Redneck Orchid Flasking

[Jensine449]

I've gobbled up this thread, thanks everyone!! Redneck or not!!

[10010100102]

Here's a link to an extensive and very detailed pod-ripening-time list for anyone who needs it:

Pod times

Great thread and thanks to everybody who posted!

[girishgkulkarni]

hi ... Can u give us a pic of the latest development of those protocrom?

[OzPhal]

G'day Gin,

I'm a microbiologist by trade - and also have a love of Orchid Culture

I looked at your sterilisation cycle with interest... the standards from a microbiological perspective are:

121oC for a hold time of 30mins. What that means is that any autoclave will take time to get up to operating temperature - when it does reach operating temperature then it needs to maintain that temperature for 30mins minimum (unless it's a dry heat autoclave which isnt the case with a pressure cooker). You could do one of two things:

1) modify your pressure cooker to incorporate a temperature gauge and, once it reaches 121oC, start your 30min timing
2) Purchase some autoclave tape which changes colour when the cycle has met the required temperature/sterilisation times - i would definitely extend your sterilisation time though

15mins will kill of most vegetative cells but you'll find that the media wont get to sufficiently high temperatures to potentially kill some of your mold or bacillus spores. I would suggest erring on the side of caution - 15mins for the most part may work but you may also partially sterilise some of the spores and these may germinate a few weeks down the track once they have recovered.

Also, once you have completed your cycle let the pressure cooker cool down to room temperature without taking the lid off. This will take sometime but it will ensure that only sterile air is sucked in to the jars during the cool down period. It will also prevent the media from boiling. If you find that it is boiling and you take the lid off and there's media halfway up the side of your jars then it means that the pressure cooker hasnt sealed properly. Always leave your caps loose (slightly) though as there is nothing worse than exploded glass jars in your autoclave/pressure cooker.

Hope i'm not coming across as a know all coz i'm certainly far from that - i hope i've helped...

Originally Posted by Gin

I have the same problem the box is to small it is a 10 gal . aquarium maybe a 20 would be better, yard sale
I read and read a lot of different sites , tried to sort out what I might be able to do
vmax ...I use the pressure cooker with 2 or 3 inches of water in the bottom 15 lbs. pressure for 15 minutes .
Thanks for the info. on the tape could be I used the wrong type or not enough of it . Gin

[OzPhal]

Purchase some isopropyl alcohol - mix it with 30% distilled water (so 70% isopropyl alcohol and 30% water) spray all of the surfaces in your cabinet with it... anything that comes in contact with the seed pods or anything else dip in alcohol and, if it is is metal, run it through a bunsen burner flame. If it isnt metal then allow it to fully dry... wear latex gloves and similarly spray those with the isopropyl alcohol. If you can afford it make up a little HEPA filter unit for your little cabinet - you can buy the filters fairly cheap and just make up something to attach them to your cabinet then you can have HEPA filtered air blowing out of your cabinet.

Originally Posted by vmax3000

I am sad. This morning, I looked at the three flasks I had plated on Wednesday and two of them have a white fluff growing in them.... Those of you who do this more often, remind me. What are the various methods of contamination, again?? I am holding out hope for the third flask, but not so much, if I didn't sterilize the pod enough. I soaked it in hydrogen peroxide...it was dry. I have never used a dry pod. When I used a green pod, I had great success for the first and second flasks, but my culture molded when I placed them in the third flask.... still haven't confirmed my aseptic technique, apparently. Any ideas from the more experienced crowd? Thanks for the thread!

[vmax3000]

OzPhal,
Thanks! I have Ang. calceolus seed that is dry and has been sitting in my fridge for about 4 months. I may see if I can rig up a little HEPA filter with a scavenged muffin fan (I am beginning to think these things are the hammer for all of my nail-like problems ...).
I have a plastic box that I can use and I have access to gloves. I will have to set it up with some holes in the side, et cetera.
I used a pressure cooker to sterilize the media. It was sterile...up until the time I introduced not quite so sterile seeds.
So, here I go, again. to me.....!

[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

[vmax3000]

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

Hi John,
I have had access to a flow hood in the past, but I am wanting to start something up in the kitchen, so I don't have to be reliant on a fully stocked micro lab to be available (I am a chemist...physical chemist, to boot, and the biologists usually have those laminar flow hoods put to some research use and I become an interloper or a potential contaminator or just generally a pita to them). I can't use the powdered gloves, anyway.... the powder blisters my skin.... I have this large plastic storage box that is translucent, so I was thinking of employing it for flasking purposes. I am just experimenting with my hobby/addiction(....or is it that I am looking for more ways to irritate the spousal unit with my hobby ? ) Regardless, I am going to have to sterilize dry seed and hedge my bets!

[OzPhal]

Hey vmax3000, nice to hear from you... the most important part of the whole process will, as you have learnt from past experience, be the sterilisation of the dry seed. I personally prefer to work with green pods but that is just my personal preference - others may well prefer dry seed. I like the idea of being able to eliminate naturally as many sources of contamination as i can. What setup do you currently have? If you have never worked with acrylic/perspex read up about it! it is your friend in these custom sort of applications. Have a look on ebay for HEPA filters - i noticed a lot of vaccuum cleaners use nice box 'HEPA' filters... i doubt these would be the best quality but they should do the job that you need them for. Experiment with different fans - i dont know what a muffin fan is but a nice strong computer fan (the big ones not the small CPU fans) should do the job nicely. If you want any advice or to show me pictures of your setup prior to using it i would be happy to help... Also remember that isopropyl alcohol is your friend but remember to use it in that 70/30 ratio unless you're flaming metal equipment in which case dip it in ethanol and then flame it...

Hope that helps...

All the best...

Originally Posted by vmax3000

OzPhal,
Thanks! I have Ang. calceolus seed that is dry and has been sitting in my fridge for about 4 months. I may see if I can rig up a little HEPA filter with a scavenged muffin fan (I am beginning to think these things are the hammer for all of my nail-like problems ...).
I have a plastic box that I can use and I have access to gloves. I will have to set it up with some holes in the side, et cetera.
I used a pressure cooker to sterilize the media. It was sterile...up until the time I introduced not quite so sterile seeds.
So, here I go, again. to me.....!

[vmax3000]

Originally Posted by OzPhal

muffin fan

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....