Micropipettes are used for electrophysiology and fluid delivery. The pipette puller we use in the lab is a P-97 Flaming/Brown Micropipette Puller from Sutter. The manual is available here. Users need to load a glass pipette and run a program, which may have one or more pull cycles, stored on the device. Each program has its own number and will produce pipettes of different tapers, resistances, tip diameters, etc. Pipettes are then automatically pulled. Briefly:

How it works

  1. The glass pipette goes through one or multiple pull cycles.
  2. In each pull cycle the filament turns on and heats the glass at HEAT. A weak pull pulls the class until it reaches the programmed VELOCITY, which occurs exponentially.
  3. Once the velocity trip point is reached, the filament a switches off and cooling air flow is turned on.
  4. The puller (optionally) executes a hard pull that depends on either TIME or DELAY.
  5. TIME > 0, after 40 ms plus air flow for TIME, a hard pull is executed.
  6. DELAY > 0, cooling air for 300 ms and hard pull after DELAY.
  7. See image below for the effects of these programmable variables, and see the programming section below for how to set them up.

Calibration#

The device uses a filament which needs replacing on about a yearly basis. Program settings like HEAT are made relative to the filament, and so when a new filament is inserted programs may need to be edited. We typically use ‘box’ filaments. For each new filament, and periodically for old filaments, a ‘ramp’ measure should be taken with a thin/thick pipettes. This value represents the HEAT value at which the pipette glass melts sufficiently to be pulled apart. The ramp value for a new filament may deviate from a previous filament by as much as 100. A first port of call is to take the new-old ramp difference and apply that to the HEAT variable for your programs. As filaments are used and aged, their ramp value slowly changes. HEAT settings +/- 35 of the ramp are not recommended, can result in catastrophic damage to the filament.

Ramp calibration process

  1. Turn the machine on, or else hit
  2. Type the number of any program, e.g. 1 and hit
  3. Press
  4. Press 0 (i.e. don’t wipe program!)
  5. Press 1
  6. Install glass (see below)
  7. Press
  8. A ramp value will be calculated. When completed, extract and bin the glass.
  9. Record the ramp value for your thin or thick pipette type on a piece of paper stuck to them machine and date it, for other users to see.

Creating a Program#

Users can create and store their own programs on the machine. Each program is identified by a number. On top of the machine we have a paper table of programs and their users. Please update your entries here so that people do not accidentally overwite others’ in-use programs. Up to 100 programs may be stored. There are two sorts of variables you can edit. Cycle variables, i.e. HEAT, PULL, VELOCITY, TIME, DELAY, which apply per pull cycle of a program, and program variable, e.g. AIR PRESSURE or AIR MODE, which applies to the whole program.

Program Process#

  1. Turn the machine on, or else hit
  2. Type the number of the program you wish to edit, e.g. 56 and hit
  3. Edit program variables:
  4. Press
  5. Press 0 (i.e. don’t wipe program! Or 1 if you do want to wipe extant program)
  6. The screen will slowly show all the options you can edit along with a number. Hit the number for the option you wish to edit, e.g. 2 for AIR PRESSURE and 3 for AIR MODE, which decides whether you use TIME or DELAY. 1. For thick walled glass, AIR PRESSURE of 500 is recommended, for thin walled, 300. 1. With AIR PRESSURE, select TIME for multi-cycle programs for pipettes and/or pipettes of <1.2 mm outer diamater. Use DELAY for single cycle programs and/or pipettes of >1.2 mm outer diamater.
  7. Enter new value, hit
  8. Hit or to go back
  9. Edit cycle variables:
  10. steps through cycles, steps back.
  11. You can have one or multiple cycles.
  12. One each cycle, the ‘cursor’ is blinking on the HEART value. You can enter and new value, then hit to move to the next variable, else just hit to accept what’s there and move to the next variable.
  13. Once you are done, hit

Example programs#

  • Patch pipette
    • Use Thick Walled Sutter instruments BOROSILICATE GLASS w/ Filament
      • OD. 1.5mm ID. 0.86 mm
      • ITEM # BF-150-86-75 “fire polished"
    • P= 500 (pressure)
    • Cycles:
      • 1  H: (RAMP) p- V: 16 Del:1
      • 2  H: (RAMP) p- V: 16 Del:1
      • 3  H: (RAMP) p- V: 16 Del:1
      • 4  H: (RAMP) p- V: 16 Del:1
      • 5  H: (RAMP) p- V: 15 Del:1
      • 6  H: (RAMP + 11) p=10  V: 19 Del:1
  • Large cleaning pipette
    • Use Thin walled Glass 1.5OD. 1.12 ID 8WPI brand
    • Pressure = 500
    • 1: H: (Ramp - 15) V:18 TIME: 250
  • Medium cleaning pipette
    • Use Thin walled Glass 1.5OD. 1.12 ID 8WPI brand
    • Pressure = 500
    • 1: H: (Ramp - 9-10) V:26 TIME: 250
  • Alternative cleaning pipette
    • Use Thin walled Glass 1.5OD. 1.12 ID 8WPI brand
    • Pressure = 500
    • 1 H: (RAMP - 27) p- V: 27 Time:250

Pulling a pipette#

When pulling a pipette make sure it is secure at all steps of the below process, else it might slip and contact and damage the filament. Replacing the filament is a pain because it requires everyone to edit their programs. First, select your pipette taking care to know you have the desiered outer diamater, inner diamater and glass type. Your choice depends on the micropipette holder you have, the type of experiment you are doing, etc. Then:

  1. Open the machine’s hood
  2. Place your glass pipette (at this stage more just a glass tube!) into the groove in the silver holder to your left, and leave about 3 cm poking out the end, past the tightening screw.
  3. Tighten the screw until finger tight, so that the pipette is secure.
  4. Push the silver meal plate to unlock the holders on both sides of the device.
  5. Push both arms towards the centres. Your g lass tube is now in the outer box containing the filament, but not yet through the filament.
  6. Loosen the left side tightening screw. It should be still tight enough that when you push on the glass tube in the next step, you feel your thumb indent uncomfortably.
  7. Push the glass tube through the filament box gently to the other side.
  8. Tighten the tightening screw now on both arms.
  9. Close the hood.
  10. Turn the machine on, or else hit
  11. Type the number of the program you wish to edit, e.g. 56 and hit
  12. Hit
  13. Wait! If the program works, your glass tube will be automatically melted and pulled cyclically and then separated in two.
  14. Once the program cycles are done, open hood and exteact your two pipettes.

Pressure polishing#

To obtain a small patch pipette tip that is ideal for recording from small Drosophila neurons, it can be useful to ‘pressure polish’ the pipette tip with the CPM-2 microforge.  This also tends to increase the amount of glass surface available for sealing onto a neuron, and so can yield higher seal resistances (up to ~50 MOhms, versus 10-fold less for non-polished pipette). The technique of ‘pressure polishing’ was first developed by Goodman & Lockery for recording from C. elegans neurons. For Drosophila neurons it’s not essential, but it can be helpful, especially if you’re targeting particularly small cells and/or you need a tight seal. To do this:

  1. Insert your pipette onto the holder and tighten.
  2. Set microforge to POLISH on medium high.
  3. Using the 1.5X objective and looking through the eye pieces, place the filament at ~7 tick mark and the electrode at ~5 tick mark.
  4. Turn ON the air pressure (40-50 psi target) that runs thru the pipette tip. 
  5. While watching through the eye pieces touch the petal for a short period (1-3 seconds) to polish the tip to the desired size. Then move the pipette away from the filament.
  6. Turn OFF the air pressure (IMPORTANT for safety!!!!!) and then remove the pipette from the holder.

Note: Be sure to add a small bit of glass to the polishing filament prior to using. This is a suggestion in the manual which helps to avoid mysteriously bad seals due to particles of platinum / iridium flying off and landing on the end of the pipette causing poor seals.

Filament Maintenance#

Filament Replacement#

  1. Remove the chamber that encloses the heating filament (remove the black thumbscrew at the lower left corner of the cover place & pull the chamber straight off)
  2. Loosen the two clamp screws (D) that hold the filament in place and slide the filament out
  3. Slide in the new filament & center it over the air jet before retightening the two clamp screws
  4. The air jet should be 2-3 mm below the center of the filament, if this isn’t the case loosen the screw holding it in place and reposition it. 

Positioning the new filament#

  1. Slide the glass along the V-groove in the puller bar & look at it’s position relative to the filament
  2. Loosen the locking screws (A’ & B’)
  3. Turn the locking screw A on the vertical face to adjust the vertical position of the filament & the chrome screw B on the horizontal face to adjust the front-to-back position of the filament 
  4. Tighten the locking screws (A’ & B’)
  5. If the vertical range available with the vertical chrome screw (A) isn’t enough to center the glass you will need to reposition the upper and lower heater jaw assemblies by loosening the brass screws holding the jaws to the black nylon (D). Reposition the jaws & retighten the brass screws & reposition the air jet.

Testing the Position#

  1. Run a RAMP test with the new filament 
  2. Install a one line program: 
  3. Example program to test pipette lengths:
HEATPULLVELOCITYTIMEPRESSURE
ramp7070250500
  1. Pull a pair of pipettes using this program, remove them from the puller bars and hold them side by side to compare the length of the pipette tip shanks. If the shanks are not equal between the two this means the filament is not centered left to right relative to the air jet, one is being more cooled than the other. If this is the case then loosen the filament clamping screws and move the filament slightly towards the side that made the shorter pipette, tighten the clamps and try again.

Puller Maintenance#

Disposing of drierite#

  1. Drierite is a toxic chemical reagent (composed of Calcium sulfate >95%; Cobalt (II) chloride <2%, see safety spreadsheet) that:
  2. May cause an allergic skin reaction or serious eye irritation.
  3. May cause respiratory irritation, allergy or asthma symptoms, or breathing difficulties if inhaled.
  4. Suspected of causing genetic defects.
  5. May cause cancer by inhalation.
  6. May damage fertility.
  7. When replacing and/or disposing drierite from the puller airline, It must be handled under the chemical fume hood wearing personal protection equipment (gloves and mask).
  8. Drierite waste must be stored in the white waste container located in the satellite accumulation area in the fume hood (labeled as “Calcium sulfate >95%; Cobalt (II) chloride <2%”).
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