Prepare your own tissue culture medium. Microclone Mult-BA contains 4.43g MS nutrients and vitamins, 4.4 micromoles BA multiplying hormone, 1ml PPM plant preservative, and 3.90 g of our exclusive Microclone Gel/agar mix. Mix with two TBS (30g) sugar to make one liter multiplying tissue culture medium.One liter fills 50 Microclone Flip-Top vials, 30 baby food jars, 100 24mm tubes, or 20 MK5/GA7 vessels. Average production per liter is 200 new clones every 4-6 weeks. Please search Microclone Tissue Culture videos and photos online for mixing, plant preparation and handling videos.1) Add 940ml RO water to mixing container. 2) Pour in Mult-BA liquid. 3) pH to 5.8 if preparing liquid media or 6.3 or higher if preparing gelled media. pH will fall about 0.5 point after adding gel powder. pH to 5.8 before dispensing into vessels and sterilizing.
Media prep is best done in a clean kitchen. Media is mixed, distributed into vessels, and
sterilized by pressure cooker or microwave.
Extra water, paper towels, and instruments are sterilized for use in
transfer. Give yourself enough counter
space to move and mix freely.
The kit contains MS media salts-hormone-PPM preservative
mix, Gel-agar powder, pH control kit, and special jar lids. Home items that are needed are baby food jars
(BFJs), 40-50 brown or white commercial paper towels, and a pressure cooker. Additional flip-tops are available from planttcdotcom. A microwave will do in
place of a pressure cooker, but is not as good and less likely to completely
two tablespoons sugar into 900 ml cool purified water in a clean container with
a one-liter mark. A one-quart Pyrex cup
makes mixing and dispensing easy.
add MS-hormone-PPM liquid. The most
common establishment and multiplication media is called B1 and is made from 1
mg BA, 0.1 mg NAA, and 1 ml PPM per liter.
This is the Mult
formula in this kit and is the most common starting media.
purified water to the 1-liter mark and pH correct to greater than 5.8 using the
test strips and pH up and down. Adding gel-agar will decrease the pH a few 0.1
of a point.
may wish to prepare a rack of liquid tubes at this point. See “Liquid culture”. If so, pH to 5.8 before adding agar and
dispense 7ml into each of the 16 screw-top tubes and prepare the vials with
gel-agar in a gentle sweeping motion while stirring. Avoid dry “balls” that are difficult to
to 5.8 before distributing into vials.
Agar is special because it must be added to cold water. Like gelatin, it will ball up and not dissolve
in hot water. When added to the
solution, the powder will sink to the bottom of the container and must be
stirred while dispensing.
Alternatively, heating is an optional method to disperse the
gelling agent evenly in the solution.
Heat in a microwave or on the stovetop gently just until the solution
goes from cloudy to clear, usually about 10 minutes.
Dispense approximately 20 ml into each flip-top vial or 33
ml into each of the BFJs. Small
measuring cups or a turkey baster make measured dispensing easier. If using culture tubes for gel culture, fill
to 10 ml and set them at a slant for hardening while still hot from the
cooker. The tubes are slanted because
water that is formed during cooling drips down the side of the tubes and may
drown a young explant.
Cool Tool A magnetic stirrer is a handy tool for
mixing the media and again later for cleaning plants for culture. It spins a magnetic bar in the bottom of the
mixing cup. Magnetic stirrers are
available from lab supply stores and on eBay.
Remember to remove the stir bar before microwaving.
The vessels now need to be pressure-cooked or microwave
sterilized. The pressure cooker works
best because it can hold the high temperature needed without the jars boiling
over. Also metal lids and tools can be
cleaned in the pressure cooker but not in the microwave.
Pressure cookers are available in many sizes, but the 22-24
qt size works best. Some pressure
cookers come with a wire rack and lifting handle to keep the jars off the
bottom and make handling easier. They
should all have a canning rack to keep jars off the bottom. If your cooker did not come with a rack; you
can make one from galvanized wire and hardware cloth. First you will need to know the arrangement
of the jars that will fit into your cooker.
If they all don’t fit in one batch, split them into two even batches for
uniformity. Jars can be double-stacked
for space. Save room for the purified
water, paper towels and instruments.
Wrap the paper towels and instruments loosely in aluminum foil for
sterilizing. The foil will keep them
clean when moved to the transfer hood.
Fit foil over the top of the water jars or fit caps loosely. Foil should reach two inches below the neck
of the Mason jar. Fill the jars 70
percent full to avoid splashing.
Follow the instructions your pressure cooker manufacturer’s
manual. Begin warming the cooker before
loading. Use only distilled or purified
water in the pressure cooker. A load of
30 jars and 1 or 2 pints of water will take 20-25 minutes just to begin
building pressure. You can save some
time by preheating the cooker while you prepare the media.
The Presto cooker has an aluminum vent button that
allows the air to escape until it has built a good head of steam. It will automatically pop up and seal when
the steam is venting fast enough.
Pressure cookers without an auto-vent should be heated without the
pressure weight on until a steady stream of steam is coming out. When the vents are closed, the pressure gauge begins to climb. Heat
on full to 15 psi, and then adjust heat lower to keep pressure steady.
At 15 psi, the weight is lifted and a small bit of steam
begins escaping. That is the sound of
proper pressure. Use a cooking timer and
watch the pressure. Do not leave the
cooker unattended. After 20 minutes,
turn off heat and allow cooker to cool.
Do not attempt to open cooker early.
The Presto vent button drops when the pressure is gone.
Note: The best
tool for sterilizing is an autoclave.
Countertop autoclaves with their own steam are available secondhand from
dentists and tattoo-tool sterilizing or laboratory supplies. Autoclaves have a pressure gauge, timer, and
sometimes, automatic cycles. A German
company called Gallenkamp makes a portable electric autoclave that does not
need a stove. Try a trial run with water
only to learn the heat settings and timing.
Do not run dry or use non-autoclavable plastics.
cooker is filled to the recommended level with reverse osmosis-filtered,
distilled, or deionized water to prevent dissolved materials from precipitating
jars and instruments in a stable pattern on the protective bottom plate.
the lid tightly and add heat at a reasonable rate. Leave the valve open until the steam is
steady, and then close the valve until pressure is reached.
must be maintained at 15 psi and 250°F (121°C) for 20 minutes.
Pressures fluctuate, so do not leave cooker alone.
cooker must be allowed to cool without lifting the pressure weight or releasing
jars can still be at boiling temperature and boil over. Leave the pot until it is below 90 degrees C
or about 20-30 min.
Take this time to clean the hood and bench area for media
cooling. That makes about 50 minutes for
a cycle or just over an hour to make media.
Label the corner of the media rack with the media type and hormone code
and take it to the clean hood to cool.
Microwave treatment depends on the energy and age of
the oven, the vessel, volume, and water.
The original procedure called for two Pyrex bottles of water to run near
the agar to keep them from boiling over.
The jars would then be run for either 7-½ minutes with 250ml water in each jar or 10
minutes with 900ml. A new procedure is
to do away with heat sinks altogether.
a paper towel on the microwave carousel and arrange jars in a single
layer. Use only polypropylene or
microwave-safe lids, no metal lids.
sure lids are loose to prevent tops from boiling off. Add a microwave-safe plate or baking dish
across the tops as a weight.
on high until the first jars boil but before they boil over. Pause for ten seconds and repeat. If the jars are heating unevenly, rearrange
them and continue.
Three or four boiling cycles are usually recommended. Microwave ovens cannot produce the steady
heat and pressure of the pressure cooker.
can be pulled into jars as contents cool.
Make sure air is clean.
vessels must be allowed to cool before transplants can be put in them. Allow the gel to harden. Moving around while hardening breaks the gel.
towels will have condensate in them.
Transfer them to a warm oven for a few minutes to dry.
sterile water to cool before using it on plants. This takes a while.
may be stored for up to two weeks.