• Prepare workstation for slime collection.
  • Turn on and set microscope stage chiller to 10 degrees Celsius (prevents convective currents within the experimental chamber)
  • Fill a deep dissection pan with a small layer of ice. Place the Clove Oil Bucket on top of this layer and pile the ice up around the bucket. This ice will have to be periodically replaced as it is melted. A thermometer should also be placed in the aneasthesia bucket to monitor the temperature of the water (it should remain around 10 degrees Celcius)

• Collect and anaesthetize hagfish using the slime collection protocol.
• Place and line up the black pvc pipe on the stir plate.
• Thoroughly dry the experimental chamber with a kimwipe. Using 70% ethanol thoroughly clean both the inside and bottom of the experimental chamber. The bottom must be absolutely dry to insure ease of movement on the microscope stage.
• Place 10mL of 10:1 deionized water, poly-L-lysine solution in the chamber. After ten seconds pour back into holding beaker (this poly-L-lysine solution may be reused for one set of experimental trials only before it should be replaced)
• Add 75 mL of cold seawater (10 degrees Celcius) to the chamber, followed by the 2 inch stir bar.
  • Note: You do not want the seawater to warm up. So make sure the hagfish are completely anaesthetized before completing this step.

• Place the experimental chamber on top of (and lined up with) the pvc pipe.
• Bring the pipette guide down into position (marked on ring stand), and line up with the inside edge of the chamber.
• Turn the stir plate on to desired mixing rate.
• After thoroughly drying the area surrounding a number of slime gland pores stimulate the hagfish and use the P2 micropipette to collect 0.5 uL of fresh exudate. Note: The exudate should be collect from the interior of the puddle (it is imperative that the area from which the exudate is collected has not contacted the air)

• Note: If the slime is being collected from pores posterior to the anus they are best stimulated from the dorsal side of the pore. If using pores anterior to the anus they are best stimulated from the ventral side of the pore. A slight movement toward the pore (after the probe has contacted the skin) aids in slime deployment.

• Before removing the stir bar place the pipette partially into the guide. Turn off the stir plate at the same time the timer is started. Remove the stir bar quickly once it has stopped moving (the stir bar should be approached by the marked side) lower the pipette the rest of the way into the chamber once the five second timer is up. Eject the exudate into the chamber and hold the pipette in position for 5 seconds.
• Before removing the pipette tip from the water ensure that no threads are attached to the pipette tip. If so, use a pair of sharp scissors to cut the thread.
• Place the hagfish back into the anesthesia bucket after wiping any of the remaining exudate from the skin and thoroughly rinsing the area with deionized water.
• Carefully move the chamber to the microscope for viewing.

• If staining a few minutes may be required for the desired stain contrast to be achieved.