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  1.  

    Construction:

    The easiest way to begin constructing the flow-through system is to begin with the ring stand components …

    1. Place the ring stand in an open desk area such that it is as near as possible to the vacuum in the hood

    2. Attach two claw-like attachments to the ring stand.

    3. Remove the plungers from two 30ml syringes and secure the syringes in the claw. Ensure that each of the syringes is level with one another (use a leveler or a straight edge to determine that they are level).

    4. Twist a two-way stopcock onto the end of the tubing attached to the syringe (see picture).

    4a. It may be necessary to tape the inflow tubing into place in the stopcock. If so, simply wrap the tape around the stopcock and the tubing so that the tubing does not move or slip out of the stopcock.

    5. Attach a 20G x 1 ½ needle (Yellow) to the other end of the two-way stopcock.

    6. Attach the 16.5” length of the plastic tubing to the end of the yellow needle.

    7. Attach the other end of the 16.5" piece of tubing to one of the bent needles sticking through the PVC pipe **You may want the needles to be held by clay so that they do not move on their own [The clay setting can be made by molding clay around the area where they pass through the PVC pipe]

    7a. If the flow-through system has not been used for some time, it may be useful to create a new pvc pipe holder. If that is the case, find a piece of pvc pipe that will fit snugly around the current petri dish size that you are using. cut the pvc pipe so that roughly 0.5" of pipe remains above the dish when the pipe is placed around the dish. Next, drill holes in that 0.5" section of the pvc pipe such that new inflow needles can be placed and bent according to your desired dimensions and inflow preferences.

    8. Slide the PVC pipe over a p35 dish (it should fit loosely, but hold the dish enough so that it doesn’t fall out when lifted into the air. Once you are ready to begin your experiments, you can tape each side of the PVC to the microscope stage to prevent it from moving.

    9. Attach one end of the 37” plastic tubing to the needle on the opposite side of the PVC pipe that you used already.

    10. Run the other end of the plastic tubing through a 1ml syringe to its tip and seal the opening of the syringe through with clay so that the tubing does not move and air cannot enter or leave.

    11. Attach a 22G x 1 ½ needle (Grey) to the other end of the stopcock.

    12. Push the needle (carefully so as not to bend the needle or stab yourself) through the cork of the flask.

    13. Make sure there is a plain needle (no colored attachment end) in the cork so that it pokes out inside and outside of the flask.

    14. Attach the longest length of tubing to the plain needle.

    ***IF THE SUCTION FORCE IS TOO STRONG. Use another 20G x 1 ½ needle (Yellow) to puncture the rubber cork sealing the flask to dilute the vaccuum. Also, slide a 27G X 1 ¼ needle (Gray) into the yellow needle to decrease some of the dilution.

    23. Run the tubing (with whatever length is necessary) to the vacuum nozzle.

    24. Place a 1000p pipetteman tip (tip first) into the vacuum nozzle and run the plastic tubing through the large end of the pipetteman tip.

    25. Turn on the vacuum so that the tubing feels like it seals into the tip.

     

    Testing the Flow-through System:

    1. First open both stopcocks and turn on the vacuum.

    2. Give the vacuum pump a minute or two to create a vacuum in the flask so that it begins sucking air from the needle in the PVC pipe.

    3. Adjust the suction needle in the dish so that it will suck from the level at which you want the media to remain the entire duration of your experiment.

    4. Pour your media into one of the 30 mL syringes.

    5. The media may not begin, initially, to flow into from the inflow reservoir/syringe into the dish. If so, use the syringe plunger as needed to initiate the flowing of media into the Petri dish.

    6. If the media level in the dish reaches the suction needle and then is sucked through the needle and into the outlfow reservoir/flask your system should work.

     

    Experimentation: You want your cultures to remain in media constantly while using the system so be very careful not to let the system dry up your dish. To prevent drying up of cultures, ensure that both your inflow and outflow (suction) needles are positioned correctly in your dish. If the needles move too much, try molding the clay to hold them tighter. Also remember that once you put sterile media into the 30ml syringe it is no longer sterile and bacteria may enter your culture. Depending upon the length of your experiments, this issue may not be of great concern. Be sure to consider its implications for your specific experiment.

    1. After uncovering the inverted microscope, fill the syringes with media. If you need to use two solutions, you can use both syringes; however, if you are only using one solution, use one syringe.

    2. Ensure that the Petri dish is in the PVC ring and the inflow and outflow tubes are positioned appropriately. Go ahead and place the dish surrounded by the PVC pipe on the microscope stage in its correct location.

    3. Before locating a growth cone, you must calibrate the flow-through system. Once your growth cone is found, any fiddling with the flow-through system is likely to cause you to lose your image and will ruin any time-lapse imaging that is presently taking place.

    4. To calibrate the system, turn the stopcock lever at the intersection of the two tubes coming from the syringes to the on position. If there are two solutions, be sure that the dial is in the off position for the solution not being used.

    5. Media will most likely not be entering the Petri dish. This will require you to use a syringe plunger and apply pressure to the syringe opening to initiate the flow.

    6. Once you see that the fluid is being delivered into the Petri dish, turn the dial to slow the flow down (if necessary) so as not to disrupt the eye buds.

    7. Turn the vacuum on gently and make sure that the outlfow needle is positioned at the desired level of culture media.

    8. Allow the flow-through system to equilibrate for at least 2 minutes before beginning experiments.

    9. After the media is flowing at a constant rate in the Petri dish, turn the inflow AND VACUUM off.

    10. Now you can find the growth cone(s) to be studied.

    11. Turn on the inflow.

    12. Turn on the vacuum.

    13. The rates of inflow and outflow should already be set; however, you may need to adjust the inflow and outflow stop cock dials throughout the experiment.

     

    Maintenance/Troubleshooting (Possible issues and their solutions):

    • THE DISH DRIED UP! If the dish dried up, this means two things. First, your suction needle was aimed to low in your dish, causing it to be capable of sucking up all of the liquid. Secondly, your inflow of media is moving slower than the outflow (suction) of media from the dish. The easiest and really only solution to this problem is to aim your suction needle higher in the dish.

    • THE DISH OVERFLOWED!!!* ****Be sure to prevent this from occurring, even at the expense of your experiment, given that an overflow would cause damage to the microscope***** Try closing the stopcock to adjust inflow. Also, make sure you are getting suction. Try replacing the dish, because it may be leaking.

    • THERE’S NO INFLOW AT ALL! Make sure your stopcock is open. Use a plunger to force the liquid in the 30ml syringe to flow through the inflow line

    ***In all maintenance cases, these are the issues MOST LIKELY to occur. They are not the ONLY possibilities.***

  2.  

    The best way to sterilize tubing for reuse between trials is to autoclave old tubing by looping it (lasso style) and wrapping it in cheese cloth. Your lab technician should be able to help with this.

  3.  

    The tubing we have been using (2009-2010) can be found at smallparts.com

    Tygon S-50-HL Medical Tubing, 1/32" ID x 3/32" OD x 1/32" Wall

  4.  

    Note that inflowing culture media does disperse through the entire dish. However, if you would like to test that fact, you can begin with a culture dish full of water. Next, use the flow through system with culture media. As the culture media flows into the dish, the pH indicater in the media will show you how the media is dispersing in the dish.

  5.  

    The flow through system produces an inflow rate that is very difficult to control (may range from 16-30 mL/hr). As such, it is necessary to record the inflow rates and volumes so that you may statistically assess variable media rates in your study