Orchids do very well on plain coconut water with 1,5% agar added to it. It contains all the right hormones, and together with some activated charcoal, it should work just fine.
I never tried orchids though. Well.. I did, but ebay sellers ripped me off and kept selling me seeds of some kind of giant grass species. That was an awfully funny joke, but at the same time a poor waste of effort and money.
Oh well, let's just get this thing started.
General things
I will use the metric system. If you want to do conversions, go ahead and try to find a erlenmeyer flask with pints and gallons noted on it. Lab work requires math, if you lack that skill, go to wolframalpha and let it do the work for you. I use it to check my calculations on a daily basis - better to be safe than sorry.
Every medium I use has some fixed components/ingredients. My medium contains at least the following ingredients unless specified otherwise:
- MS salts with vitamins (66% strength, available from a lot of places, I got mine from a biochemistry supplier) -> basic nutrient mixture with added vitamins
- PPM (plant preservative mixture, you can find this on ebay or at a tissue culture webstore) -> Antibiotic against most - but surely not all!!!!!! - contams.
- 1.5% - 2% agar agar (99% pure, ebay, grocery store) If you can get your hands on some Daishin Agar, that's better, but not necessary.
- Every part of plant is taken with a sharp, clean, sterilized razorblade. Pinching with fingernails is savage in this in vitro world and will be punished by legions of contams.
- Every contamination - no matter how small - is contained (since it became antibiotic resistant!) and autoclaved before disposal. -> skip this part and your family / housemates / you are in great danger. Don't fuck around with this. Don't. If you can't handle that rule, GTFO!
- Procedures take place in a still air box, I'm wearing nitrile gloves (straight from the box, unwashed or whatever).
- All materials like tweezers, knives, plastics etc. are autoclaved while packaged in tinfoil. This is the same as with shrooms. For the sharp things, I use some tissue paper between the material and foil to keep the stuff from punching holes in the foil.
- All media are prepared in unsterile condition, boiled until dissolved in a microwave, and then packed and autoclaved for sterilization. This means that some hormones and organic stuff get lost in the process. Deal with it. I add hormones in 20-45% excess and I calculate that on the go. Over here, I mention the final concentration only, since it would be quite tough for readers to understand otherwise. The math wouldn't make sense. If you have filters with meshes below 45 micron, then you could filter sterilize your hormones. If you decide to do that, use the original concentrations. If not, add 30% to the concentration.
Things to have in stock before you even start thinking about tissue culture
Hormones
- 6-benzylaminopurine (BAP or 6-BAP) @ 1mg/mL -> dissolved in 1M KOH solution by dripping it on powder untill dissolved (usually it takes around 1 mL) then water is added to make the final concentration. Store solution in the fridge for 3-5 months.
- Naphtalene Acetic Acid (NAA) @ 1mg/mL -> Dissolved in 10% HCl solution by dripping it on the powder until dissolved (usually takes around 2-3mL) then water is added to make the final concentration. Store solution in the fridge for 3-5 months.
- Indole Butyric Acid (IBA) @ 1mg/mL -> Dissolved in Ethanol, added water to make the final concentration. Store solution in the fridge and keep dark(!) for 2 months.
NOTE: NAA and IBA are interchangeable (is that really a word? English keeps amazing me!) that means that one could replace the other. Both do have some different effects, but in the broad sense they do the same.
- 2,4-dichlorophenoxyacetic acid (2,4-D) a herbicide that "orders" cells to start dividing uncontrollably. This stuff causes cancer, it's a thing I keep in mind all the time when handling things. Prepare outdoors or in a safe place with a dust mask and gloves. You'll smell it. Once in solution, it is fairly safe to use if you don't spill. Yes, it's dangerous stuff. But I use it. You could do without, it would just take some more experimenting. I added a dye (food coloring) so that my media containing this stuff, can be recognized from the outside. Use with caution.
Liquids, Hard- and software
- A Pipette + tips (ebay has some cheap ones that have uL (micro-litre) amounts, those are the ones you'll need if you want to make anything under 100 samples). Pipetting 200uL with a dropper is possible, but far from accurate. If you are good at using wolframalpha.com or you're good at maths, then do whatever suits you. You could change stock solutions to meet your requirements at any time, I mean, it's not a law or anything.
- Agar Agar (99%) pure. Go for at least 10 grams, otherwise you'll come short. Daishin agar is better, because it solidifies better and doesn't interfere much with nutrients. Regular agar is fine. NEVER use the vanilla flavored stuff.
- Demineralised water (enough to make some batches, I keep a stock of around 5 litres). RO water, ionated water, activated water, whatever, nah. That's NOT going to work. End of story. Demineralised or nothing.
- Plastic/Glass wares -> Petri's, tubes, tissue culture vessels (or just polypropylene/PP5 plastic containers). Keep enough in stock to do everything a third time in a row. If something needs to be recultured away from a contamination, your work will be gone if you'll have to wait 5 days for the delivery man. Say, you're doing 5 petri's, then keep another 15 in stock just in case.
- Bleach
- Glassware for soaking things, Glassware to prepare media.
- Tweezers, knives, handy metalworks
- Ethanol / IPA / High percentage alcohol but below 90% Vol. Otherwise the ethanol can't do it's job. It needs water for transportation. You could always dilute high vol. alcohols of course.
- Transparent wrapping foil on the roll. Cut these in 3cm rolls with a sharp knife (do this while using your brain, you'll cut yourself otherwise, no really. It just takes a few minutes to do this tedious task but it you'll be rewarded if you take that time). Otherwise, use the expensive Parafilm. I never used it for anything in my home lab, since the stuff costs an arm and a leg and I'd use a roll within a week.
Hormones + pipettes + other materials will cost around 100 bucks to get started from scratch. The pipette will last a lifetime, the hormones probably too if you buy 10 grams of the stuff, the other materials like glasswork as well. Other things are plain throw-away materials. PP(5) plastics can be autoclaved and re-used up to 40 times. Don't throw those out immediately if something goes wrong. Check if it can handle re-use, because that could save you a looooot of money.
Cost reduction comes in numbers in this case. Also, if you've been working with fungi, some of this stuff might already be in your closet tucked away somewhere. I suggest you run the dishwasher and clean that stuff up to use it. It would be a waste to buy thing you already have.
Nutrients
- MS salts with vitamins -> that stuff is expensive, yes. It is the main stuff used in literature, and I recommend using it for ease's sake. It makes things easier to understand, easier to reproduce protocols and do the awesome stuff others do.
- If you want to go experimental, you could use coconut water, liquid nutrients, solvable nutrients, whatever, but this complicates things since you'll need to calculate concentrations yourself. I cannot guarantee anything. Every brand has different concentrations, and therefore different results. Vitamins can come from multivitamin pills, or vitamin B pills. Use a quarter pill per litre at most.
- Cannabis can do a while without nutrients, but not for long. A week or so at most.
That's all I am giving you guys for today. Next up is sterilisation, but I think I have talked about that in the botanist files V1. In the episode after that, I'll explain more about the procedure.
After that, it's just a waiting + updating results game.
Good to have some readers already, I hope you'll enjoy the show!
