Preparation of Mammalian Teeth for Age Determination by Cementum Layers: A Review

Author(s): S. G. Fancy
Source: Wildlife Society Bulletin, Vol. 8, No. 3 (Autumn, 1980), pp. 242-248
Published by: Wiley on behalf of the Wildlife Society
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 PREPARATION OF MAMMALIANTEETH FOR AGE
DETERMINATION BY CEMENTUM LAYERS: A REVIEW
S. G. FANCY, LGL Alaska Research Associates,P.O. Box 80607, Fairbanks, AK 99708

Abstract: Age determinationof mammals fromtooth cementum layer counts is widely used as
a management technique. Standard methods used by researchers to prepare dental material for
cementum layer analysis are reviewed. Choice of tooth and plane of sectioning forcarnivores
and ungulates is also discussed. The accuracy of aging mammals by counting tooth cementum
layers can be improved if standardized methods and reference collections are established for
each species and fordifferentpopulations. Recommendations are made forresearch needed to
improve the accuracy of this technique.

The tooth annulation method is the
most accurate means of age determina-
tion formany mammalian species (Thom-
as 1977). Over 40 species of mammals
have been aged by this method since
Laws (1952) firstshowed that internal
dentinal and cemental annulations were
correlated with age. Klevezal and Klei-
nenberg (1967) gave an excellent review
of age-related stratificationin the struc-
ture of teeth and bones, and Morris
(1972) gave a general review of mamma-
lian age determaination methods. How-
ever, there have been many recent im-
provements in the techniques used to
prepare dental tissues for age determi-
nation, particularly with staining meth-
ods. In this paper I review some of the
standard methods and improved tech-
niques used by researchers to obtain
sharp, easily interpreted annulations. A
great deal has been published on this
topic, and minorvariations in toothprep-
aration methods will not be discussed.
Most earlier studies, particularlythose
dealing with marine mammals, con-
cerned growthlayers in dentine. Dentine
is deposited within the tooth,and is thus
spatially limited in its growth (Morris
1972). Dentinal increments tend to get
smaller and more difficultto count with
age (Mitchell 1967), and growth of den-
tine ceases in most mammals soon after
242
the animals reach maturity(Harris 1978).
Dentinal layers have been useful in
aging many species, including many ma-
rine mammals and carnivores. Lord et al.
(1976) found dentine growth lines to be
clearer and more easily read than cemen-
tum growthlines in determiningthe age
of vampire bats (Desmodus rotundus).
This paper is limited to a discussion of
cementum layers, which are used more
commonly with terrestrial mammals.
Layering in cementum is less confused
by accessory bands than is layering in
dentine (Klevezal and Kleinenberg 1967).
The techniques used to prepare teeth
for age determination by cementum an-
nuli counts result in 3 major types of sec-
tions: (1) histologicallyprepared sections
cut with a microtomeor razor blade after
decalcification of the tooth, (2) transpar-
ent sections cut with a fine saw and
ground thin enough to be observed with
transmitted light, and (3) sawed and
ground,or ground sections observed with
reflectedlight.
HISTOLOGICALLY PREPARED
SECTIONS
Histological procedures produce the
most consistent results, but may require
more time and effortthan the othermeth-
ods, particularlywith larger teeth. Teeth

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 PREPARING MAMMALIAN TEETH FOR AGE DETERMINATION * Fancy 243

rangingin size frombatincisorsto molars followingdecalcificationis necessaryto

of large ungulateshave been processed ensuregood stainingreactions.This may
byhistologicalprocedures.Althoughsev- be done by rinsingtheteethin tap water,
eral sectioning methods will be dis- or by soakingin a lithiumcarbonateso-
cussed, all methods require extraction lution(Low and Cowan 1963).
and decalcification
ofthe tooth.Low and
Cowan (1963) thoughtthat boiling the
mandibleto extractteethwould alterthe Razor Sectioning
staining properties,but Turner (1977) Razorsectioninghas been used on red
foundno significant loss in annulardefi- fox(Vulpes
fulva) canines (Allen 1974)
nitionbetweenincisorsextractedbyboil- and graysquirrelmolars
(Fogl and Mos-
ing and those extractedby soakingthe by 1978). Althoughrazor
sectioningis a
mandibles. veryrapid process,sectionsmay be un-
even or slanted, making photomicro-
Decalcifying Teeth graphsalmostimpossibleto obtain(Fogl
Formicacid or a formicacid/formalinand Mosby 1978).
decalcifyingsolutionhas been used by
mostresearchers.However,Conley and
Jenkins(1969) did notfindit to be com- Microtome Sectioning
pletely effective.Miller (1974) experi- The standardparaffin techniqueusing
mentedwithvariousdecalcifyingagents the rotarymicrotomewas described in
on caribou (Rangifer tarandus) teeth, detailby Low and Cowan (1963),and was
and found that formicacid resulted in used withslightmodification by Adams
less clarity in the annulations, while and Watkins(1967) and Thomasand Ban-
formic acid/sodium citrate solutions dy (1973). The freezingmicrotomesec-
proved too costly and time-consuming tioningmethodis morerapidthanthero-
and resultedin poor sections.Millerrec- tarymicrotome procedure,and eliminates
ommendeda 5% solutionof 67% nitric dehydrationof the tissue. Miller (1974)
acid, which has been used by otherre- thought thatmuchoftheseparationofthe
searcherswith good results(Grue and cementallayerfromthe dentinethathe
Jensen 1973, Jensenand Nielsen 1968, encounteredoccurredas the resultofde-
Lockard1972,Marksand Erickson1966, hydration.Frozen sectionsmustbe han-
Willey 1974). Turner(1977) foundnitric dled individually,whereas sectionsob-
acid unsatisfactorybecause it obliterated tained by the paraffintechnique are
otherwisedistinctannuli.Fogl and Mos- attachedto each otherin ribbons,making
by (1978) preferred a formicacid/sodium it easier to obtain serial sections of a
citratesolutionto nitricacid due to short- tooth.Frozen tissue sectionshave been
er decalcificationtimes and bettersec- made fromteeth of black bear (Ursus
tioningresultson graysquirrel(Sciurus americanus) (Sauer et al. 1966, Willey
carolinensis)molars.Other decalcifying 1974), European red fox (Vulpes vulpes)
agentsused withsuccess include5% HCI (Harris 1978), coyote (Canis latrans)
(Novakowski1965),commercialRDO de- (Linhart and Knowlton 1967, Roberts
calcifyingsolution(Croweand Strickland 1978), bobcat (Lynx rufus) (Crowe
1975),and Hartman-Ledden fluid(Adams 1972), vampire bats (Linhart 1973), and
and Watkins1967). Neutralizingthe acid caribou (McEwan 1963, Miller 1974).

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244 Wildl.Soc. Bull. 8(3) 1980
Staining
Staining tooth sections after affixing
them to slides provides better contrast
between the light and dark cementum
layers. Hematoxylin has been the most
common stain used, but other stains may
be superior forcementum layer analysis.
Thomas (1977) experimented with 20
metachromaticdyes on teeth of 10 differ-
ent species, and ranked toluidine blue,
crystalviolet, cresyl violet, Ehrlich's he-
matoxylin,and methylene blue as the 5
best dyes for cementum layer analysis.
The advantages of metachromatic dyes
over hematoxylinincluded ease of prep-
aration, longer shelf life, better delinia-
tion of rest lines in a color that contrasts
with the background cementum, more
consistent staining reactions between
batches, and lower cost.
Stone et al. (1975) found that sections
stained with Giemsa, Field, and Maxi-
mow solutions were superior to sections
stained with hematoxylin and eosin.
Giemsa stain is used by G. Matson's com-
mercial microtechnique company (Box
308, Milltown, MT 59851) for section
analysis. Matson found thatgreaterstain-
ing variation existed among individual
teeth than among differentbatches when
using Giemsa stain (G. Matson, pers.
commun.). Paragon multiple stain has
been used successfully by Linhart and
Knowlton (1967), Crowe (1972), Linhart
(1973), Allen (1974), Turner (1977), and
Roberts (1978). While Paragon stain is no
longer commercially available, its main
component is toluidine blue.
Interpreting the stained sections is
usually done with a light microscope, al-
though Lockard (1972) recommended a
microprojector.Low and Cowan (1963)
found that polarized light gave better
contrast than transmitted light when
viewing sections. Interpretationis easi-
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est while the sections are still wet be-
cause the sharpness of the opaque rest
lines and the color contrastbetween rest
lines and background cementum are
often impaired by dehydration to per-
manent mounts (Thomas 1977).
GROUND SECTIONS VIEWED
WITH TRANSMITTED LIGHT
Saw and grind techniques have been
used mainly with ungulate and grizzly
bear (Ursus arctos) teeth. Transparent
sections are obtained by firstsawing the
tooth with a circularjeweler's saw made
of carbon steel (Fisher and MacKenzie
1954, Sargeant and Pimlott 1959), dental
diamond wheel (Hemming 1969), Niclas
bone and tooth cutting machine (Arm-
strong 1965, Mundy and Fuller 1964), or
other thin-sectioningsaw (Erickson and
Seliger 1969).
The sawed sections are then ground
using the technique outlined by Frost
(1958), or with a fine carborundumstone.
Gasaway et al. (1978) obtained transpar-
ent sections of moose (Alces alces) inci-
sors by grinding alternate sides with a
water-bathgrinderequipped with a fine,
abrasive wheel. Fisher and MacKenzie
(1954) designed a special grinding ma-
chine, but McEwan (1963) had poor re-
sults with this technique. Craighead et al.
(1970) softened the ground sections of
grizzly bear teeth with nitric acid, then
stained the sections with hematoxylin
and eosin. Erickson and Seliger (1969)
described a rapid, simple technique that
required no polishing, using hematoxylin
and eosin to stain the sections.
Hemming (1969) embedded Dall sheep
(Ovis dalli) teeth in plaster of paris to fa-
cilitatehandling and observed the ground
sections with a fluoroscope, thus elimi-
nating decalcification and staining. Sar-
geant and Pimlott(1959) and Mundy and
 PREPARING MAMMALIAN TEETH FOR AGE DETERMINATION * Fancy
Fuller (1964) observed unstained sec-
tions under a microscope.
GROUND SECTIONS VIEWED
WITH REFLECTED LIGHT
Cementum layers observed with re-
flected light have been exposed by saw-
ing and grinding teeth or by grinding
alone. Novakowski (1965) and Douglas
(1970) used a bench grinderon ungulate
teeth to obtain cementum layer counts.
The ground surface was polished by
hand, and decalcified in 5% HCl for2 h.
Differential decalcification of opaque
and transparent bands produced ridges
that could be seen under a microscope.
Wolfe (1969) used a diamond lapidary
saw to cut sections thatwere then ground
smooth with abrasive paper. A thin coat
of oil or nail polish was applied to the
ground surface to aid interpretation.The
grindingprocedure was repeated several
times until the maximum number of dark
lines was exposed. Mitchell (1963) cut
teeth with a fine hacksaw, polished the
cut surface with a fine carborundum
stone, and examined the surface,dry,un-
der a dissecting scope. White-tailed deer
(Odocoileus virginianus)teeth fromar-
cheological sites have been aged using
the cut and grind procedures outlined by
Ransom (1966) following vacuum im-
pregnation of cavities in the teeth with
epoxide (Kay 1974).
SELECTION OF TOOTH AND
SECTIONING PLANE
There has been much debate on which
tooth and plane of sectioning gives the
best results forcertain species. Although
many of these argumentsmay apply only
to a particular species, several generali-
zations can be made.
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245
Carnivores
The best teeth for aging are those in
which the layers are wide enough to be
easily counted under magnification,but
not so wide that numerous supplemen-
tary streaks are seen within them (KIev-
ezal and Kleinenberg 1967). Canine teeth
have been used by many researchers for
aging carnivores.The apex of the roothas
the thickest cementum and is the best
area for counting annuli (Linhart and
Knowlton 1967). Roberts (1978) recom-
mended using canine teeth foraging coy-
otes after comparing longitudinal sec-
tions of the canines, premolars, and
incisors. Linhart and Knowlton (1967)
found longitudinal sections of coyote ca-
nines preferable to transverse sections
because layeringis more distinctin some
areas. Conley and Jenkins(1969) report-
ed that longitudinal sections of bobcat
canines were superior to transverse sec-
tions. Cross-sections are easier to cut,
and more sections can be taken froma
single tooth (Morris 1972). Transverse
sections expose the layer around the en-
tire circumference of the root; however,
where cementum is deposited irregularly,
some transversesections may be mislead-
ing. Harris (1978) found that transverse
canine sections of European red foxwere
easier to read than longitudinal sections,
although sagittalsections ofthe premolars
were easiest to interpret.Annuli in canine
sections tend to split intosubsidiarylines,
making interpretationdifficult;premolar
annuli were clear, uniformlyspaced, and
did not show any subsidiary lines. Mon-
son et al. (1973) also found fox cheek
teeth to be superior to canines. The first
premolar is used by most researchers to
age black and grizzly bears, and is as
reliable as canines forage determination
(Craighead et al. 1970, Willey 1974).
246 Wildl.Soc. Bull. 8(3) 1980
Ungulates
Cementum layers in both incisiform
and molariformteeth have been used to
age ungulates. The cementum complex
of molariformteeth is generally thicker
than that of incisors, thus facilitatingthe
interpretation of growth layers (Wolfe
1969). Counts fromthe enlarged cemen-
tum pad between the roots of molariform
teeth are often more reliable than those
from other portions of the cementum
complex (Wolfe 1969). Wolfe also found
thatgrowthlayers of maxillaryteeth were
more regular and distinct than those of
mandibular teeth. Most ungulate re-
searchers favor a sagittal section of mo-
lariform teeth (Miller 1974); however,
Wolfe (1969) got the best results when he
cut teeth on a slightly oblique plane to
the medium axis in the jaw.
The first incisor of ungulates is the
most commonlyused incisiformtoothbe-
cause it is the largest, the firstto erupt,
and is easy to remove and decalcify (Gil-
bert 1966, Sargeant and Pimlott 1959).
Hemming (1969) found sagittal sections
of the entire root most satisfactorywith
Dall sheep incisors.
ACCURACY
The interpretationof cementum layers
is a subjective process, and trained ob-
serversoftendisagree on the age of a par-
ticular section (Gasaway 1978, Monson et
al. 1973, Nellis et al. 1978, Utsler 1972).
Many researchers have reported com-
plete agreement between age estimates
from cementum layer analysis and the
known ages of the animal (Erickson and
Seliger 1969, Keiss 1969, Kleinenberg
and Klevezal 1966, Lockard 1972, Low
and Cowan 1963). Others have foundthat
the cementum layer counts differedfrom
the known age of the animal formany of
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their specimens (Gasaway et al. 1978,
Monson et al. 1973, Ransom 1966, Sauer
1973, Sargeant and Pimlott 1959, Wolfe
1969).
The accuracy of the cementum layer
techinque depends largely on the meth-
ods used to prepare the material. Fogl
and Mosby (1978) had only 75% accuracy
aging dry-storedgraysquirrel molars,but
aged 96% of their formalin-storedspeci-
mens correctly.Lockard (1972), Stephen-
son (1977), and Low and Cowan (1963)
had poor results with cut and ground sec-
tions,yetobtained excellent results using
the paraffintechnique.
The interpretation method varies
among species and regions. G. Matson
(pers. commun.) has found that white-
tailed deer fromGeorgia are incorrectly
aged by criteriaused fornortherndeer.
RECOMMENDATIONS
Standardized preparationand interpre-
tation methods should be developed for
each species. Histological procedures us-
ing the paraffintechnique or a cryostat
have produced the most consistent re-
sults. Standardized interpretationproce-
dures such as those described by Matson
(unpubl. manuscript)for16 species should
be developed.
Problems with population and regional
differences can be overcome by estab-
lishing reference collections for each
area. These collections should be estab-
lished using the standardized methods
recommended above.
Time and effortcan be saved if juve-
nile animals can be eliminated by some
aging method before teeth are prepared
forcementum layer analysis. Aging meth-
ods thatwill easily separate juvenile from
adult animals need to be described for
each species.
 PREPARING MAMMALIAN TEETH FOR AGE DETERMINATION * Fancy 247

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Received 31 August 1979.
Accepted 17 December 1979.