Mounting aphids (and other small arthopods) in Canada balsam
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.All steps should be performed in a fume hood.
- 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.
- Pierce the body wall with a sharp needle to allow easy access of reagents.
- Heat (95–99°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.
- 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.
- Transfer to chloralphenol (a 1:1 mix by weight of chloral hydrate and liquified phenol)
(very noxious, toxic fumes!: use a fume hood).
Leave at room temperature for 15 minutes to half an hour,
or, if your are in a hurry, heat for 2 to 5 minutes.
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.
- 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).
- Transfer to pure alpha-terpineol.
Leave for 5 to 15 minutes (depending on size of aphid).
- 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. ✎
- Dry for 2 weeks at 50°C.
Hint numbering corresponds to the steps in the above protocol.
All steps should be performed in a fume hood.
- 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.
- It is very difficult to puncture the aphid after maceration in KOH due to the increased flexibility of the cuticle.
- Lower concentrations of KOH (as low as 5 or 10%) may be used and the times extended correspondingly
(in fact, overnight in cold 5% KOH often gives acceptable results).
In general the higher concentration results in more complete clearing of embryos contained within the parent body,
and provided that the maceration time is kept short, there is no undue cuticular damage.
If you experience damage to wings or distortion of appendages before full clearing
has occurred, go to a lower concentration.
Do not clear so long that cuticular pigmentation patterns are lost.
We use 1.5 ml microcentrifuge tubes and a dry-block heater to heat specimens.
This gives good temperature control and minimizes volumes used.
This also facilitates the monitoring of the maceration process - you can easily
remove the tube from the block for a quick check under a binocular microscope.
For very small specimens we use 0.2 ml tubes in an old repurposed PCR thermocycler.
- DO NOT CAP small tubes, unless there is a locking mechanism (such as the locking lid of a thermocycler) to keep the cap in place under pressure.
The cap will tend to open explosively, dispersing the contents - a danger to both the specimen and the user.
If you have a locking method available, do not open tubes until they have cooled.
- Do not let the specimen boil.
It is difficult to get bubbles out of the specimen
and boiling reagents tend to deposit specimens on the side walls of the container.
- A longer series can be processed if, after heating, the tubes are put on ice (or program the heating block to go to a low temperature hold).
This will allow more handling time without the risk of over-maceration.
- It is always a good idea to add some cold water or ethanol to the hot KOH before
inserting tools into it to prevent sudden boiling of superheated KOH solution.
- We use a microspoon made from a wire flattened and bent at the end to transfer specimens.
Commercial versions of this are available (e.g. from BioQuip).
A fairly broad surface is useful for specimens with long legs; otherwise, the legs tend to wrap around the tool.
To get a specimen out of a narrow tube (or to find minute specimens),
it may be easier to flush the contents into a wider container with a pipette or submerge the entire tube in the rinse container.
- If you are in a hurry use 70% ethanol for 5 or 10 minutes, then a few changes of water.
- You can leave specimens in chloralphenol overnight if refrigerated.
- If the specimen does not become clear after an hour in chloralphenol,
transfer to 70% ethanol to remove the choralphenol, then repeat the KOH treatment for a short time.
- We perform this and the following steps using a few drops of the reagent in a depression plate.
You need only enough to cover the specimen.
- 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.
- 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.
- As above, rearrange the appendages after transfer.
- The balsam should be fairly thin so that it flows easily around the specimen
and spreads under the coverslip, but not so thin that it cannot fill the gap
between the slide and coverslip over a large specimen.
If the balsam is slightly on the thick side, spread a layer of terpineol over the
centre of the slide before applying the balsam; this facilitates flow over the glass.
- The specimen should be mounted dorsal side up with the appendages spread (forelegs
forward, midlegs and hindlegs back, according to the natural rest position of the
When arranging the specimen, take into consideration the characters you will need
to see; if you are mounting a series from the same collection, some variation in arrangement may be useful
(for example, pull the rostrum to one side in some to permit observation of rostral characters,
but centre it on others so the the specimen
does not tend to rotate when the cover slip is applied).
- Use a fine needle to reposition appendages.
Try to take advantage of the natural movement patterns of the appendage.
Use the viscosity of the medium to move wings and fine body parts rather than
directly manipulating them.
If a specimen is stubborn, give up; you are likely to do more damage than good if
- It is sometimes useful to precrush the highly arched thorax of some winged forms along the centre line with a needle;
this reduces the tendency of the specimen to roll onto its side when the coverslip is applied.
- Final arrangement of appendages can be achieved with judiciously applied gentle pressure on the cover slip.
- The final mount should be relatively thin to facilitate observation of ventral characters
even with the short working distance available under high power,
but not so strongly compressed that ventral and dorsal surface approach each other
so closely that characters on the respective surface are difficult to differentiate.
- We do not recomend mounting more than one aphid on a slide.
A collection may prove to contain more than one species, and multiple
specimens tend to be difficult to keep in place when the cover slip is applied.
If it is desired to do so (for example to associate copulating pairs
or parent and offspring) either mount under separate small cover slips
or arrange the specimens in a thin layer of balsam,
allow it to dry partially (under a dust cover!) to fix them in position,
then add more balsam and apply the coverslip.
- 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.
- For drying a large number of slides, use a vented laboratory dying oven, or a food or jerky drier.
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 2017-09-19
R.G. Foottit and E. Maw