Recent Changes

Monday, July 31

  1. page Traditional culture method edited ... Dish (φ10 cm x 5 cm) {dish-pipette.jpg} ... pipte (φ 8 10~12 mm) to Setting up {beak…
    ...
    Dish (φ10 cm x 5 cm)
    {dish-pipette.jpg}
    ...
    pipte (φ 810~12 mm) to
    Setting up
    {beaker_overview.jpg}
    (view changes)
    1:47 am

Thursday, February 26

  1. page Metamorphosis edited ... {Inducing_metamorphosis-002-copy.JPG} Leave over a night. ... slides to a culture tank.…
    ...
    {Inducing_metamorphosis-002-copy.JPG}
    Leave over a night.
    ...
    slides to a culture tank.
    ...
    is safe out of sea water for short
    ...
    (5~10 seconds) to movefor transferring from the petri dish
    {Slides_(primary_polyps)-002 copy.JPG}
    ...
    eat 1~2 days after metamorphosis.
    ...
    polyps for 2~3several days. Once they eat,Normal feeding will be OK once they form bigger polyps.
    Important note: Primary polyp is smaller than normal polyp. They often degrade the polyp body after eating for up to a few times. This is because the primary polyp cannot simply change its body size and a colony makes bigger polyp through degrade-reform process. Reforming new (and bigger) polyp however fails quite often. Only small population of primary polyps, for example 10%,
    will grow so that they eat living artemia.
    to colonies more than 2 polyps.
    (view changes)
    8:05 am
  2. page Fertlization edited ... This can be done to collect as many eggs as possible or as quicker as possible in order to mic…
    ...
    This can be done to collect as many eggs as possible or as quicker as possible in order to microinject into a lot of eggs. Jellyfish swimming keep eggs suspended in sea water, making egg collection a bit painful. Start egg collection under binocular as soon as you see the first several eggs released into the dish. Normally we use mouth pipette to collect if allowed. Try not to pick jellyfish with the pipette.
    b) Collecting eggs after taking out jellyfish from the dish back to the culture tank.
    ...
    dish to collect eggs quietly. You canculture tank. Then collect eggs into other dishes.dishes quietly. If there
    ...
    can fertilize eggs without collection eggs in this dish (without collection eggs) by adding
    ...
    water (see step (3).) YouIn this case, you can collect fertilized eggs/embryos 2- to 4-cell stage.after confirming the first cleavage.
    You can
    ...
    sea water.
    The
    The concentrated eggs
    c) Mixture of a) and b): for a lot of injection (600 or higher)
    ...
    When you hen you fished itfinish the injection to these eggs (15~20 minutes
    ...
    the injection chamber.chamber, to continue microinjection. The point of this mixed approach is to save time to collect eggs to spend more time to do microinjection.
    3) Fertilization
    Fertilization has to be done with in 60~70 minutes after spawning. (3 hours ~ 3 hours 10 minutes after the light ON.) Rate of cleavage error goes higher if you wait longer.)
    Take out male jellyfish from dish and quantify approximately the amount of sperm with dark-field illumination with the dissecting microscope. Take sea water (male seawater) from the bottom of the dish and put it into egg dishes. Use plastic transfer pipette for cell culture.
    Amount of sperm is OK when you estimate 20~40 spermatozoids are swimming around an egg. Amount of sperm will be dependent on the number of eggs, size of the dish etc. Usually 3~5 ml of sea water taken from the bottom of 10 cm dish with 10~12 male jellyfish is optimal for 300~400 eggs in 3 cm dish. Too much spermatozoid results in polyspermea and cleavage error. (Eggs from old jellyfish (= 3 weeks after maturation or older) tend to have polyspermea.)
    4) SimplyIf you simply need to get fertilized eggseggs.
    Put 10
    ...
    approach is also useful if
    (view changes)
    7:15 am
  3. page Fertlization edited ... 5~8/dish for overnight incubation in a dish 2) Collecting eggs (for microinjection purpose) …
    ...
    5~8/dish for overnight incubation in a dish
    2) Collecting eggs (for microinjection purpose)
    ...
    (b) is a lot more easier if
    ...
    the shaker.
    a) Collecting eggs during ovulation (jellyfishes are still in the dish).
    This can be done to collect as many eggs as possible or as quicker as possible in order to microinject into a lot of eggs. Jellyfish swimming keep eggs suspended in sea water, making egg collection a bit painful. Start egg collection under binocular as soon as you see the first several eggs released into the dish. Normally we use mouth pipette to collect if allowed. Try not to pick jellyfish with the pipette.
    b) Collecting eggs after taking out jellyfish from the dish back to the culture tank.
    Once ovulation is over (or there is enough eggs released into sea water), you can take out jellyfish from the dish to collect eggs quietly. You can collect eggs into other dishes. If there is no manipulation/microinjection is necessary, you can fertilize eggs without collection eggs in this dish by adding male sea water (see (3).) You can collect fertilized eggs/embryos 2- to 4-cell stage.
    ...
    sea water.
    The

    The
    concentrated eggs
    ...
    injection chamber.
    c) Mixture of a) and b): for a lot of injection (600 or higher)
    ...
    injection chamber.
    3) Fertilization
    Fertilization has to be done with in 60~70 minutes after spawning. (3 hours ~ 3 hours 10 minutes after the light ON.) Rate of cleavage error goes higher if you wait longer.)
    (view changes)
    7:07 am
  4. page Fertlization edited ... Collecting eggs and sperms and fertilization 1) Transferring jellyfish into dish ... and s…
    ...
    Collecting eggs and sperms and fertilization
    1) Transferring jellyfish into dish
    ...
    and sperms.
    Sperm: transfer male jellyfish (5~10) into a deep 10 cm dish with 200 ~ 300 ml sea water within 1 hour after the light is on.
    Eggs: transfer female jellyfish (max 30/dish) just before the ovulation. (90~100 min after the light is on.) This is not to leave jellyfishes for long time in a dish at high density.
    ...
    5~8/dish for overnight incubation in a dish
    2) Collecting eggs (for microinjection purpose)
    ...
    culture tank. Please remember that rate of irregular development increases if eggs are fertilized > 60~70 minutes after spawning. It is therefore often necessary to collect eggs as quick as possible if microinjection (or other manipulation before the fertilization) is planned. Procedure (a) is recommended for this purpose. It is also used to collect as many eggs in the dish as possible. Procedure (b) is a lot more easier if you don't do microinjection and if you don't need to collect all eggs in the dish. Procedure (c) is mixture of (a) and (b); begin with procedure (a) first. Then once you get enough eggs to inject in 15~20 minutes, switch to strategy (b) and go for injection while you are waiting to concentrate eggs on the shaker.
    a) Collecting eggs during ovulation (jellyfishes are still in the dish).
    ...
    bit painful. Start egg collection under binocular as soon as you see the first several eggs released into the dish. Normally we use mouth pipette to collect if allowed. Try not to pick jellyfish with the pipette.
    b) Collecting eggs after taking out jellyfish from the dish back to the culture tank.
    ...
    ovulation is over,over (or there is enough eggs released into sea water), you can
    ...
    eggs quietly.
    You
    You can collect eggs into other dishes. If there is no manipulation/microinjection is necessary, you can fertilize eggs without collection eggs in this dish by adding male sea water (see (3).) You can collect fertilized eggs/embryos 2- to 4-cell stage.
    You
    can concentrate
    ...
    10~15 minutes. This technique is particularly interesting if you needThe speed of rotation to inject a lotefficiency concentrate eggs depends on dishes and volume of eggs. (see below)sea water.
    The concentrated eggs are ready to transfer into an injection chamber.

    c) Mixture of a) and b): for a lot of injection (600 or higher)
    ...
    (a) approach to collect eggs just enough for microinjection in the first 300 eggs then start injection. Meanwhile injection concentrate eggs usingnext 15~20 minutes (3~400 if you are experienced). Then switch to (b) approach.approach, meanwhile you perform the first series of injections . When you hen you fished 300 injections, a lot more of eggsit (15~20 minutes later), there are concentrated eggs in 10 cmthe center of the dish and ready to continue injection.transfer to the injection chamber.
    3) Fertilization
    Fertilization has to be done with in 60~70 minutes after spawning. (3 hours ~ 3 hours 10 minutes after the light ON.) Rate of cleavage error goes higher if you wait longer.)
    (view changes)
    7:01 am

Wednesday, February 25

  1. page Metamorphosis edited ... GLWamideII (GNPPGLW-NH2) 1 mg/ml Normal (1 x 3 inch) or wide (2 x 3 inch) glass slides, prop…
    ...
    GLWamideII (GNPPGLW-NH2) 1 mg/ml
    Normal (1 x 3 inch) or wide (2 x 3 inch) glass slides, properly marked with diamond pen (not a marker pen).
    10 cm plasticpetri dish
    1 ml syringe with 25G needle (to smash artemia)
    Procedure
    ...
    Quietly pour 2~3 ml of GLWamideII-MFSW for a normal slide (4~5 ml for double-width slide) using a transfer pipette.
    Collect planulae with mouth pipette and put on the glass slides. Planulae will stop swimming immediately (5~10 seconds) and will undergo metamorphosis.
    Leave quietly{Inducing_metamorphosis-002-copy.JPG}
    Leave
    over a
    ...
    culture tank. Primry polyp is safe for short time (5~10 seconds) to move from petri dish to the tank.
    {Slides_(primary_polyps)-002 copy.JPG}

    Some primary polyps start to eat 1~2 after metamorphosis. Feed smashed artemia, passed through the 25G needle 2~3 times, to primary polyps for 2~3 days. Once they eat, they will grow so that they eat living artemia.
    (view changes)
    8:54 am
  2. page Nursery tank edited ... The speed has to be strong enough not to let the baby medusae staying on the bottom but not to…
    ...
    The speed has to be strong enough not to let the baby medusae staying on the bottom but not too strong to avoid the damage. Usually choose minimum speed enough to rotate baby medusae.
    We normally keep baby medusae up to 3~4 mm in diameter in this tank. Once babies are grown to this size, any speed of bubbles will be harmful. It is then necessary to transfer them to Kreisel tank.
    Nursery tank (dram) model 2010
    Dimenstion
    φ250 (240) x 80
    Target of culture
    Collecting baby medusae and grow-out (up to 3~4 mm).
    Nursely for very young medusae.
    Weight
    Capacity
    Note

    (view changes)
    6:36 am
  3. page Villefranche-culture_Tanks edited Villefranche culture system - Tank set-up - Nursery Tank This tank will be used to collect bab…

    Villefranche culture system - Tank set-up -
    Nursery Tank
    This tank will be used to collect baby medusae released from polyps and to grow them out to certain size (3~4 mm).
    We collect baby medusae by leaving colonies in this tank up to 2 nights. This is because…
    Polyps eat quicker than baby medusae. If there are babies and polyps in the same small tanks, babies can never eat their first food.
    Leftover of the food (dead artemia) will stay inside and attach to the polyps, which accelerate algae growth.
    You may want to collect babies for more than 2 days, if you couldn't get enough. In this case, try either of these two.
    Simply set up another nursery tank and transfer polyps to it.
    Take out all babies using a pasteur pipette and grow them on a dish at least for a few days. Clean the nursery tank and re-install the sea water.
    {1.jpg}
    Setting up
    Connect the aeration tube.
    Fill the tank with sea water to 90~95%.
    Install colony plates
    Fill the tank to 100% (to maximize the water circulation).
    Adjust the aeration speed.
    2 Days later, take out the polyp plates and fill up with sea water
    5. is of particular importance. See below the criteria
    Aeration
    (first 3~4 days or for baby medusae up to 2~3 mm): Relatively strong. Some bubbles will travel 10~15 cm on the surface before they disappear. See right tank of the photo above.
    (Until 7~9 days or for baby medusae up to 3~ 4 mm): Relatively weak. Bubbles do not travel and disappear at the edge of the tank. See left tank of the photo above.
    The speed has to be strong enough not to let the baby medusae staying on the bottom but not too strong to avoid the damage. Usually choose minimum speed enough to rotate baby medusae.
    We normally keep baby medusae up to 3~4 mm in diameter in this tank. Once babies are grown to this size, any speed of bubbles will be harmful. It is then necessary to transfer them to Kreisel tank.
    Kreisel Tank

    {Kreisel.jpg}
    Kreisel tank for polyp
    ...
    8 mm thick plexiglass is unnecessarily thick and heavy. Change to 5 mm thick plexiglass.
    Sub-chamber can be smaller. The bottom of the sub-chamber will be just below the window between main and sub camber.
    Nursery tank (dram) model '10A
    Dimenstion
    φ250 (240) x 150
    Target of culture
    Collecting baby medusae and grow-out (up to 3~4 mm).
    Nursely for very young medusae.
    Weight
    Capacity
    6.4 liter
    Note
    Prototype
    Future modification
    Diameter -> 300 mm, Width --> 75 mm (4.5 liter)

    Colony plate + suspender
    Large plate
    (view changes)
    6:34 am

More