Flea Mounting Procedures

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 Procedure

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 structures.

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 xylene.

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 as possible.

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:

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.