A Method for Mounting Small Insects on the container.

This solution may be prepared any time

Microscope Slides in Canada Balsamx before needed,but it turns slightly dark with age; only
fresh solution should be used in preparing the phenol-
balsam mixture.
WILLIS W. WIRTIt AND NORMAN MARSTON 3
2. A bottle of phenol-balsammixture, preparedby mix-
ing eqnal parts (v:v) of liquified phenol and Canada
Traditional methods for mounting insects in Canada
balsam have often proven unsatisfactory because of balsam of the consistencynormally used for making
shrinkage of delicate structures such as the antennae and slides or a little thicker. This mixture must be pre-
pared at least a day in advance of use and no more than
palpi in the highly viscous mounting medium. Clearing
in potassium hydroxide and subsequentgradual dehydra- 3 veeksahead, since it turns dark with age.
tion in a series of alcoholsare very time consumingand 3. Canada balsam for replenishingthe phenol-balsam
mixture in mounts as they are dried in the oven.
often require isolation of individual specimens,and sep-
arate treatment of the wings when adults are being 4. (For Procedure2 only.) Ten percentpotassium
hy-
mounted. While satisfactory mounts may easily be made droxide preparedby placing 1 part of a saturatedsolution
of relatively large and heavily sclerotized insects,mounts of potassiumhydroxide and 9 parts (v:v) of distilled
water in a Stender dish or vial.
of fragile insectsare often inadequatefor detailed taxon-
omic study. This problem has led many workers to try PROCEDURE1. ItOUNTING SPECIMENS COLLECTEDIN ALCoHoL
other mounting media, but none has proven entirely
satisfactory. Sinceinsectscollectedin alcoholdie ;v{th their append-
Carter et al. (1920) and Lutz (1922) long ago called ages fully expandedand relatively relaxed, they may be
attention to the advantagesof dehydration and clearing mounteddirectlyinto a phenol-balsam mixtureafter being
insectsin carbolicacid (phenol). A methodusingphenol- cleared and alehydrated{n phenol. (Dry-collected or
balsam for mounting specimensof Ceratopogonidaeand heavily sclerotizedspecimensalso may be mounteddi-
Chironomidae(Diptera) with little or no shrinkage has rectly from phenol,althoughsuchmountsare generally
been developedby the senior author (1961) and adapted lesssatisfactoryfor detailedtaxonom{cstudyand may be
for mounting Cecidomyiidae by the junior author. The mountedaccordingto Procedure2.)
a. Place liquefied phenol in a Stender dish or vial and
specimensare placed in an alcoholic solution of phenol
for dehydrating, clearing, and relaxing which allows transfer to it the specimensto be mounted. As many
them to be mounted in a phenol-balsam mixture. The specimens as desiredmay be placedin eachdishor vial up
phenol then evaporatesor polymerizesduring the drying to of the volumeof the solution. The specimens should
process,allowing a gradual transition into a permanent be left in the solutionfor 1-24 hr dependingon the length
balsam mixture. Additional major adavntages of this of time necessaryto relax and clear them. It may be
method are that the chitin remains elastic and easy to necessary to placeunusuallyfragile specimensin a phenol-
dissect,hairs and scalesare not easily detached,wings 70% alcoholmixture for about5 rain beforeplacingthem
do not have to be treated separately, and as a result a in pure phenolto preventshrinkage. Specimens may be
large nmnber of specimensmay be handled at 1 time or left in the phenol indefinitely;if the phenolcrystallizes,
add 100% alcohol.
held indefinitelyin storage during the process.
b. Place a drop of phenol-balsammixture on a micro-
SOLUTIONS REQUIRED scopeslide. Transfer the specimenfrom the phenolsolu-
1. Liqu{fiedphenol,preparedby diluting pure phenol tion to the slide. Dissect and arrange the specimenon
(carbolicacid) crystalswith enoughabsolute (i00%) the slide as requiredfor studyin eachgroupof insects.
ethyl alcoholto form a saturatedsolution,with a layer of For femalesof Ceratopogonidae and Chironomidae,cut
about by volume of phenol crystals in the bottom of off the headwith dissectingneedlesand orient the face up-
ward. Cut off 1 wing and the abdomen,orientingthe lat-
ter ventral side upward. For males, cut off 1 suingand
Accepted for publication December 14, 1967.
* ResearchEntomologist,EntomologyResearchDivision, ARS, the head and orient as for the female; separatethe ab-
U.S. Department of Agriculture, Washington, D.C. domen just ahead of the genital segmentsand orient the
aResearch Entomologist, Biological Control of Insects Re- latter ventral side upward.
searchLaboratory,ARS, U.S. Department of Agriculture, Colum-
Bia, Missouri. For Cecidomyiidae,
removeboth suings,the head, and
784 ANNALS OFTHE ENTOMOLOGICAL
SOCIETYOFAMERICA [Vol. 61, No. 3

the genitalia (if a male). Place the vings at the upper
right, the head at the upper left with the face upward, and
the body at the bottom with the legs inward. Male geni-
talia should be placed in the upper half of the slide with
either side upward.
A small fragment of broken cover slip shouldbe placed
beside the thorax to prevent flattening and crushing the
specimenas the mixture contracts, through capillarity,
during drying. The wings or other structuresmay be flat~
tened under a separate cover slip if desired. Place only
I specimenon each slide.
Place the cover slip on the specimen. Circular cover
slips of 18 mm diam are most satisfactoryfor Ceratopo-
gonidae and Chironomidae, 15 mm diam for Cecidomyi-
idae. Cover slips shouldbe rested with 1 edge on the slide
to the side of the droplet and sloxvly lowered onto the
specimen,taking care that the small parts are not drifted
out of position. Drifting can be forestailed by adding
phenol-balsammixture to that side of the cover slip with
a pipette. The mount should taper in thicknesswith the
head, xving,etc. on the thin side and the main body on
the thick side of the mount.
For delicate specimensit may be necessaryto dilute
the phenol-balsammixture with additional phenol. In
extreme cases the specimenmay be mounted in pure
phenol to prevent shrinkage, in which case it may be
necessaryto add additional100% alcohol to the phenolto
prevent crystallization as the mount is prepared. Phenol-
balsammixture may then be addedalongthe edgeof the
cover slip and will gradually replacethe phenolas the
slide dries.
c. Label the slideand placeit in a drying ovenat about
135F for 1-2 weeks, or until the balsam has hardened
sufficientlyto prevent slippageunder moderate lateral
pressure. Replace the evaporatedmixture with pure
balsamor phenol-balsam mixtureeveryday or two until
no further evaporationtakesplace. Carter, H. F., A. Ingram, and J. W. S. Macfie. 1920.
PROCEDURE2. 5rOUNTING SPECIMENS COLLECTEDDRY
Observationson the ceratopogoninemidges of the
Gold Coast, with descriptionsof new species. Part
In all groups
of insects
it is oftendesirable
to mount
specimenswhich have been preserveddry or placed in
alcoholafter desiccation.Specimenswith a rigid exo-
skeletonmaybe mountedusingtheinethodjust described, Wirth, W.W. 1961. Instructionsfor preparingslidesof
but thosespecies
whichcollapse
upondryingmayrequire Ceratopogonidae and Chironomidae. Studia Entomol.
treatment with potassiumhydroxide to restore them to a
suitable condition. Potash treatment also may be nec-
essary to macerate and give better visibility to species
with opaque, thick, or dark-colored integument. Since
the musclesand other internal tissues are well preserved
(though cleared) in specimensmounted by Procedure 1,
it may be necessary to treat them also with potassium
hydroxide if the scleroized internal structures are to be
studied or if the tissues interfere w-ith phase-contrast
microscope examination.
a. Place the specimensin a solution of 10% potassimn
hydroxide preheated to 200F for 5-10 min. It is very
important not to leave xvingedspecimensin the solution
too long, since the wings will lose their rigidity and will
wrinkle during the mounting procedure. Sometimes it
may even be necessary to dissect off the wings and
mount them separately before treating the specimenwith
potash.
b. Transfer the specimensfrom the potassiumhydroxide
solution to 70%-75% alcohol. In about 30 min the in-
ternal pressureof the specimenswill cause the abdominal
segments,head, and appendagesto expand beyond their
normal extended positions. This feature is desirable with
normally telescoped structures such as the female ovi-
positor.
c. Transfer the specimensto a solution of phenol anti
70% alcohol for about 10 min and then into phenol-100%
alcohol solution. They should not be left in phenol for
more than 3 or 4 hr, sincethe phenol vill causethe wings
to becomeadhesive,making mounting extremely difficult.
d. Arrange the specimenin a drop oi pure phenol as
describedunder Procedure 1. Apply the cover slip, label.
and dry, using phenol-balsam mixture rather than balsam
to replenish the evaporating phenol.
REFERENCES CITED
I. Ann. Trop. Med. Parasitol. 14: 187-210.
Lutz, A. 1922. Contribution aux methods d'observa-
tions microscopiques de biologiques. Ann. Biol.
Lacustre 11: 90-102.
4: 553-4.

Reprinted [rom the

ANN.L$ OF THE ENTOMOLOGICALSOCIETY OF AMERICA
Volume 61, Number 3, pp. 783-784, May 1968