Techniques for Preparing
Fleas for Identification
Fleas can be processed by two methods: serial alcohol solutions
and xylene, or cellosolve only. The use of cellosolve is not
a permanent means of preservation, whereas the use of serial
alcohol’s and xylene produce mounts that will last for
many decades. The former requires less preparation time and
provides an adequate specimen for routine identification.
Fleas are usually preserved in 70-80% ethanol, thus they are
soft, pliable, and ready for standard mounting procedures.
Avoid using isopropyl, methyl, or higher concentrations of
ethyl alcohol that will cause specimens to become brittle.
Such preparations may be inadequate or impossible for preparing
a suitable mounted specimen.
Serial Alcohol and Xylene Mounting
1. Fleas must be cleared of all soft tissues. This is best
accomplished with a 10% solution of potassium hydroxide (KOH).
Although there are other methods, the author mixes a saturated
solution of KOH (stock solution is good for years) and add
three (3) drops (standard disposable Pasture pipette) of stock
solution to 0.75 ml distilled water in a 1.5 ml screw-cap
vial. These small vials save material and space compared to
using standard stender dishes. Using more than three drops
of KOH is unnecessary and will result in destruction of delicate
surface sculpturing and may make the edges of sclerites thin
and difficult to see. Place the flea (in alcohol) in a petri
dish of hardened paraffin wax. Puncture the flea in the right
side approximately between the first or second abdominal segments
(penetration of the pin is permitted by the wax substrate)
with a minuten nadeln mounted on an applicator stick. The
hole is necessary to allow KOH to enter the flea. Allow poked
specimens to remain in the KOH solution for 24 hours. Small
or light colored fleas may require less, while very large
or darkly pigmented specimens, or those engorged with blood
may require longer. Internal contents should be completely
liquefied before attempting evacuation.
2. After ca. 24 hours (+/-), place the specimens in distilled
water (a few drops of 70% ethanol may aid in breaking the
surface tension if fleas will not “sink”). With
the flea on its left side, carefully evacuate the liquefied
tissues in the abdomen by placing a minuten between the thorax
and abdomen and applying pressure with a minute spatula (home
made) until the contents are expulsed. Occasionally all the
contents are not evacuated on the first “squeeze”.
Allow the flea to remain in the distilled water for a few
minutes (while squeezing other fleas) to refill with water
(by osmosis) and re-squeeze repeatedly as necessary. Great
care should be taken to avoid damaging setae and other surface
3. Transfer the “cleared” flea through serial
solutions of ethanol (70%, 80%, 95%, and 100%) for 30 minutes
each (minimum). Some advocate using acidulated water to neutralize
KOH crystals, however, this “fixes” remnant membranous
tissues which may become brown and obstructive. Material that
has been mounted in excess of 30 years that were not treated
with acidulated water, or other rinsate show no sign of KOH
crystallization, or over-clearing with time.
4. Transfer to oil-of-wintergreen (methyl salicylate), creosote,
or clove oil for a maximum of 20 minutes (longer will make
the specimens very brittle resulting in loss of setae and
even appendages). This step renders the chitin more translucent,
permitting better observation of structures.
5. Transfer to xylene for one hour (minimum, do not leave
overnight). If desired, dissections are best conducted in
6. Mount in Canada balsam. Do not use synthetic resins.
Use round or square cover slips (round are best), 12-15 mm,
No. 1 thickness or less (0, or 00).
7. Orient specimens with head to the right and legs away
from technician. Spread legs so as many segments are visible
8. Place specimens in drying oven (37-41°C) for 4-6
months for complete curing of Canada balsam. Even the largest
(thickest) fleas will be cured after 6 months.
9. Label cured slides as follows:
|Det: J.Q. Doe
1 SEP or IX
Record localities as: City, Political Subdivision (county,
providence, department), State or Country, and use standard
grid coordinates when possible. When recording dates the month
should always be written out or specified as a Roman Numeral,
e.g., SEP or IX and also the year should be written out as
1999, not ’99.
Cellosolve Mounting Procedure
1. After completing steps 1. and 2. above, transfer specimens
directly to cellosolve and soak for a minimum of 30 minutes.
Steps 1 and 2 may be omitted, but many morphological features
will be obstructed and difficult to see in final preparation.
2. Mount fleas directly from cellosolve into Canada balsam