tissue culture question!

[digdugdank]

I made up 25 jars of agar based medium, and was wondering how long the jars without explants (un-opened) are viable? From what I've read the jars with explants last two weeks! But read nothing about jars without! Can anyone help me here! This whole concept of propagation really fascinates me. This is my first batch (got kit for Xmas):) I took explants 1/3/13 and so far I've had to toss 4 jars out of 12 that I made in my dirty kitchen!! Lol! I can't wait to move!!! The jars that are good look very clean:) I'm very excited :o any info helps! Thanks, digdug!

 

[digdugdank]

Thanks dankherbs, I'm working on a February 1st move in to a bigger house, with plans for clean room! I wanted to do some cultures here just to see just how clean u gotta be! ;) I'm just wondering if I need to make fresh jars, or could I use the unopened jars that are already made up? They would be 2weeks old! I'm excited about learning more about TC! Strain saver for sure! Thanks, digdug!

 

[paulycali]

Not clean. STERILE or a big waste of time. I'd like to see a diary on your tissue culture setup. If you are able to give them the proper environment and go forth with it please share your experiences. Not much on tissue culture here at the farm. Thanks and good luck with your project :)

 

[digdugdank]

I will start keeping records when I move! The house I'm in is way too small! I was successful with 8 explants! :) and my jars did come out sterilized, and I am very pleased! I took explants and placed them with success in a not so perfect environment! Not bad for a newbie, just lucky I'm thinking! Lol. I couldn't think of a better place to share experiences, paulycali thank you I will keep the farm updated! Thanks, digdug!

 

[Animal Chin]

Not clean. STERILE or a big waste of time. I'd like to see a diary on your tissue culture setup. If you are able to give them the proper environment and go forth with it please share your experiences. Not much on tissue culture here at the farm. Thanks and good luck with your project :)

Maybe you should look up the defination of a "clean room" as in what labs use. I'm not referring to a room that's clean lmfao I'm talking about an actual clean room.

 

[outwest]

Maybe you should look up the defination of a "clean room" as in what labs use. I'm not referring to a room that's clean lmfao I'm talking about an actual clean room.

I built a few in a college for the EE department.

outwest

 

[Animal Chin]

Im a PIMP...

outwest

word!

 

[rootsnshoots]

If you're pouring plates in open area you're always going to contaminate. You will never achieve a "clean room" you need a laminar flow hood dude. Not sure if you've seen my thread on the topic....

 

[Animal Chin]

If you're pouring plates in open area you're always going to contaminate. You will never achieve a "clean room" you need a laminar flow hood dude. Not sure if you've seen my thread on the topic....

this thread is becoming a joke..for one if that flow hood isnt in a clean room your fucked...ps you also need some outlets, light bulbs, light switch, light switch and outlet cover plates...im sure Im forgetting a grip please forgive my ignorance
___________________________________________________________________

Cleanrooms are facilities designed for conducting research or manufacturing products that require extremely clean environments. Typically, cleanrooms employ a broad range of techniques to prevent air particles, bacteria, and other contaminants from entering the workspace, often by means of employee dress code and washing, pass-thru lockers and chambers, and intensive detail to cleaning. However, one of the major forces keeping a cleanroom particle free is the air filter system. Cleanrooms employ many different types of filters, including HEPA filters and ULPA filters, but there are two standard air flow patterns that are consistently used: laminar flow and turbulent flow.
Cleanroom Basics
Cleanrooms are necessary for various kinds of scientific research that require particle- and bacteria-free environments. For example, when scientists grow cultures, it is important to reduce the introduction of other bacteria so that results will not be compromised. Manufacturing various kinds of products like microprocessors also requires particle-free environment, because even a human hair contacting the small chips of a microprocessor can inhibit or destroy functionality.
Cleanrooms are either hard- or soft-walled. A hard wall cleanroom is a permanent structure or part of a larger permanent structure, while a soft wall cleanroom can be transported or augmented depending on requirements, and primarily exists within a larger, permanent structure. Modular, soft wall cleanrooms are needed for medical emergencies or when smaller runs of environment-sensitive materials are produced within a larger facility.
Cleanrooms are graded depending on how clean the air in the facility is. There are two standards used for this determination: the ISO and United States federal standards. ISO grades are numbered sequentially, advancing from 1. A cleanroom graded ISO 1 contains ten or fewer particles per 0.1 micrometer cubed area. A cleanroom graded ISO 2 contains 100 or fewer particles per 0.1 micrometer cubed area. The rest of the series feature the amount of particles rising by a factor of 10 per level. US federal standards are numbered 10, 100, 1000, etc., with the lower class number representing a cleaner facility. Class 1 cleanrooms have one or fewer particles per 0.5 micrometer cubed area. Class 10 cleanrooms have 10 or fewer particles per 0.5 micrometer cubed area. Ascending class grades rise by a factor of 10.
Because people often work in cleanrooms, they are required to follow dress and behavior guidelines to limit the amount of particles they will bring into a cleanroom or particles they will shed while working in the environment. Workers must change from street clothes into specially designed outfits, often with full hood coverings, gloves, and breathing masks. Workers must also enter through an air shower to eliminate remaining particles on the cleanroom suit, and then pass items into the cleanroom through a small chamber that prevents outside air from entering the clean environment.
Cleanroom Air Filtration
Cleanrooms employ air filtration to limit the particles in the environment air. Typically, this is through the use of either a highly efficient particulate air (HEPA) or ultra low particulate air (ULPA) filter. These filters can remove roughly 99.9 percent of all microparticles in room air by applying either laminar air flow or turbulent air flow techniques to the environment air.
Laminar air flow refers to air that flows in a straight, unimpeded path. Unidirectional flow is maintained in cleanrooms through the use of laminar air flow hoods that direct air jets downward in a straight path, as well as cleanroom architecture that ensures turbulence is lessened. Laminar air flow utilizes HEPA filters to filter and clean all air entering the environment. Laminar filters are often composed of stainless steel or other non-shed materials to ensure the amount of particles that enter the facility remains low. These filters usually compose roughly 80 percent of the ceiling space. Cleanrooms employing laminar air flow are typically referred to as Unidirectional Airflow Cleanrooms.
Non-unindirectional airflow cleanrooms utilize turbulent airflow systems to clean particulate air and maintain a clean environment. While laminar air flow filters are often a component of turbulent airflow systems, they are not the only systems employed. The entire enclosure is designed to use laminar flow and random, non-specific velocity filters to keep the air particle-free. Turbulent airflow can cause particle movement that can be difficult to separate from the rest of the air, but non-unidirectional airflow systems count on this random movement to move particles from the air through the filter.

 

[digdugdank]

Thanks farmers! Dankherbs, loads of great information, thank you! Popcorn, outwest, ;) got it thanks!! I have successfully kept 8 out of 12 explants (sofar!!) And all 25 jars of agar are sterile! I have 12 empty jars, unopened and sterile! My question, Are the unopened jars still good after two weeks? I'm sure there is someone that can answer this question! I learn more from failure then success!! Anyone can do things if all goes well!!!!! Life throws curve balls, adapt!! Any positive information is greatly appreciated! :) I will continue to post on my success and failures ,with positive feedback! Thanks digdug! :)

 

[rootsnshoots]

A joke for sure... Good luck with your ventures

 

[neverbreak]

theoretically they should last indefinitely, as they should be sterile.

neverbreak

 

[rootsnshoots]

Look at some mushroom cultivation sites. There is great info on how to achieve sterile environments and techniques on a budget. A clean room is a long shot. As when YOU open the door to enter the room, you're going to fill it with contaminated air. And YOU being In the room breathing/moving shedding hair and skin, these things will find their way into your cultures. The only way to achieve consistent results is to use a laminar flow hood. But it can be done in shmuv or still air box. Start with a still air box as they are cheap.

You're blank plates should last a very long time un opened. Store them in the fridge if its going to be a while before using them.

Check out my thread. It should give you SOME direction. And get you off the kit and save you money on further ventures

 

[green punk]

I wish someone would develop a commercial lab. That we can bring our chosen ones to, and have them cultured for a fee. Seems like a no brainer for some of you ballers in CA to invest in this.

 

[Phillthy]

what food mix are you using?

 

[digdugdank]

Rootsnshoots, is your thread on tissue culture? If so can you direct me to it, as I am not experienced enough with forums and how they run! This is only my second forum I've ever posted too. The other was monster fish keepers! Lol! I've got some crazy aquariums! :o! Off track sorry!!! Thanks rootsnshoots I have jars in fridge and will only b putting brand new explants into them. My transplants will go into fresh made jars :) philthy! Is this philthy phil from the scv? I'm using a mix from a kit I got from internet. My local hydro store recommended it. Its pre measured, all I add from home is purified water and a couple tbs sugar! I'm learning as I go, any positive input is much appreciated, thanks digdug!

 

[rootsnshoots]

Yes it is. It's under this section (advanced teks) as "in vitro micropropigation" (misspelled lol). I've never seen or used one of those kits so I don't really know much about how they tell you about doing things.

How are you sterilizing the media? And does it have you mix up the media then sterilize it and then pour it into your vessels?

Feel free to post any questions on my thread as you browse through.

 

[digdugdank]

Thanks rootsnshoots, I've started reading your thread, with interest! In the kit I got it tells you that a clean room with flowhood is best but not necessary! I cook my media in the microwave then pour into jars. From there I pressure cook the jars for twenty minutes at 250 degrees(15 psi)!let cool and you have sterile jars! (Hopefully) ;) all 25 jars I made came out sterile, from what I can see. Only time will tell. I love experimenting! I took explants from two strains (banana kush &grease og) I took tips &stems with nodes, and placed them into the media! I have 8 jars with explants that are still super clear with no signs of contamination! The explants with tips seem to be swelling at the base of my cut like it would if you take clones conventionally. I'm really looking forward to getting this technique mastered! The possibilities are endless! The explants that have failed were due to not cleaning the explants as well as they stated! I cut cleaning times down to a third! I washed with 10% bleach&water for only 5 minutes, and just splashed alcohol for a few seconds then clean water rinse! I did all this on purpose expecting 100% failure, but over half my goods are still looking clean.as I get more into it I will keep better notes!:) my methods are not professional at all! I'm just having lots of fun! I will keep posting for those who wish to read and b positive! Thanks digdug!

 

[rootsnshoots]

Nice man, if you're pressure cooking the jars for 20 min at 15 psi you are most definitely sterilized. Anyway you could post some pics of the kit? Does it tell you what's in it? Cytokinins or auxins?

That's been most my struggle is not getting the explants sterilized or killing them.

I guess there is a product called ppm that you mix into your media that helps battle contams..

I'm pretty much on a stand still until I have the funds to build a flow hood. I'm tired of muddling around with still air boxes or the shmuv.

 

[rootsnshoots]

Im not at a stand still because I built a cheap clean room with a tent with negative air pressure some hepa filters and a flow hood. Go to CC and look up Dr GVZ. He taught me and hes da man on the topic. I never said it has to be expensive I just said you need a clean room first and foremost. Im battling broad mites in my mother room so Im preoccupied right now but just take my advice. Im not just making shit up. PS I started with the kit too;)

Pics? Let's see some of yer werk? I'm having success just can't crank out the numbers. And set up time sucks.

I got a tile floor and can of oust....