Maney Publishing

Pollen Extraction for Arid-Land Sediments

Author(s): Anne I. Woosley
Source: Journal of Field Archaeology, Vol. 5, No. 3 (Autumn, 1978), pp. 349-355
Published by: Maney Publishing
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Pollen Extraction for Arid-land Sediments

Anne I. Woosley
SouthernMethodistUniversity
Dallas, Texas

Criticalexaminationof traditionalextractionproceduresemployedto recover

pollenSroma varietyof archeologicalcontextsdemonstrated the inadequacy°f
manysuchtechniqaes.Particularlyin aridzones,wherepollenpreservationis
notoriouslypoor, inappropriatelaboratoryanalysisacts to distortgrainsSurther.
A satisSactorymethodologySor pollensamplesrecoveredSrom areassubjectedto
suchdeterioratingetSectsas oxidationandweatheringis essentialSor
archeologistsattemptingto reconstruct prehistoricenvironments. A newextrac-
tionmethodologyharmlesstopollengrains,yieldingexcellentpollencountsand
preservationSrom archeologicalsoils was,thereSoreJ
experimentally developed
Sorarid-landsediments.

Pollen analysis is a fundamental tool for arche-
ologists who reconstructpast environmentsor for those
wishing to understandman's impact upon his physical
surroundings.Often, however, the ideal natural con-
ditions for the preservationof pollen, i.e., slightlyacidic
soils with little or no oxidation, simply do not exist in
areaswhere a knowledgeof ancientpollen sequencesis
necessary. The perfectly preserved pollen grains
recoveredfromthe water-loggedbogs of NW Europeare
in strikingcontrastto the collapsedand distortedgrains
characteristicof depositsfromthe aridNear East or sw
United States. Because weathering does have highly
detrimentaleffects on pollen grains, laboratorytechni-
ques that recover pollen from weatheredsoil samples
must be both efficient and exceedinglygentle in the
treatment of already delicate pollen grains. Initial
methodologies developed for pollen extraction in NW
Europeare consequentlynot appropriateto a diversity
of geographicareasrangingfromaridland sedimentsto
tropical rain forests.' What nature and man have left
for interpretationmust not be destroyedby inadequate
laboratorytechniques.
The following controlledextractionprocedureswere
conductedto determinethe optimal processfor extrac-
tion of pollen from archeologicalsamples.Four groups
of artificalpollen samplesweretested.One group of ar-
tificial pollen samples was made by subjecting soil
samplesof differentweights(TABLE 1) to 440° C for 45
minutesto destroy any organic materialthe soil might
have originally contained. Care was taken to utilize
1. J. Iversen' Landnam i Danmarks Stenalder-En Pollenanalytist
UnderXgelse over det fXrste Landrugs Indvirkningepaa tegetationsud-
viklingen(Copenhagen 1941).
soils most closely resemblingthose found in arid-land
contexts.Theseconsistedof colluvialclays and sands.
Pollen of Pinus sylvestrisand Pseudotsugamacrocar-
pa, chosen in part for their abundanceand availability,
was then added to the sterile soil in specific amounts.
Both Pinus and Pseudotsugawere propitious choices
becausetheirpollen grainsare not resistantto corrosive
chemicals(NaOH, KOH, HNO3) as are those, for ex-
ample,of Eucalyptas.2They will, therefore,more readi-
ly demonstrateany detrimentaleffectsproducedby the
extractionprocessitself.
In addition to the Elrstgroup of samplescontaining
sterile soil plus a speciElcamount of pollen, a second
groupof sampleswas producedconsistingof sterilesoil,
specific amounts of pollen, as well as charcoal and
other detritus.This sort of organicmaterialcausesmost
confusion when scanning slides. If too much of this
materialcomes through the extractionprocess, pollen
grainsbecometoo obscuredto identifyor count.
A third group of untreatedsoils was also analyzed.
These samples consisted of archeological deposits
recovered in Khuzistan and the Deh Luran Plain in
Iran and from HovenweepNational Monument in SE
Utah and sw Colorado.3
A Elnalgroupof samplesartificallypreparedand sub-
jected to high temperatures,to which pollen and, in
some cases, organicmaterialincludingplant Elbersand
2. Personalcommunicationfrom SherwinCa.rlquist.R. J. Stanley,
and H. F. Linskens, Pollen: Biology, Biochemistry,Management
(Springer-Verlag,
New York 1974)142-44.
3. For a discussion of Hovenweep pollen results?cf. Anne I.
Woosley, "Farm Field Location ThroughPalynology,"Hovenweep
1976, ArcheologicalReport No. 3, J. Winter, ed. (San Jose State
University1977)133-50.

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 ..

350 PollenExtractionSorArid-landSediments/Woosley

Table 1. Comparativepollenextractiontechniques.

Weightin Grams Grains/

Pollen Debris Extraction Pollen Sq. Cm.
Soil Added Added Technique Type Count Preservation
Sample
55 .5 - MEH Pinus 947 Falr
.

2
55 .5 - CHV sylvestris 1795 Good
3
55 .5 - HL ,,
1512 Falr
.

4
55 .3 - MEH 565 Falr

5
55 .3 - CHV 1525 Good
6
55 .3 HL 1299 Fair

7
55 .1 - MEH 56 Falr
.

8
55 .1 - CHV 326 Good
9
55 .l - HL 312 Fair

10
55 .01 - MEH 8 Falr

11
55 .01 - CHV 142 Good
12
55 .01 - HL 136 Fair

13
40 .2 - MEH 452 Falr
.

14
40 .2 - CHV 1130 Good
15
40 .2 - HL 1017 Fair

16
40 .05 - MEH 48 Fair
17
40 .05 - CHV 852 Good
18
40 .05 HL 500 Falr

19
40 .01 - MEH 6 Fair
20
40 .01 - CHV 160 ,!

wooa
.

21
40 .01 - HL 152 Fair

22
25 .2 - MEH 395 Fair
23
25 .2 - CHV 2130 Good
24
25 .2 - HL 1960 Falr

25
25 .05 - MEH Falr
.

26
25 .05 - CHV 600 Good
27
25 .05 HL 575 Fair

28
25 .005 - MEH
29
25 .005 - CHV 20 Good
30
24 .005 - HL 15 Falr
.

31
50 .5 15 MEH Pseudotsuga 365 Fair
32
50 .5 15 CHV macrocarpa 1595 Falr
.

33
50 .5 15 HL ,,

1395 . Halr
_ .

34
40 .5 20 MEH 200 Falr
.

35
40 .5 20 CHV 1230 Fair
36
40 15 20 HL 834 r alr
37
30 .1 20 MEH 56 Fair
38
30 .1 20 CHV 277 Falr
.

39
30 .1 20 HL 164 Fair
40
20 .05 20 MEH 42 Falr
.

41
20 .05 20 CHV 169 Fair
42
20 .05 20 HL 85 Fair
43
10 .01 30 MEH 6 Fair
44
10 .01 30 CHV 45 ralr

45
10 .01 30 HL 37 Falr
.

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 -- .

Journalof FieldArchaeology/Vol.521978 351

Weightin Grams Grains/

Pollen Debris Extraction Pollen Sq. Cm.
Sample Soil Added Added Technique Type Count Preservation
46 15 .01 20 MEH Pseudotsuga 2 Falr
.

47 15 .01 20 CHV macrocarpa 85 ralr

48 15 .01 20 HL
,,
73 rS
r alr
-

49a 30 MEH -

50a 30 CHV 141 Falr

51a 30 LIT
11L
113 Poor
52a 25 MEH Poor
53a 25 CHV 42 Poor
54a 25 HL 40 Poor
55a 40 MEH 6 Poor
.

56a 40 CHV 119 Falr

57a 40 HL 90 Poor
58b 30 MEH 28 Poor
59b 30 CHV 169 ralr

60b 30 HL 138 Poor

61 MEH
62 20 CHV 54 Fair
63 20 HL 34 Poor
20
64 .5 CHV Pinus 1695 Good
65 40 .5 MOD sylvestris 1695 Good
40 ,,

66 .01 CHV 156 Good

67 50 .01 MOD
,,

185 Good
50
68 .01 CHV Pinus 147 Good
69 30 20 151 Good
.01 MOD sylvestris
30 20
70 .01 CHV
,,
142 Good
20 10
71 .01 MOD ,,
153 Good
20 10
72a 40 CHV 56 Fair
73a 40 MOD 113 ralr

74a 30 CHV 20 Poor

75a 30 MOD 42 Poor

All samplenumberswithouta letterindicateartiElcially

madepollensamples.Numberswith "a"
indicate archeological samples from Chogha Mish, Iran. Numbers with "b" indicate
archeologicalsamplesfromthe Deh LuranPlain.

MEH = Mehringer Method CHV = ChevronMethod

HL = Heavy Liquid Flotation Method MOD = ModifiedChevronMethodwith Sieving

charcoalwas added, was used to demonstratethe most
efficientrecoverytechniqueof all. It comparedthe best
extractiontechniqueas establishedby the resultsof the
firstthree seriesof experimentswith a modifiedversion
thereof;the optimal extractionprocedurewas then es-
tablished(TABLE 1).
The first extractionprocess,the Mehringermethod,4
to be tested experimentallywas developed specifically
to analyze sw United States alluvialsamples(TABLE 2).
4. P. J. Mehringer,Jr., "LatePleistoceneVegetationin the Mojave
Desertof SouthernNevada,"J. Ariz. Acad. Sci. 3 (1965) 172-88.

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352 Pollen Extraction for Arid-land Sediments/ Woosley

Table2. The MehringerMethod. Table3. The HeavyLiquidMethod.

-

1. Selectapproximately50 g. of a sediment. 1 Select50 g. of sediment

2. Swirlin HCL in a 750 ml. beaker. 2 Treatwith 10%HCL
3. Passthrough100meshscreen(brass). 3. Washin H2Othreetimes
4. Discardremainingsediment. 4 Add HF to coversample,allow to stand24 hours.
5. Swirlin HCL in 250 ml. beaker. 5. Add fresh HF if all silicatesnot removed,allow to
6. Transferto test tube. stand 12-20hours.
7. Discardremainingsediment. 6. Washin H2° threetimes.
8. Treatwith 10-15%HCL. 7 Add 61 4%HNO3and 0.05 g. KC12to residue.
9. Treatwith 25-30%HCL. 8. Washsampleuntilclearin 100ml bottlesat 2,200
10. Washin H2O. rpm
11. Add 50%HF, allow to stand6-24 hours. Note: Acceleratecentrifugespeedslowly to 2,
12. Add 70%HF, allow to stand6-24 hours. 200 rpm,switchoff, and applybrakesat
13. Washin hot H2Otwice. 1,500rpm
14. Treatwith 20%HNO3for 10 minutes. 9 Mix residuewith 10%KOH solution.
15. Washin hot H2Otwice. 10 Washsampletill clear
16. Treatwith concentratedHCL for 2-3 minutesin a 11. Mix residuewith heavy liquid,e.g., Zinc bromide
boilinghot H2Obath. at a specificgravityof 2.1 or 2.0.
17. Washin hot H2Otwice. 12. Centrifugeat two minutes,3,000rpm.
18. Treat with 5-7%KOH for 2-3 minutes in boiling 13 Collect surface material in beaker together with
bath. heavy liquid, check and discard residue in cen-
19. Washuntilclear. trifuge
20. Transferto vial. 14. Decant material diluted five times with distilled
21. Mounton slide. H2Oand leaveovernightto settle.
15. Allow organic material to settle, decant upper
Distilled water is used throughout.
half, centrifugelower portion in 10ml.test tubes,
and washseveraltimes.
16. Check drop of sample under microscopeand if
The secondrelieson heavyliquidflotationand has been organicdebrisis too thick, treatwith HNO3for 5-
extensively used by Soviet scientists in palynological 10 minutes.Wash in 10%KOH solution and then
studies of the fRoraof India (TABLE 3).5 Chevron Oil washin H2O.
scientists developed the third process to recover fossil 17. Add 5 cc. distilled H2° and several drops of
pollen and micro-organismsin their quest for fossil safranineO.
fuels(TABLE4). 18 Centrifugesampleafter approximately30 seconds
The Elrststeps in all of the extractiontechniquesare and wash untilclear(untilwaterfreefromstain).
19. Prepareslides
based on a series of acid and water washes to break
down the carbonatesand silicates in the soil. As is evi- Distilledwaterusedfromstep15to endof theprocess
dent from the tables, the number and strengthof the
acid washes may vary, as does the length of time a par-
ticular sample is immersed in the acid solution. The
functions of the washes, nevertheless,are the same: the bottom of the beaker. The smaller, lighter pollen
hydrochloric (HCL) and hydrofluoric (HF) acids grains remain suspended for a longer period and are
remove carbonates and silicates respectively; water decanted into another beaker. This procedure is
washesremoveboth acids. repeatedseveraltimes until that portion of the sample
The Mehringer method is distinguished from the to be run throughthe remainingsteps of the processis
other two by what is termed the HCL "swirl."6Ap- transferredto test tubes. The remainingsedimentin the
proximately50 grams of sample is placed in a solution beaker is discarded. In both the Heavy Liquid and
of HCL and distilledwater,stirreduntil all particlesare Chevron methods, on the other hand, entire soil
in suspension,and then allowed to "swirl"for a short samplesto be tested for pollen content are run through
time while the larger,heavierparticlescome to rest on the completeextractionprocess.No soil is initiallydis-
carded.
Proponentsof the Mehringermethod maintainthat
5. PersonalcommunicationfromDr. S. K. Srivastava. no significantamount of pollen is lost by discardinga
6. The HCL "swirl" is sometimes used in conjunctionwith the major portion of the sediment because all micro-
HeavyLiquidflotationmethod:G. Weir,unpublishedPh.D. disserta- organic particles go into suspension during the HCL
tion (TexasA & M University1976). "swirl."Since pollen grainsare lighterthan sand, grit,

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Table4. ChevronMethod.
1. Add concentratedHCLto 50 g. of sediment.
2 Washin H20 one or moretimesas needed.
3. Add HF to sampleand place in hot waterbath for
one hour.
4. Washin H20 to removeacid.
5. Add Calgonto sample,beginuse of distilledwater.
6. Allow to settleand decantuntilclear.
7. Stainwith safranine.
8. Panto concentratepollen.
9. Mount on slides.
Distilledwaterusedfromstep fiveto end of process.
or most organic/inorganicdebrislikely to be presentin
the solution, they are allegedlypoured off to be subse-
quentlyrecovered,identified,and counted. Meanwhile,
supposedlyonly the heavierfractionhas been discarded
thereby making it more feasible eventuallyto remove
pollen grains for analysis. Comparison of the
Mehringermethod with both the Heavy Liquid and
Chevronproceduresdoes not, in termsof actual pollen
counts, appearto supportthis claim(TABLE 1).
After acids have broken down the carbonates and
silicates, the three extractiontechniquesproceed along
different lines leading to the ultimate separation of
pollen grains from the sample residue.Both Mehringer
and Heavy Liquid methods then utilize nitric acid
(HNO3) and potassium hydroxide (KOH) or other
equivalentbasic solutions to rid the sample of organic
debris. The Mehringermethod continues with an ad-
ditional HCL wash, in some cases repeatingthe HNO3
and KOH washes, while the Heavy Liquid method
relies primarilyon gravitationalseparationtechniques,
i.e., flotationto isolate pollen and spores.
In the Chevron method, once the carbonates and
silicates have been removed, an exceedinglymild base
known commerciallyas Calgon (a water softener) is
added to the sample.The sampleis well mixed,allowed
to settle, and decantedseveraltimes until the solutionis
clear. The Calgon action removes much unwanted
organic debris including carbonized plant remains
without resortingto harsherKOH or sodiumhydroxide
(NaOH) solutions. The remaining pollen-containing
residueis then stainedand finallymountedon slidesfor
identification.7
On the basis of the comparativeresultsobtainedby
testing the above extractionprocedures,it was shown
7. All extractionproceduresdiscussed were conducted in a well
equipped laboratoryat Rancho Santa Ana Botanic Garden. Such
facilitiesareessentialfor successfulpollenanalysis
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Journalof FieldArchaeology/Vol.5, 1978 353
Table5. ModifiedChevronMethod.
1. Add concentratedHCLto 50 g. of sample.
2. Washin H2Oto removeacid.
3. Add concentratedHF.
4. Washin H2Oto removeacid.
5. Add Calgonto distilledwater.
6. Decantand allowto settleuntilclear.
7. Stainin safranineT, O, or Y.
8. Sieve sample in lOO,u,60,u,40,u, 20,u, lO,umesh
screens.
9. Panin a watchglass to concentratepollen.
10. Prepareslides.
Distilledwaterusedfromstep fiveto end of process.
that both the Chevron and Heavy Liquid techniques
returnedgood preservationand recoveryratesof pollen
grains from artificially prepared samples. Overall,
Chevrondemonstratedhighercountsin everycase. This
was not so obviouswhen largequantitiesof pollenwere
added to samples. Th'e variation in recovery rates
become particularly apparent, however, when only
small amounts were added; Chevron yields were ap-
preciably higher (TABLE 1). The difference in
preservationand recoveryrates betweenChevron and
the other two extractionmethodswas even more strik-
ing when actualarcheologicalsampleswereprocessed.
After HCL and HF were added to a sample no
matterwhat the extractiontechnique-the remaining
pollen-bearingsamplewas placed in a watch glass and
examined under a stero-microscopeat 600x. Pollen
grains, their density and state of preservation,could
then be monitored. HCL and HF are not harmfulto
most pollen grains. Indeed,pollen is remarkablyinert,
reactingwith relativelyfew substancesas demonstrated
by its exceptional longevity in fossil or archeological
deposits.However,as is truewith most fossil remnants,
there are chemicals(e.g., KOH and NaOH) that will
cause pollen to deteriorate.8As varioussteps duringthe
extraction procedures were checked microscopically,
one could clearlysee that whenevera basic solutionwas
added to a sample, pollen preservationsuffered.Even
with weak solutionsof KOH and NaOH for only brief
periods, it seemedthat the action of the basic solution
was dependenton the degree of oxidation reachedby
HNO3. With modern or very well preservedmaterial,
e.g., pollen from Danish bogs, the problemof oxidation
is a minor consideration.9If, on the other hand, pollen
8. Stanleyand Linskens,op. cit. (in note 2) 129-42.
9. J. Troels-Smith,"PollenanalytischeUntersuchungenzu einigen
schweizerischen
Pfahlbauproblemen," in DasPhaflbauproblem
(Copen-
hagen1955)269-72.
 354 PollenExtractionfor Arid-landSediments/Woosley
Figure1. Pollengrain(bottom)distortedby 5%KOH solutioncom-
paredwitha grain(top) not subjectedto KOH. X500.
has already been subjectedto oxidation before being
collected, any further oxidation resulting from inap-
propriatelaboratoryprocedurescauses additionaldis-
tortion of pollen grainsmakingidentificationextremely
difficultor even impossible(FIG. 1).
Because the Mehringer method results in lower
pollen counts than either of the other methods even
when large amountsof modernpollen are addedto soil
samples,apparentlymuch pollen is lost when the bulk
of the sample is discardedafter the HCL "swirl"(FIG.
2). Pollengrainsare eitherclumpingtogetheror sticking
to other particlesin the sample makingthem heavy so
that they settle and are pouredout with most of the soil.
Very likely all or even most of the pollen grainsare not
suspendedin the HCL "swirl."Where pollen is abun-
dant this loss may not be significant if the relative
proportionsof plant species representedare the same.
Whenpollen is scarceit is importantto recoveras much
as possible. Archeological pollen samples often are
poorly preservedand contain small numbersof pollen
grains.
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Additionally, some researchersadd Lycopodium or
Eucalyptus tablettes to individual samples to
demonstratethat their method does not in fact affect
pollen counts or pollen preservationduringlaboratory
analysis.'° Lycopodium spores and Eucalyptus pollen
grains are two of the most resistantpalynomorphsand
can surviveconditionsthat would distort or completely
destroy other pollen grains, e.g., Pinus, Pseudotsuga,
Quercus, Zea. They are certainlynot accurateindicators
of pollen counts or preservation during extraction
procedures.
As has been noted previously, the Heavy Liquid
method is extremelyeffectivein recoveringpollen from
artificiallypreparedsamples.This is no doubt because
fresh pollen grains added to the soil sampleshave not
previouslybeen oxidizedand are, therefore,not unduly
affected by a chemical oxidizing agent. But even in
these fresh samples appreciablymore distortion and
collapsing of grains occurred than in the Chevron
process. The Chevron method does not use KOH or
NaOH and is considerably less corrosive to pollen
grains. Recovery and preservationwere consequently
much betterwith the Chevronmethod in both artificial
and, moreimportantly,archeologicalsamples.
Because laboratory procedures developing from
methodologies employed to analyze water-logged
sedimentswere also applied to other soil types, KOH
and NaOH washeswereconsiderednaturalsteps in any
extractionprocess.In a 50-gramsamplethe overwhelm-
ing amountof materialis not pollen and is of no use for
analysis. Besides soil, most of this materialconsists of
organic debris, i.e., plant and woody bits, small par-
ticles or carbonizedwood, ash or other detritus.The
presence of this material can make the isolation of
desiredpollen grains and/or spores very difficult.Such
debriscan completelyobscurepollen grainswhenone is
attemptingto identify them microscopically.A variety
of basic solutions do rid samplesof many useless sub-
stances thereby making the sample smaller in volume
and easierto work with. Unfortunately,these chemicals
destroymany kindsof pollen grains,especiallyif preser-
vation is poor at the onset.
A preferablemethod to the use of a basic solution
treatmentthat is more effectivefor pollen recoveryand
less damaging to grains is ( 1) employment of non-
corrosivechemicalswith respectto pollen, and (2) siev-
ing with a series of micron-sizedmesh screens. The
combinationof these two elements results in least dis-
tortion of individualgrains and producesbest overall
recoveryrates in archeologicalsoil samples(TABLE 1).
Most pollen grains range in size from 35,u to 60,u.
10. Jens Stockmarr,"Tabletswith Spores Used in Absolute Pollen
Analysis,"Pollen et Spores, 13 (1974) 616-21.
 0 :::&n :+f0 LLf w Cti:t$S;SAtT:00 v

Journalof FieldArchaeology/Vol.5, 1978 355

awareof secondaryconsideratlonssuch as time and ex-

pense needed to conduct successful pollen analysis.
From the standpoint of time only, the Modified
Chevron method is most desirablesince it takes about
half the time necessaryto complete the Mehringeror
Heavy Liquid methods. Because it utilized less
chemicals,it is less expensiveto processsamples.
Reswitsof comparativeextractiontechniquesclearly
demonstrate that pollen methodologies employed in
archeological contexts must be flexible. The
palynologist cannot be unalterably wedded to one
favorite procedure.Not only must methodologies be
frequentlymodified from one geographicregion to the
next, but sometimes even from site to site wlthin a
localized area, depending on physical clrcumstances.
Our interpretationsof the past will be limited by ex-
: : : :::zE:sf
f :0005 0q0ff;: a
t00::;fff::f00:
;:::0:S700:::
0000000X
t :: :f ;; ff00000000
00
000:0
0000
00f : 00 t :fff00
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000000
::
isting environmentalconditions, but, to a great extent,
they will depend upon the sophistication and ap-
propriatenessof our methodologies.
fw t$#:00::
: O 0-
Anne1. Woosleyis AssistantProfessorof Anthropology
at SouthernAfethodistUniversity,Dallas, Texas.She is
currentlyworkingin theAmericanSouthwestandwestern
Asia. Herprimaryresearchinterestsincludeland
managementandchangingsubsistencepatternsthrough
time.

Figure2. Recoveryof pollengrainsfromartificiallyprepared

samples,Mehringermethod(bottom)versusModifiedChevron
Technique(top). XlOO.

Organicand other debris is often much larger.Screens

decreasingin size from 100, to 60, to 40, to 20, to 10,
not only removemuch unwanteddebris,but also serve
to separate the pollen grains into specific size
groupings.The separationof grains accordingto size
categories is very helpful in identiElcationof grains.
Moreoever, any organic material other than pollen
remainingafter both the Calgon wash and sieving is
usually not enough to obscureor interferewith pollen
identification. Consequently, a Modified Chevron
technique employing mechanicalsieving produced the
best resultsof all extractionprocedurestested.11
In evaluatingextractiontechniquesone must also be

ll. Experimentsemployingthe scanningelectron microscopewere

conductedto establishwhetherpollen grains could be identifiedin
soils without benefit of chemical extraction procedures. Such
microscopydid not prove feasiblebecauseof problemsin mounting
and coating materialsto be scanned.For detaileddiscussion,see A.
Woosley,unpublishedPh.D. dissertation(UCLA 1976).

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