Whitefly Collection and Specimen Preparation
Aleyrodids are found on the aerial parts of a wide range of terrestrial
plants. They often inhabit
the underside of leaves, but sometimes can be found on the upper leaf
surface. Whiteflies have
six life stages: egg, crawler (1st instar), two sessile nymphal instars
(2nd and 3rd instars), the pupa
(4th instar) and adult. Identification of aleyrodids is largely based
upon characters found in the
pupal (4th instar) stage.
A hand-lens is useful when searching for and collecting whiteflies
in the field. Collect leaves
infested with whiteflies, wrap them in a paper towel to avoid excess
humidity and subsequent
fungal growth, place the sample in a zip-lock plastic bag, maintain
the sample refrigerated or in a
cooler until it is ready for slide-mounting. Identify the host plant
or collect a sample of the plant
sufficient for identification by an expert.
In the Laboratory
Examine the infested leaves under the microscope and select the largest
nymphs for specimen
preparation. Place a few drops of 10% potassium hydroxide (KOH) in
a small vial. Scrape off
several of the largest nymphs from the leaf surface and place them
into the vial. Sometimes it is
helpful to place a drop of KOH on the whitefly pupa to help loosen
it from the leaf surface.
Allow them to soak in KOH overnight. The process can be expedited by
heating the specimens
on low-heat on a hot plate for 10-20 minutes; however, the specimens
are more likely to be
damaged, especially if all of the KOH evaporates, and the specimens
are left on the hot plate.
Slide Mounting in Canada Balsam
1. Pour the specimens out of the vial into a small petri dish. If the specimens
are very dark brown or black, clear them in bleach for several minutes.
2. Gently press on the top of the whitefly pupa with a small spatula or
similar tool to extract the internal contents of the whitefly.
3. Place the pupae in stain for about 2 minutes.
4. Transfer specimens to 70-75% ethanol for 2-3 minutes. Do not use isopropyl
alcohol in this process.
5. Remove the excess stain from each pupa by gently pumping the upper surface.
6. Transfer specimens to 95% ethanol for 2-3 minutes.
7. Submerge the pupae in clove oil for several minutes. When the specimen
is first placed in the clove oil, its surface will shine as the clove oil
replaces the alcohol in and on the specimen. The specimen is ready for
slide mounting when it loses its shine.
8. Place a small drop (about 3/4 the diameter of the cover slip) of Canada
Balsam at the center of a clean microscope slide. Dilute the balsam with
a drop of xylene, if necessary.
9. Transfer the specimen from the clove oil and place it in the drop of
Canada Balsam. The specimen should be positioned so that it is facing upward
(the operculum is on the upper surface) and the head of the specimen faces
10. Place a coverslip on top of the specimen, apply gentle pressure to
the top of the coverslip until the balsam extends to the edge of the coverslip.
11. Label the slide with the following data: Locality (Country, State,
City, other), date of collection, collector and host plant, sample number,
mounting medium (Balsam). For example, the label on the left is the species
identification and the label on the right is the collection data:
24 VII 2002
G. A. Evans