Redneck Orchid Flasking - Page 10 - Orchid Board

OzPhal · #91 08-17-2009, 07:56 PM

Absolutely correct John..

Basic steps of sterilisaton

1) Start with a sterile environment - whether you're using a glove box, fume hood, class II biocabinet the principle is the same. Use Isopropyl Alcohol! mix up a 70/30 blend: 70% Isopropyl Alcohol & 30% distilled water (referred to as IPA from here on in). Make sure you use gloved hands - never try and do this without gloves (it may work but it's poor aseptic technique and will increase your contamination risk - dead skin cells which, no matter how good your aseptic technique is, contain millions of bacteria and spores. Always use good quality sterile gloves (they come individually packed and are usually gamma irradiated. After having put the gloves on coat your hands liberally in the 70/30 IPA and allow to air dry (your body heat will evaporate the IPA off pretty quickly) Have a look at this tutorial for good aseptic gloving technique:

YouTube - Sterile Gloving

. Liberally (with a fine mist) coat all of the interior surfaces of your 'cabinet'.
2) Spray the bench near your cabinet with the IPA and use a sterile wipe

to wipe it down
3) Place all of the equipment you need to use on that sterile bench ensure that a bunsen burner is operating near the cabinet and have a container of alcohol nearby (use common sense - alcohol is flammable). Give all of the equipment a good spray with the IPA, turn it over after having coated one side, allow it to air dry. Give your hands a liberal spray of IPA (allow to dry fully. Ensure you have a resting block that you can put in the cabinet - prior to flaming anything pick up that block with a sterile wipe (think of the wipe as a barrier between contamination and sterility - your hands are contamination - the block is sterility) and give the block a good wipe with the sterile wipe as you place it in to the cabinet. Then pick up the metal equipment that can be dipped in alcohol and flamed. Dip the surfaces you desire to be sterilised i.e. with a scalpel it would be the blade. Get it glowing white hot and then place it on the resting block so that the sterile surface is not contacting the walls. Do that will all of the equipment that you can flame sterilise
4) Then you have the items you cant flame sterilise - you need to wipe these as you place them in to the cabinet. Use the barrier technique utilising a sterile wipe to keep you from the sterile equipment and give them a good wipe as you place them in to the cabinet
5) Last but not least sterilise in the media - some may laugh - but you definitely need to take care of the outer surfaces of the media containers as you place them in to the cabinet. These should have been sitting on your bench with your other equipment when you gave it a good liberal spray with IPA. A technique that i found worked for me is to double wrap things. When you make the media and pour it in to your containers make sure you leave the lid a little loose. Wrap in two layers of aluminium foil. With a freshly IPA glove take the container and, as your placing it in the cabinet, peel off the outer layer of aluminium foil. This should leave your media jar with one layer of foil on it. Prior to your using the container you peel that final layer of foil off. After everything is sterilised in take your sterilised seed pod and again wipe it in to the cabinet with a sterile wipe
6) the very last step, after having taken 6 hours to get setup, is to take off your gloves, put a fresh pair on and let the fun begin...remember to IPA them well before you put your hands in to the cabinet. Once they are well wetted put your hands in the cabinet and let them dry in there as this is now a 'sterile' area

Oh, also, 70/30 isopropyl alcohol is not highly flammable as such - you could spray it in to a bunsen burner flame and you will get a flare up but not an explosion - just use common sense

Other equipment you will need:

Quote:

Originally Posted by John

Ok I am a little confused. I don't know if you are talking about a glove box or flow hood. If you are using a flow hood, then spray the inside with alcohol and run it for 15 to 30 minutes and let it dry before lighting the bunsen burner, if you are not careful, you may get a flash from the alcohol. If you are using a glove box, then you probably can't use a burner and everything will need to soak in alcohol or bleach solution. I was also told not to use gloves that are powdered. The powder will fall off and contaminate the culture. So either wash well with the alcohol or don't use them at all and use good proceedure to avoid contamination.
John

Royal · #92 08-18-2009, 11:28 AM

Quote:

Originally Posted by vmax3000

is a computer fan!! I think it refers to the size/shape, but I have scavenged them from many a computer tower and have a variety of sizes. I usually wire them into an AC/DC transformer and "VIOLA", an air moving device. I'll have to see about a HEPA filter. I have these little 'sun' filters (it's a Japanese brand name of some sort) that I got from Sigma Chemicals. I might be able to place them in between the fan and the box. I have been using the filters to permit air flow into/out of the flask...well, jelly jar, for me...via a hole in the lid. They block particulate.

OOOO....

Hey Vanessa,

A key factor which influences the efficiency of a LF hood is the volume of air moved. I don't think the muffin fan has what it takes to create a true laminar air flow. I'd suggest either going to a real blower unit or just making it work just as a glove box and eliminate the fan.

My glove box isn't even fully closed and I get less than 10 % contamination. It's just a clear storage tote with the top sealed with plastic wrap and a long narrow slot cut into the front for my hands. I never use gloves but I spray alcohol in between each transfer. Gloves aren't sterile either (once opened). The key, IMHO, is to minimize air movement inside the box by moving slowly and deliberately. Your kitchen is dirty, your hands/gloves are dirty, air is dirty. Assume that nothing is sterile and use good aseptic technique and you'll do fine.

vmax3000 · #93 08-19-2009, 01:44 PM

Thanks, Royal.

In the past, I have worked with green pods. Seed is new to me, especially dry seed. The last time I worked with it, I tried sterilizing with h peroxide. It didn't work....which may be more indicative of me not knowing what I am doing rather than insufficient h peroxide. Is there a way to sterilize the seed so I don't loose so much of it, or to retrieve it from the solution (I am seeing a funnel with some filter paper, here, but this is all trial and error and I love learning from the more successful, so as not to attempt reinventing the wheel...or fail at it, again

). How long should the seed be left in contact with the sterilizing solution? The pod is dehisced, but most of the seed is still attached. I captured it in a plastic baggie and enclosed a desiccant just for good measure. OzPhal, thanks for the video!

Royal · #94 08-19-2009, 03:16 PM

Hydrogen peroxide does work according to many - I've just never used it. I just stick with the standard:

Household Bleach (~6%) diluted 1:10.
10 minutes is good contact time to use as a starting point.

Getting the seed out is the tricky part.

I've used pipettes and droppers, and even just dumped them. There are some good sep funnel options. OSP sells glassine filters, funnels, and wash bottles for pretty cheap - all autoclaveable.

I know others have used coffee filters with some success.

vmax3000 · #95 08-19-2009, 03:22 PM

Royal,

Dumped them into what from what?? Love the quote, btw!!

John · #96 08-19-2009, 06:18 PM

Vanessa I use syringes and bleach solution to soak the seeds and then I push out the bleach and pull in sterile water to rinse and then squirt the seeds on to the media. From what I remember, flow hoods need between 60 and 90 feet per minute air flow. So if your hood filter was 2ft x 2ft, the blower would be sized to produce 240 to 360 cubic feet per minute air flow. The computer fans probably can not meet this flow rate.
John

Royal · #97 08-19-2009, 06:20 PM

I use small test tubes, maybe 15ml. I put small amounts of seed into the tubes ahead of time. When I'm ready, I add a few ml of solution. Expose seeds to this solution for your preferred time (10 min), agitating periodically.

Hopefully, the seed will sink and drawing the bleach sol. off the top is no problem. If it floats, you need to draw the sol. from underneath which is harder (and floating seed may indicate incomplete infiltration of sterilant into the seed testa). I try to rinse with a dropper full of sterilized water at least once, but some have success with no rinse.

If you don't have access to pipettes or droppers you're reduced to just decanting and dumping.

Aquaculturekenya · #98 08-21-2009, 06:30 AM

Hello all, I am from Nairobi,Kenya and I do plant tissue cultures here with exotic ornamental plants and also grow great African species of orchids from seeds. I have just joined your forum and hope to find interesting friends and discuss a lot of orchids with many who knowledge on growing and keeping orchids.