Friday, December 28, 2012

Some tissue culture tips and success secrets

I was browsing through the stats on the blog recently to see what subject seems to be the most popular. It appears as though there are a lot of would-be amateur tissue culture hobbiests out there with a keen interest in how to get started but also how to get things done properly with little fuss. I have read some interesting articles on the internet about home tissue culture of orchids and what methods to follow. Most are basic repetition of the blah-blah basics with some useless stuff perpetuated. Some are good though, especially those which dispense with the drivel about useless or over-complicated techniques. One such "technique" is the tea-bag or waterproof paper technique for surface sterilising dry orchid seeds. Equally irritating is the syringe-method which I threw out many moons ago (in fact about a week after I started). So... since I am abviously irritated by things that challenge my impatience I have decided to share some simple techniques with you that actualy work. In fact, so much so that my entire seedling collection is based upon these since most were cultured as dry seeds.  

Dettol hand sanitiser
In previous and slightly aged posts, I indicated the importance of understanding the concept of sterility. This is a crucially important first step. Sterility does not mean clean or disinfected, it means absolutely sterile - ie - nothing living! Now consider that everything that is not sterile is covered in bacteria and fungal spores and other microscopic organisms that are just waiting for an opportunity to spread and to multiply. Your basic view now should be that of an obsessive compulsive with a phobia of germs! Possible transmission routes include YOU (your skin, hair, breath, clothes etc.), the air, all surfaces, your tools and kit and the orchid seeds themselves. Some of the most stubborn of bacteria I find are those associated with the actual seeds! Ok, so most of you would be aware that absolutely everything would need to be sterilised before success can be achieved with germinating orchid seeds in vitro. My archived post on building and using my laminar flow hood should provide you with some basic and useful information on building your own hood for orchid seed use. Although you can do seeds in a glove box or sterile still-air cabinet which I also did at the very beginning, you will never achieve the rate of success that you will with a laminar flow hood. It's worth spending the time and the bucks to make it. Mine cost approximately R2000 for all the materials and construction. For the US readers this equates to about $250. All my tools are cleaned before being sterilised in a 10 quart 15/16 bar pressure cooker for 30 minutes. I wrap all my tools in aluminium foil first so that I can remove them without actually touching them and can transfer them to the laminar flow hood where they can be carefully unwrapped under sterile air and placed into a sterile jar ready for use. I also try not to store too much ready-made plates of media in the fridge and prefer to make a batch of new media the same day or the day before and to allow these to set in the laminar flow with the Hepa running until I use them (so plan ahead - it helps avoid unnecesary non-sterile exposure). Wash your hands and forearms well before working in the hood. Use a good non-sticky hand sanitiser like the one made by Dettol (kills 99.9% germs). Use this liberally on your hands, making sure you get it under your nails and remember to remove your rings and watch before working. I routinely wipe down the inside of the hood working chamber with neat sodium hypochlorite solution before commencing with any work. When the hood is not in use I have a hinged door that seals off the front of the chamber to prevent incidental aerial contamination that might threaten the viability of my Hepa. 

Holstered auto-pipette and pipette box
Now for the good stuff. Buy yourself an automatic pipette like the one in the photo. These often come in standard starter packs supplied by almost all of the lab consumable companies. They are a standard feature of molecular laboratories. However, ensure that the one which you get is fully autoclavable. The unit can be wrapped in foil and autoclaved as for all the other tools. Those which are not fully autoclavable can be used but you will need to familiarise yourself with all the internal parts and how to dismantle and reassemble them. All these parts must be placed into a plastic container and completely covered with a 40% hydrogen peroxide solution for 30 minutes before rinsing in sterile water and reassembled ready for use (so... just get those which are fully autoclavable - it is MUCH easier). The starter packs usually also come with pipette tips supplied in an autoclavable box. If not, just get a box of tips and a bag of replacement tips from the same supplier to fit your pipette. These are dead-cheap and disposable and fully autoclavable. I suggest that you get yourself either a 200µl or 1000µl pipette and tips. These two sizes are often the most common and are also the most appropriate for the job depending on the working space which you have. I use 200µl pipette and tips.

Now purchase a bag of eppendorf tubes from the same lab consumables supplier (see pic). There are various volumes to choose from but get the graduated 1500µl or 2000µl tubes with lockable caps. Your next investment will be a vortex mixer (see pic) which will ensure that the seeds will be continuously aggitated and mixed with the sterilising agent for the correct amount of time and will avoid dead-spots associated with other techniques. Vortexing in the eppendorfs also concentrates the seeds nicely allowing better control for rinsing and pouring of seeds onto plates. Eppendorfs are also autoclavable and I place a fistful into a polypropylene tub with a sealable lid dry into the pressure cooker for sterilising. Once sterile, I remove this tub of eppendorfs to the laminar flow. You can include the vortex mixer inside the laminar flow if you have the space but you can also have it next to the hood on the working bench if you wish. In the hood you should also have a sterile flask of RO water and a flask of pre-made sodium hypochlorite solution to which some surfactant has been added. Use the following concentration of non-perfumed liquid sodium hypochlorite: 120ml tap water to 20ml neat household sodium hypochlorite solution (3.5% m/v). Add a few drops of Tween20 or household dish-washing liquid (not too much or it will foam too much!). The surfactant facilitates good soaking by allowing the sodium hypochlorite solution to "stick" to the surface of the seeds.

Vortex mixer
Sterile eppendorfs
When ready to start, put the dry seeds onto a folded piece of paper and funnel into a sterile eppendorf tube. Inside the hood under full flow, push the pipette onto a tip in the box and close the box again. Do not allow the pipette or tip to touch any surfaces unintentionally while working. If this happens discard the tip by depressing the eject button on the pipette and begin again with a new tip. Take up a metered solution of sodium hypochlorite (200µl if you are using a 200µl pipette and tip; the pipette is adjustable to volume - pre-set the volume before you sterilise) and add repeatedly to fill the eppendorf to about 3/4 full. Holster the pipette and close and lock the eppendorf cap. Vortex on high for 5 minutes. Shake initially with your hand before vortexing to loosen any seed clumps. At 4 minutes remove from the vortex mixer temporarily to view the buoyancy of your seeds. If they are nearly neutrally buoyant/slighly heavier than the solution, allow the final minute for the seeds to settle at the bottom of the eppendorf tube. If they sink quickly, you can allow them to settle at the end of the 5 minutes. Some seeds with float, especially those of terrestrial orchids. Allow these to collect at the miniscus. For seeds that collected at the bottom of the eppendorf carefully pour off the sodium hypochlorite solution inside the laminar flow hood into an awaiting empty waste receptacle which was also previously sterilised in the pressure cooker. The seeds will remain at the bottom of the tube after the solution has been poured off. Hold in one hand inside the hood and with the other hand charge the pipette with a new tip (expel the old tips into the waste receptacle) and take up sterile RO water and add to the tube to 3/4 full again. Close and lock the cap and vortex again for 30 seconds. The seeds will appear heavier in the RO water (sinking faster) because of the reduced specific gravity of the solution. Allow seeds to collect again at the bottom of the tube and pour off all the water. Repeat again with a new tip and RO water and vortex to mix all the seeds again. This time, pour the entire contents onto your awaiting plate and seal. Floating seeds are a bit tricky because you have to work them to the bottom of the tube by holding the tube horizontally while carefully twisting it from side to side between your fingers. You will lose some seeds during the above process but with experience you will get a feel for it and get to a point where you lose almost nothing.

I do all of my dry seed work as above. I have found it produces superior results and minimises contamination. I hope you found this post useful. I would love to hear your thoughts and your experiences.

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