Slime Collection Protocol – Myxine glutinosa

Materials:

• 3.5 L fresh, artificial seawater, 10 degrees
• clove oil anaesthetic stock (1 part clove oil: 9 parts anhydrous ethanol)
• bucket of ice
• squirt bottle of dH₂O
• 100 mL beaker of dH₂O
• narrow spatula for scooping slime
• vial(s) of stabilization buffer on ice
• paper towels
• Kimwipes
• Large dissection tray filled with ice
• Small dissection tray lined with clean J-cloths soaked in s.w., wrung out
• Electrical stimulator and stimulator wand
• Plastic bag with ice for regulating water temperature

Procedure:

• Add 1.75 mL of clove oil stock solution (1:9 co:ethanol) and mix
• Transfer to white bucket for trip to Aqualab
• Bring a spare bucket for recovery water
• Rinse dipnet thoroughly (*very important*), collect hagfish as gently as possible
• Transfer hagfish into anaesthesia bucket, close lid, note time
• Collect a couple of litres of water from hagfish tank into recovery bucket
• Bring hagfish and recovery water back to lab
• Set up dissection trays

Make sure stimulator settings are correct: Frequency: 60 Hz, Delay: n/a, Duration: 0.8 ms, Volts: 18 V, Mode: repeat, Output: bi *NOT DC or mono*

• Hagfish should be knocked out after 10 minutes or so in bucket
• Once hagfish stops moving, wait another couple of min. to ensure complete anaesthesia
• Place hagfish in chilled tray, ventral side up
• Rinse area where slime will be collected
• Blot dry with Kimwipes
• Press firmly with stimulator wand at ventral midline between opposite slime glands
• Remove wand before exudate contacts it
• Scoop up exudate with spatula, transfer to stabilization buffer, twirl to disperse

Wipe off spatula, rinse in dH₂O, wipe again to dry *This is important to ensure that stabiliation buffer does not contact the hagfish’s skin*

• When finished with slime collection, return hagfish to recovery bucket, massage skin with gloved hand to remove any adherent slime. Return to Aqualab ASAP.