Mounting aphids (and other small arthopods) in Canada balsam

Go here for Hoyers gum mounts

The basic requirements for a good mount are that the body contents, including all muscle and fat tissue be removed, and the water replaced with an organic solvent miscible with Canada balsam. The following is but one method for achieving this goal, but has proved relatively quick, consistent and easy to apply.

Go here for hints
  1. Heat the specimen in 95% ethanol for 5 to 10 minutes. This step is not required for material previously preserved in ethanol for a month or more.
  2. Pierce the body wall with a sharp needle to allow easy access of reagents.
  3. Heat (near 100°C) in 40% potassium hydroxide for 2 to 5 minutes, until the body contents are liquified. The duration depends on the size of the aphid, prior preservation history (length and medium) among other factors.
  4. Transfer to a large volume of distilled water. If the potassium hydroxide has done its job, you should see brownish fluid stream from the body through the needle hole, anus and genital opening. Leave in water for an hour or more. We prefer overnight to give consistent results with larger aphids. Change the water at least once during this time. If you are in a hurry use 70% ethanol for 5 or 10 minutes, then a few changes of water.
  5. Tranfer to chloralphenol (a 1:1 mix by weight of chloral hydrate and liquified phenol). Leave at room temperature for 15 minutes to half an hour, or, if your are in a hurry, heat for 2 to 5 minutes (very noxious fumes!: use a fume hood). Any residual fats and oils should disappear and the specimen should be quite transparent. Lactophenol (1:1 lactic acid and phenol) can be substituted for chloralphenol if obtaining chloral hydrate is a problem, but sometimes gives inferior results.
  6. Transfer to a mixture (1:1) of acetic acid and alpha-terpineol (oil of lilac). Leave for 5 to 15 minutes (depending on size of aphid).
  7. Transfer to pure alpha-terpineol. Leave for 5 to 15 minutes (depending on size of aphid).
  8. Place a drop of balsam on the center of a microscope slide. Place the aphid in the drop, arrange the appendages, and gently lower a cover slip over the specimen.
  9. Dry for 2 weeks at 50°C.

Hints

Hint numbering corresponds to the steps in the above protocol.
  1. Specimens preserved in formalin-containing fluids and some other preservatives may require longer treatment with ethanol and generally give inferior results. Specimens which have dried up should be heated in water for several hours.

  2. It is very difficult to puncture the aphid after maceration in KOH due to the increased flexibility of the cuticle.

  3. The specimen begins to harden and become more brittle in this and the following step. Do not leave in this reagent too long, especially if the specimen is already fragile. It is advised that the appendages be arranged more or less in their final position to facilitate the final mounting.

  4. As above, rearrange the appendages after transfer.

  5. Check the slides after a day of drying. The balsam may withdraw as it dries if the specimen is large. Apply a drop of balsam to the edge of the coverslip to replenish it.
When tranferring the specimen from one reagent to the next, we often pass the specimen through a drop of the second reagent to remove most of the previous reagent from the surface. This reduces reagent carry over and increases number of specimens we can process with a given volume of reagent. However, this is not recommended for very delicate specimens (such as those with long legs), due to the extra handling involved.

Hoyer's gum mounts

Follow the above procedure as far as step 5 (chloralphenol). Transfer directly to a drop of Hoyer's mount on a slide and apply the cover slip.

last update 2014-06-27

R.G. Foottit and E. Maw