray my flasks down with H2O2 as they go into the hood so they are sterile on the outside. This is a very important step. A laminar flow isn't very helpful if you are bringing filthy items into it. This is doubly so for fingers.

If you are working in a laminar flow or glove box and at any time can't recall where your hands have just been then stop and resterilize your hands.

 

[Trithor]

I might not remember what I had for breakfast most days, but generally I can remember where my hands have been, ..... picking up my glass of wine that I can take an appreciative self congratulatory sip! More disturbing is that often I cannot remember how I came to be sitting in front of the lam flow in the first place!:rollhappy:

 

[Dane]

Hahaha yeah I need to sterilize my gloves more , it's all about technique to minimise contamination. I gave replating a try again to see what will happen