tissue culture experiment

[fleshgear]

i am trying for the second time to grow plants with tissue culture. the first time did not work, it just went moldy. i did a control one on both trys. the control did not get any mold. so it was the plants that were not sterilized. the first try i tried many different plants. crypt, java fern, anubias, bacopa, aponogeton, African violet, echinodorus rose. the violet was the last to get mold, but it had no sign of growth. the E.rose and crypt started to grow but shortly after got moldy. it wasnt the same kind of mold either. there was several different types.

i used glass containers that i got from the dollar store, and am putting them in a zip lock bag.

the growth medium is made of

* 1/8 cup table sugar

* 1 cup water

* 1/2 cup stock solution (Miracid diluted 1/4 tsp in 1 Gallon water) i substituted this with a few drops of miracle grow

* 1/2 inositol tablet (125mg)

* 1/4 vitamin tablet with thiamin

* 2 Tablespoons agar flakes

i added 2ml of quick grow solution part 1 and 2 ml of part 2. these are for vegetative growth

for plants this time i used crypt "wendtii" cause i have lots and nice thick rhizomes. and one jar i put and orchid in

to clean the plants i put the rhizome under running water for 5 min and scrubbed the rhizome with a brush.

then everything went into a 20gallon tank turned on its side to keep dust and contaminates out.

then soaked for 5- 10 min in 1 part bleach and 9 parts water.

then soaked in 70% rubbing alcohol for 5 min.

then soaked in hydrogen peroxide for 2-3 min.

the rinsed in distiled water twice.

i then cut the rhizome with a scalpel and forceps that were soaked in rubbing alcohol after each cutting. i cut the plants on a paper towel that was soaked with rubbing alcohol.

one of the jars i put some methylene blue into the jar.

one of the jars i dipped the plant in methylene blue

then i put the plants into the medium and put the jar with medium into a new zip lock bag and sealed it.

i have done a few things different this time round. last time i put a lid on the jars. and there was quite a bit of condensation on the glass. i think this may have accelerated the mold.

i will put the jars under a florescent light that is on for 14 hrs per day on a timer.

i took a few pics. the close ups of the plants in the medium are blurry because i took the pic through the bag.

[Baos]

Most culture kits that I'm aware of use an extract from coconuts. Easily purchased on ebay. Able to grow from a single cell or combine plants.

[Wingin' It]

WHOA! That's a lot of work! I hope it pays off for you! It'd definately be interesting to see if this go round works! :thumbs:

[fleshgear]

Most culture kits that I'm aware of use an extract from coconuts. Easily purchased on ebay. Able to grow from a single cell or combine plants.

there was one variation of the recipe that called for coconut milk instead of some of the water.

[fleshgear]

update.. all the crypts went moldy... the orchid just got some mold tonight but it is very little. the control has no mold at all. i even made a few cuts in it with the scalpel i used to cut the plants..

so i am not getting the plants disinfected enough. i am not sure how long i can leave it in the bleach without killing the plant. and can i use a higher concentration of bleach?

[Baos]

perhaps formaldehyde would work better? It is naturally part of some of our fresh water streams in the mountains. I know that bleach leaves a film behind due to the time I washed out an aquarium with bleach and killed all my plecos when they sucked on the glass. I washed it out ten times after using the bleach but it didn't get rid of the film. Finally a vinegar bath fixed it.

[fleshgear]

perhaps formaldehyde would work better? It is naturally part of some of our fresh water streams in the mountains. I know that bleach leaves a film behind due to the time I washed out an aquarium with bleach and killed all my plecos when they sucked on the glass. I washed it out ten times after using the bleach but it didn't get rid of the film. Finally a vinegar bath fixed it.

when you used the bleach did you use a mix of bleach and water or just bleach? i have washed several tanks with a bleach water mix and have never had a problem

[Baos]

It was many many years ago and I don't remember. Sorry. It was most likely a very strong mixture.