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Bol. Depto. Geol. Urn-Son, 1992,Vol. 9, W 2, p. 101-107
TECHNIQUE OF SAMPLE PREPARATION
FOR PALYNOLOGICAL ANALYSIS
Salvador ENCISO-DE LA VEGA, Instituto de Geologia, UNAM
Ciudad Universitaria, Delegaci6n Coyoacan, 04510 Mexico, D. F.
ABS1RACf
The techniqueof samplepreparationfor palynologicalanalysisis described.The essentialprocessing
proceduresof acetolysis,cleaning,stainingand mountingare listed and discussedstepby stepfor calcareous,non calcareousand coal samples.
The basicequipmentand suppliesof a palynological laboratory
arelisted.
RESUMEN
Se describenlas tecnicasque se empleanen la preparaci6nde una muestrapara su analisispalinol6gico. Los procesos basicosde acetolisis, limpieza, tenir y montar sondescritosparamuestrascalcaTeas,no calcaareasy para carbones.Se provee una lista de 10sequipos basicos y de 10sproductos
necesariosparael funcionamientode un laboratoriopalinol6gico.
GENERALSTATEMENT
The proceduresdescribedin thepresentreport
are those employed in the preparation of six
lithologically fifferent samplesmadeat the palynologica1laboratory of the Stanford University
by S. Davidson, graduate student, under Dr.
W.R. Evitt's direction. Although pollen and spores liberation literature is extensive,no attempt
was madeso far to describedifferent techniques
or processvariations.Other specialtechniqueas
well as additional equipment, supplies and
chemicalreagentswere alsoomitted.
In general,palynological proceduresrequires
skill, ability and experience,and therefore data
suchasreagentamount,chemicalconcentrations,
and time of cooking are not given in detail.
However most of the times the appearanceof
the samplebeforeand after eachprocessingstep
gives a criteria to work with.
BASIC PREPARATION
TECHNIQUES
Recording and numbering
Most of the sampleshavea definiteprocessing
schedule, and thus, before any mechanical or
chemical treatment,each sampleshouldbe properly registeredunderthe laboratoyregisterbook
and lab number assigned.Care must be taken to
record data such as lithologic type, geographic
location, age, collector's name, geologic formation, and code number.All of thesedata as well
as processingschedulesare necessarydetails in
S. Enciso-De la Vega
102
order to keep good permanentrecords of each
processedsample.
damage to the man, they also attach optical and
laboratory instruments.
Crushing and contamination
Centrifugation
The first step of the routine processingconsistsof crushingthe material.Obviouslythereare
samplesin which crushing will not be necessary.
However in consolidatedrocks the surfaceof the
sampleis cut away and only its centralportion is
taken.
This phaseis usually carried out in the palynologicallaboratoryover a steelblock; in addition
hammerand aluminum pie dishesare used.Generally different samplesof distinct lithology are
processedat the sametime. Therefore, extreme
careand someprecautionsshouldbe takenalong
the complete processin order to eliminate contaminationand transposingof samples.Keep out
wind that can blown pollen by having closed
windows;cleanequipmentconstantly;usefiltered
water in the completeprocess;control the numbered beakers,plastic cups and the centrifuge
tubes.Use of aluminumpie dishesand styrofoam
cupsis also required
This part of the processingprocedurerequires
to take into accountvariablefactorSasdensityof
the liquid, centrifugespeed(!pm), centrifugation
time, size and specific gravity of the grains.
Normally, running testsof different time-speed
combinationsare necessarywhen a brandnew or
unknown centrifuge is used. In addition, well
balancedtubes during the centrifugation are required. That will be obtained using approximately the sameamountof liquid. The centrifugationprocessis carriedout on completecapacity
of the tube head centrifuge; when less than
required samplesare fUnned, additional water
will be used.The generalprocedurefor centrifugation is asfollows:
Maceration and safety
As soon as the sample has been crushed it
shouldbe transferedto glassbeakeror styrofoam
cupswherethe main chemicaldissagretationwill
take place. This process is usually known as
maceration. It consists mainly of the dissagretation and matrix dissolving of the sampleto
be processed.Generally it is carried out with
corrosive acidSsuch asHCL, HF or HNO3. The
time necessaryfor this treatmentvaries~gularly
from one or two hours to 24 hoursor even more
than two days. During this stage some sample
needsto be periodically agitated.Previousmoisterizing and soaking with water or having samples covered by water helps during the maceration stage.
Extreme cautionsshould be takenduring this
stepbecausedangerouschemicalcompoundsare
being handled constantly. In addition, security
equipment should be wear. The vapors usually
produced for these reagents cause not only
a - Stir and shake the samplein order to have
it well mixed beforecetrifugation.
b - Almost fill the centrifuge tube either with
water or dispersingsolution.
c - Centrifugeuntil a clear supernatantliquid
is obtained
d - Decant completely when water (wash) is
used;check the supernatantliquid when dispersing solutionor zinc bromideis used.
e - Repeat step a. b and c until a clear supernatantliquid is obtyained again and decant;
add more water or dispersing solution if it is
necessary.
f - Test the supernatantliquid for loose of
fossil materialor fine clay particles.
ESSENTIAL PROCESSING
PROCEDURE
1 - Washthe smfaceof the samplein waterto
removeany superficialcontamination.
2 - Crush the samplebetweenaluminum pie
dishes; get about 10 or 20 gr of good quality
grainedsample.
3 - In numberedplastic cups test the sample
by HCI; if effervescenceis produced add con-
S. Enciso-Dela Vega
centrate HCI covering the sample. If the HCI
reaction is quite strong glass beakersare used
insteadof plastic ones.
4 - When too many bubblesate producedby
the HCI reaction someacetoneis addedin order
to destroythem.
S - Mter two or three hours, the sample is
decantedand thewastesolution is transferedto
one special waste container bottle. The broken
down mineral material and fossils are removed
and transferedto centrifugetubes.
6 - Centrifugeabout 1-2 minutesanddecant.
7 - Add filtered water, stir, shake and centrifuge again.
8 - Wash and centrifuge one more time and
repeat until a neutral reaction is tested(pH paper).
Siliceous fossil material
9 - Transfer to beakerglassand let the heavy
material settle down for about two or three
minutes; take a sampleon petri dish and study it
under the microscope.Look for siliceous fossil
material. If siliceous fossils exist they will be
takenout with an eyedroperandmountedas step
33.
10 - Add HNO3 concentrate,heat over bunsen
burner, and wash until neutral reaction is obtained.
11 - Add KOH 10% solution and heat to just
warm the sample(avoiding boiling). At the same
time add one or two drops of HCI for precipitation purposes.
12 - Centrifuge,decantand washwith water in
order to eliminatetherestof HCI.
13 - Transfer the samplesto styrofoam cups,
addHF and let to standovernight.
14 - Decant, transfer the light portion to a
plastic centrifugetube. The rest of the solutionis
transferedto a wastesolutioncontainer.
15 - Wash with water until a neutralreactionis
reachedanddecant.
16 - Add sodium hypochlorite (Purex) as well
as one or two HCI drops, agitateand let to react
for about 15 minutes.
17 - Decant,add water,centrifugeand wash.
103
18 - Add two or threedrops of Amonia Hydroxide concentrateanddilute with water.
19 - Shake and let to react (1-2 minutes)
centrifuge, decant and wash with water several
times.
20 - Add a detergent (either Calgonite or
Labtone which were dissolved in water previously). This part of the treatmentproducesa
particle dispersion: the coarseparticles and the
fossil materials are settled down; on the other
hand, fine clay particles remain in suspension.
These fine clay particles are luminisens and
disturb,the normalobservationandphotography.
Thereforethey shouldbe takeout by decantation.
21 - Decant, wash and ~d previously dluted
zinc bromide. Transfer the sample to flexible
plastic tubes, already prepared (cut and mouth
immersedinto warm water); such plastic tubes
are set into centrifuge tubes with water around
them.Zinc bromidehasa specific gravity of 2.0;
thus, everything with a specific gravity of more
than 2.0 will settledown. The centrifugationwith
zinc bromidetakesabout 15 minutes.
22 - Washzinc bromide with water. The added
water will give a precipitatewhich is usuallyeliminateby addingtwo or threedropsof HCI.
23 - Before wash, a small portion of the
supernatantliquid is observedunderthe microscope. For this insert a clip acrossthe flexible
plastic tube so the supernatantliquid would be
easy to take out by pipete decantation or eye
droper.
24 - From the microscopic view of the
supernatantliquid one should decide how to
clean,run acetolysisor stain.
Acetolysis
25 - Centrifuge, decant, and add glacial solution. The acetic glacial acid takesout the water
(the next stepneedsto be without water).
26 - Add acetic anhydride and three of four
dropsof H2 504, then immersetest tube in boiling waterfor aboutten minutes.
27 - Centrifuge,decant,add aceticglacial acid
and centrifuge,this part of the treatmentis done
104
because in the next steps wash with water it will
be necessary.
28. Using water, wash and centrifuge three
times; in a shon duration at the end of this setof
washings,the samplewill be ready for cleaning
and mounting; during this stageseveral views
under the microscopeaccomplishedwith some
attempt to get mainly fossil material should be
done.
Cleaning
29 - This part of the procedurerequestsability
and practice. It is carried out using a couple of
watch glasses,an eye droper,and a small beaker.
Sometimesit is possibleto get away most of the
yunk material, particularly when the size
difference between fossils and yunk is great.
Tilting and rotatory movementsare very useful
during this stage. However, in some cases
everythingcomestogether(gumminess)andit is
impossible to obtain a separation.When this is
the case, the following procedure may be
successful:
a - Centrifugein wateranddecant.
b - Using glacial solution centrifuge and
decant two times.
c - Washwith waterandrestain.
Staining
The main purposesof staining pollen and
sporesare to get an easy observation,to obtain
betterphotographs,to remarkdetailsin structure.
Staining should be just enough, over-staining
causesto obscurestructural details. Somepractice is necessaryto determinethe degreeof color
intensity. The stain procedure is very simple,
however some grains do not need staining
specially if they have thin exine. Besides, the
acetolysisgives a translucentbrown color which
seemsto be adequatefor routine palynological
analysis; if the grains are too dark, mild chlorination destines them. During the process
observed,two types of stains were used: Safranin and Bismark brown.
S. Enciso-De la Vega
30 - After the last decantation, add the stain
previouysly choosen,and let to standfor about2
minutes,then add water. At this step somestains
often request WanDwater for staining. Repeat
until a goodquality colorationis obtained.
Mounting
31 - After stain,centrifugewashanddecanttwo
times, add water and shakewith a rod glass; at
this stageseveralviews undermicroscopeshould
be taken.
32 - Preparemounting material, clean, mark,
heatslidesandcoverglasses
upon the hot plate.
33 - Using eye droperspreadthe fossil material
on the cover glass.When the fossil material is
enough to make several slides, special care
should be taken in order to make on each one
slide a representativesample.Drop aboutthe same amount of fossil material upon each slide.
When differences in size are remarkable it is
possibleto make slides containing either fine or
coarsematerial.
34 - Add glycerine jelly, and spread close to the
border with a rod glass as soon as the glycerine
jelly is melted.
35 - Checkingwith a slide over it, let dried until
no steamwatercanbedetected.
36 - Spreadupon the slide with a plastic rod
WanDglycerinejelly-water solution.
37 - Pressure cover glass against slide,
previouslyfixed. Do not allow burblesto form.
38 - Let the slides to dry for about three of four
days.
39 - Cut away excess of glycering jelly, clean
and seal with nail polish.
Coals
Coals in generalare usually processedin a
different way. However, previous washing,
crushing as well HCl testing are usually carried
out similarly to any samplethat needsit.
1 - In a numberedglassbeakeraddjust a title
of filtered water and HNO3 concentrate,until a
strong reaction is obtained. Agitate and let to
standfor two or threehours.
105
S. Enciso-De laVega
2 - Decant and transfer the liquid to the special
waste recipient
3 - Transfer the sample to be processed into
centrifuge tubes.
4 - Centrifuge for about five minutes, decant,
transfer the supernatant liquid to the waste container.
5 - Add
down,
water
to the residue
stir, shake and centrifuge
which
was set
Maceration; oxJdize
KOH (10%)
Disentegrationand
removing particles
HF
Macemtion;eliminate
silica,demineralize
and remove
again.
6 - Decant dilute with water, centrifuge and
repeat several times until a neutral reaction is
obtained.
7 - Transfer to glass beaker, let to settle the
heavy material for two or three minutes. Now
the sample is tested for siliceous fossil material.
If there are some they will be taken out as
explained in step 9 of the general process.If there
is no siliceous fossil, the sample should be
runned as in step 10 (HNO3) and as in step 11
(KOH [10%]) of the general process.
8 - Continue subsequent treatment following
the steps 11 to 39 of the general process.
Non calcareous samples
The procedurefor non calcareousmaterialsis
basicallythe sameasthatexplainedin the general
procedureexceptfor the useof HCI. When HCI
is used in non calcareoussamplesthis is done
after
crushing and soaking with water. Then HCI is
addedin no more proportion than the sample
volume. Stir and shaketo mix well. Sometimes
heatingwill be necessaryin massiverocks.
CHART
HNO3
OF GENERAL
PROCESSING SCHEDULE
and dissolve mineral
particles
S<Xliurn
Hypoclorite
(Purex )
Bleaching
NH40H
Oxidize anddissolve
humic residue
Labtone or
Cablgonite
(Detergent)
Dispersionand
suspensionof clay
particles
Zinc bromide
Heavy liquid separation
Glacial
solution
Acetolysisand
dehydratation
Acetic
anhydride
Acetolysis;destroy
organicdebrisand
andH2SO4
removeobstructing
materials
Safraninor
Staining,only
Bismark brown if required
Glycerine
jelly
Mounting medium
Chemical
Treatment
EQUIPMENT AND SUPPLIES
HCl
Maceration, eliminate
carbonateand remove
mineral panicles
Acetone
Destroyburblesand
remove grease
Palynologicallaboratoriesshould be equiped
with an air extractorto eliminatechemicalfumes,
particularlythoseproducedby acids.
Special sinks and plumbing resistent to the
corrosive action of the reagents are requested.
Two water lines are desirable, one for filtered
106
water, and the second for flush down the sink,
becauselarge volumes of water are in constant
use.
Like in chemicallaboratoriesgasandelectricity lines arerequested.
The following list of equipment and supplies
were used for processing the samples described
here. Such material seems to be the ordinary
basic equipment necessaryin palynological techniques.
Basic palynological equipment
Centrifuge should have a minimum 4 tubes
capacity and 6 or 8 tube head with interchangeable~5 and 50 m1shells.Speedand time indicators,althoughnot absolutelynecessary,aid in
the centrifugeprocess.
Electric hot platewith temperaturecontrol.
Binocularmicroscope.
Electricalmixing device.
Chemical utensils and
accesories
supplies
Iron morter, steel block and hammer for
crushing.
Aluminum pie dishesabout6 inchesof diarn.
Glassbeakers,from 100 to 500 mI.
Glassbottles, 102oz. screwlids.
Plasticor polythyleneglasses.
Drop bottleswith drop spotandcap.
Plasticstyrofoamcups.
Bunsenburner,tripod andring stand.
Centrifugepolythyleneandglasstubes,round
bottomed
Centrifugetubes,glassand lusteroid,conical
bottomed.
Plasticflexible tubefor cutting.Funnelsof
different size.
S. Enciso-De la Vega
Watchglassesand Petri dishes,about 3 inches
diam.
Striker and beakertongs.
Plasticand glassstirring rods.
Pipettesandeyedroperswith rubbercaps.
Protectionplastic face shield.
Rubbergloves.
Polythylenebagsfor samplestorage.
Pencil glassmarker.
Glasscoverslips,squareand rectangular.
Microscopeslides,slide labels.
Watchmakersforceps.
Brushesfor cleaningtubesand glasses.
Kleenexandpapertowels.
Chemical
reagents
Acetic glacialacid (conc.),
Acetic anhydride.
Acetone.
Ammonium hydroxide.(conc.)
Zinc Bromide.
Glycerine.
Glycerinejelly.
Glacial solution.
Detergent(Calgoniteor Labtone).
Hydrochloricacid
Hydrofluoric acid (50 or 70%)
Sulfuric acid.
Nitric acid.
Potassiumhydroxide.
Safranin.
Bismark Brown.
Sodiumhypochloric (Purex5%)
AKNOWLEDGEMENTS
S. Davidsonrevisedthe fmal presentrepon
andoffered useful suggestions.
S. Enciso-De la Vega
107
REFERENCES AND NOTES
BROWN, C. A., 1960, Palynological techniques: I vol. 188 pp. Baton Rouge, La..
Brown describesdifferent methodSof sample
preparationusedby severallaboratoriesand
particulars. Completelist of chemicalreagents,
equipmentand bibliographyis included.
Evrrr.
W. R.. 1963. Eight sheets ditto
impressed for his 316 Palynology course.
Phosphoric acid. chromic anydrite and
Schulze's solution treatments are described as
well as different slide-making procedures.