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    Applied Development Engineering Practice 1: Making Embryo Manipulation Devices

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    • Introduction to practical training conducted at Nanae Fishwater Station, Field Science Center for Northern Biosphere.

    • Teacher Introduction Page ページ
    • Japanese URL
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    (1) Items to be prepared

    • ① Gas burner

      ② Pasteur pipette

      ③ Point folding tweezers

      ④ Glass wool

      ⑤ Ampoule cutter

      ⑥ Surgical needle

      ⑦ Injection needle case

      ⑧ Dramont capillary

      ⑨ Dramont capillary pipette

      ⑩ Nail polish

      ⑪ Micropipette making machine

      ⑫ Microforge

      ⑬ Micro grinder

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    (2) Gas burner


    • ① The gas burner rotates the red knob, emits gas, and then pushes the knob to ignite.

      ② When digesting, turn the knob to the opposite side to digest.。

      ③ When not in use, be sure to turn it off. Do not clog the tip of the gas burner.

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    (3) Pipette

    • ① A suitable thickness portion of the Pasteur pipette is required for the depression in the ampoule cutter to rotate the pipette.

      ② Check the pipette for cracks.

      ③ Hold both sides of the crack separately with the left and right hands, and fold it so that it is pulled.

      Flush the tip of the pipette with a gas burner. If it is too hot, the tip closes, and if it is small, the egg or embryo is caught and broken.

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    (4) Surgical needle


    • ① Warm about two-thirds of the tip of the narrow portion of the Pasteur pipette with a gas burner and pull it to the left and right to stretch it.

      ② Fold in the middle of a stretched glass tube.

      ③ Melt the tip of the thick part of the Pasteur, pinch it with a folded tweezers, and then melt the glass part.

      ④ Stretch the molten glass section at an angle.

      ⑤ Fold the stretched glass tube about 1cm from the bent portion.

      ⑥ Apply a nail nail polish to the tip, then apply glass wool.

      ⑦ Stand on a surgical needle until it dries.

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    (5) Operation needle





    • ① Pull the Pasteur pipette left and right to make a surgical needle.　　　　

      ② Fold in the middle of a stretched glass tube.

      ③ Warm the folded part with a gas burner to make the tip smooth.

      ④ For the narrower end, warm the tip with a gas burner to make the tip smooth.


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    (6) Needle for microinjection


    • ① Flush both ends of the 10 Drummont glass capillaries with a gas burner to smooth the cut. Make sure that the mouth is not blocked because it is too hot.

      ② Place the glass capillary tube in a micropipetting machine and switch on and stretch the heater.

      ③ Remove the top of both the upper and lower glass needles so as not to break them, and place them in the injection needle case. Make 20 needles from 10 capillaries.

      ④ Set each needle on a micro-forge.

      ⑤ Operate the manipulator while looking at the microscope so that the tip of the glazing passes through the center of the microheater and enters the glass pillow.

      ⑥ The micro heater is energized while observing the microscope, and the stenosis is produced in the middle of the glass needle. For infusion, the stenosis should not be narrowed too much.

      ⑦ Turn off the power to the micro heater, and then move the manipulator to remove the tip of the needle from the micro heater.

      ⑧ Place the microinjection needle that forms the stenosis in the case.



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    (7) Needle for cell transplantation


    • ① Put DW into the syringe of the micro grinder and drip water onto the grinding wheel.

      ② Switch on the micro grinder so that the whole grinding wheel gets wet with DW.

      ③ Simply set the extended needle in the holder of the micro grinder.

      ④ Lower the holder angle to 30 degrees.

      ⑤ Operate the knob on the micro grinder to lower the needle to the surface of the grinder.

      ⑥ Operate the lens knob on the micro grinder to focus the pipette.

      ⑦ Rotate the coarse lever of the needle of the microgrinder and lower the tip of the pipette just before the surface of the grinding wheel.

      ⑧ Rotate the micro-motion lever to attach the needle tip to the grinding wheel surface.

      ⑨ Lower the needle with the micro-motion lever and start sharpening the tip.

      ⑩ When the mouth of the tip is empty about 10μm, turn the roughing lever to raise the whole needle and remove the needle.

      ⑪ Attach the needle to the dramont capillary pipette.

      ⑫ Put the tip in distilled water, take in and out distilled water, and wash off the grinding wheel powder.

      ⑬ Once, transfer to a needle case to dry.

      ⑭ As with microinjection needles, a stenosis is formed at the tip. For implantation, narrow the stenosis as much as possible. Note that if it is too thin, it will become clogged.

    • Manipulation Needle Creation ファイル MP4
      224.8 MB · アップロード 23年 04月 24日 10:27
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    (8) Agarose petri dish


    • ① Put 150ml of Ringer for freshwater fish in a 300 ml beaker and put stirrer stirrer.

      ② Pour agar (Agar not for electrophoresis) to a concentration of 0.8-1%.

      ③ Cover with a saran wrap, put it on a stirrer, and stir the agar powder.

      ④ Put the Ringer's gel in a microwave oven and warm it up so that it does not boil suddenly.

      ⑤ Once warmed, place in a stirrer and stir the agar flour.

      ⑥ Repeat ③-⑤ to dissolve the agar, keeping the solution in it from boiling. When it becomes clear, put it over the light and check that no agar powder remains.

      ⑦ When the agar powder is completely dissolved, cool the agar solution a little.

      ⑧ During this time, the glass Petri dishes are placed on a desk (checking that they are level with a level gauge).

      ⑨ Pour the agar liquid into a glass petri dish. Thinner for embryo manipulation, but thicker agar for photographic purposes.

      ⑩ When the agar solidifies, cover, invert, and store in the refrigerator.。

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    (9) Culture media and agarose Petri dishes

    • ① Ringer's solution for freshwater fish: 7. 5g NaCl, 0. 2g KCl, 0. 4g CaCl2/1 l DW

      ② Egg White: Take the egg white from the chicken egg, make a merenge, and store it in a refrigerator. Allow to stand for a while and use the egg white fluid accumulated in the lower part. Necessary reconciliation.

      ③ Primary culture: Ringer's solution for freshwater fish containing 1.6% egg white

      ④ Primary culture solution for 96-well culture dishes: Add 1/1000 volume of antibiotic to the solution in Ringer's solution for freshwater fish containing 1.6% egg white (antibiotic solution stock: penicillin, streptomycin are dissolved in DW at 10% concentration each, and dispense 100-200 μl into test tubes to keep them frozen)

      ⑤ Secondary culture solution: 1. 8 mM CaCl2, 1. 8 mM MgCl2 (180 μl of 1M CaCl2, and 180 μl of 1M MgCl2 are placed in 100ml of distilled water)

      ⑥ Agarose Petri dish already described 

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    Contents of the following practical training