3.3.3. Sensory organ preparation and mounting
No universal standard exists for preparing honey bee sensory organs for electroantennographic studies (Syntech, 2004). Various mounting techniques exist and the choice of technique to use depends on the objectives and experience of the experimenter. For recording bee antennal responses over a long period of time (over one hour), whole insect preparations should be the ideal choice (Syntech, 2004). For shorter durations lasting up to an hour, excised antennal preparations can be used. Both whole insect and excised antenna preparations can be carried out using saline-filled glass or probe electrodes (Torto et al., 2005, 2007b). To prepare bee antenna for an electrophysiological recording:
- Collect insects i.e. forager, guard or nurse bees at the hive entrance or comb using an aspirator (manual or automated) as required.
- Immobilise the collected bees by placing them on ice for about 1-2 min.
- Pick individual immobilised bees and insert each at the base of an Eppendorf tube (500 µl)
- Gently blow the bee towards the pipette tip whose apical portion has been cut-off to allow only the head of the bee to go through it in order to restrain it.
- Plug the base of the pipette tip with cotton wool or paper towel to prevent the insect from crawling back inside the restraining tube.
- Prepare the electrodes (saline-filled glass or gel
electrodes) to be used for mounting.
For saline electrodes, ensure that its drawn out tips have been cut to allow both antenna base and tip to fit through under a stereomicroscope. For gel electrodes, make sure that a sufficient amount of conducting gel is applied on the electrode tip.
- Mount the excised antenna between glass micropipette
electrodes on a micro-manipulator or probe electrodes covered with conducting
gel (Syntech, 2004) For whole insect preparations, push the recording electrode
into the distal end of the organ of interest (antennal tip) and the base or
ground electrode into a body part close to the terminal end of the organ (which
is often the eye for antennal preparations).
This should always be done under a stereomicroscope.
- Ensure that saline electrodes are free of air bubbles as they can interfere with a smooth recording of EAG.
- Make sure that the mounted antenna has its basal end connected to the ground/base/indifferent electrode while its apical portion is connected to the recording/different electrode (usually the one connected to the amplifier).
- Move either the antennal preparation closer to the source of the stimulus or the stimulus dispenser close to the mounting.
- When the amplifier light signal changes from red to green, it indicates that a complete circuit has been established and the mounting process is complete.