GEOLOGICAL SURVEY CIRCULAR 830 

Palynomorph Preparation Procedures 

Currently Used in the Paleontology 

and Stratigraphy Laboratories, 

U.S. Geological Survey 

Palynomorph Preparation Procedures 

Currently Used in the Paleontology 

and Stratigraphy Laboratories, 

U.S. Geological Survey 

By L. Imogene Daher 

GEOLOGICAL SURVEY CIRCULAR 830 

1980 
United States Department of the Interior 

CECIL D. ANDRUS, Secretary 

Geological Survey 

H. William Menard, Director 

Free on application to the Branch of Distribution, U.S. Geological Survey, 604 South Pickett Street, Alexandria, VA 22304 
CONTENTS 

Page 

Introduction • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 1 

Preliminary treatment • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 2 

Cleaning the sample • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 2 

Most samples • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 2 

Paleozoic coal samples. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 2 

Breaking down the sample •••••••••••••••••••••••••••••••••••••••••••••••••• _. • • • • • • • • • • • • • • • • • 2 

Most samples • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 2 

Paleozoic coal samples. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 3 

Disaggregation. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 3 

Removal of carbonates-HCl procedure • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 3 

Removal of silica and silicates-HF procedure. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 4 

Most samples • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 4 

Paleozoic coal samples. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 5 

Treatment of coals, lignites, and organic residues................................................ 6 

Oxidation • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 6 

Paleozoic coals-Schulze's Solution • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 6 

Other coals-Schulze's Solution. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 7 

Organic residues-Schulze's Solution • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 8 

Release of palynomorphs and humic compounds. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 8 

Paleozoic coals-KOH procedure • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 8 

Weathered Paleozoic coals-KOH procedure • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 9 

Coals and organic residues-KOH procedure........................................... 9 

Coals and organic residues-Organic detergent procedure • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 10 

Coals and organic residues-Sodium metasilicate procedure • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 10 

Peats and modern soils-KOH procedure.............................................. 10 

Dissolution of cell uloses by acetylation. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 11 

Separation of organic residues by zinc bromide flotation • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 12 

Recovery of dinoflagellates.. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 14 

Final cleaning of residues • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 15 

Settling procedure A: Gravity settling in 2 tubes (removal of heavy debris) • • • • • • • • • • • • • • • • • • • • • • • • • 15 

Settling procedure B: Gravity settling with 2 tubes and dropper (removal of 

heavy debris) ••• • --:. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 15 

Settling procedure C: Gravity settling with 4 tubes and dropper (removal of 

fine debris) ••••• • ---:.. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 16 

Settling procedure D: Gravity settling with alcohol washes (removal of fine 

debris) ••••••••• :-:. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 17 

Short centrifuging (removal of fine debris) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 18 

Swirling procedure: Separation of particles according to size and specific 

gravity • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 18 

Treatment of palynomorphs.. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 20 

Staining. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 20 

Bleaching. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 21 

Sodium hypochlorite procedure • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 21 

Hydrogen peroxide procedure • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 21 

Slide preparation • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 22 

Sampling of residues • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 22 

Mounting in Vinylite AY AF................................................................... 22 

Mounting in canada balsam • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 24 

<65-mesh residue • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 24 

Translucent megaspores • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 25 

Mounting in glycerin jelly • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 25 

Most samples • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 25 

Dinoflagellates • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 26 

Single grain mounts. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 27 

Storage of unmounted material. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 28 

Storage in water • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 28 

Storage in Vinyli te A Y AF • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 28 

Storage in ethanol and xylene • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 28 

References • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 29 

iii 
ILLUSTRATIONS 

Page 

Figure 1. Sketch of separation of sediment after centrifuging... • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 12. 

2.. Sketch of filter assembly with vacuum pump.............................................. 14 

3. Sketch of settling procedure C • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 16 

4. Sketch of particles partly settled before swirling in watch glass • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 19 

5. Sketch of particles segregated after swirling in watch glass................................. 19 

6. Sketch of removal of heavier particles with a dropper • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 19 

TABLE 

Page 

Table 1. Sequence of treatment of sediment types.. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • v 

iv 
Table 1. Sequence of treatment of different sediment types 

[This is a guide for technicians and is not necessarily limiting in scope; 

variation in the sequence of treatment may be necessary or desirable for some samples] 

Sequence Treatment Page 

Silts, shales, carbonates, cherts, 

welded tuffs, and other siliceous rocks 

1 Cleaning the sample ••••••••••••• 2 

2 Breaking down the sample •••••••• 2 

3 

4 

5 

6 

7 

8 

9 

Removal of carbonates ••••••••••• 3 

Removal of silica and 

silicates •••••••••••••••••••••• 4 

Oxidation: Organic residuesSchulze's Solution ••••••••••••• 8 

Release of palynomorphs and 

humic compounds: Coals and 

organic residues-KOH 

procedure •••••••••••••••••••• 9 

Separation of organic residues 

by zinc bromide flotation •••••• 12 

Recovery of dinoflagellates •••••• 14 

Final cleaning of residues ••••••• 15 

10 Treatment of palynomorphsStaining ••••••••••••••••••••• 20 

OR: 

Bleaching ••••••••••••••••••• 21 

11 Slide preparation ••••••••••••••• 22 

1 

2 

3 

Peats and modern soils 

Release of palynomorphs and 

humic compounds: Peats and 

modern soils-KOH procedure •• 10 

Separation of organic residues 

by zinc bromide flotation •••••• 12 

OR: 

Final cleaning of residues ••••••• 15 

Treatment of palynomorphsStaining ••••••••••••••••••••• 20 

OR: 

Bleaching ••••••••••••••••••• 21 

4 Slide preparation ••••••••••••••• 22 

v 

Sequence Treatment 

Lignites and coals 

1 Cleaning the sample ••••••••••••• 2 

2 Breaking down the sample •••••••• 2 

3 

4 

5 

6 

7 

8 

Oxidation: Other coals 

-Schulze's Solution •••••••••••• 7 

Release of palynomorphs and 

humic compounds: Coals and 

organic residues-KOH 

procedure •••••••••••••••••••• 9 

Separation of organic residues 

by zinc bromide flotation •••••• 12 

Final cleaning of residues ••••••• 15 

Treatment of palynomorphsStaining ••••••••••••••••••••• 20 

OR: 

Bleaching ••••••••••••••••••• 21 

Slide preparation ••••••••••••••• 22 

Paleozoic coals 

1 Cleaning the sample-Paleozoic 

2 

3 

4 

5 

coal samples •••••••••••••••••• 1. 

Breaking down the samplePaleozoic coal samples ••••••••• ~ 

Oxidation-Paleozoic coals ••••••• ( 

Release of palynomorphs and 

humic compounds: Paleozoic 

coals-KOH procedure (see 

also: Weathered Paleozoic 

coals-KOH procedure) ••••••••• f 

Slide preparation-Mounting in 

canada balsam ••••••••••••••• 24: 

INTRODUCTION 

This manual represents a compilation of 

procedures currently in use at the U.S. Geological 

Survey pollen laboratory in Denver, Colorado. Most of 

the techniques described did not originate in this 

laboratory but have been gathered from the various 

sources referenced; techniques are subject to 

continuing revision and improvement. Subsequent 

editions of this manual are anticipated as the evolution 

of techniques progresses. 

Although the procedures have been given in 

detail for the benefit of beginning technicians, it 

should be emphasized that in both choosing and 

carrying out procedures, a large amount of flexibility 

is desirable. (See table 1). The treatment of each 

1 

sample must be tailored to its individual 

characteristics; only experience can suggest the proper 

variations. 

To organize the instructions as much as possible, 

the text is formatted as follows: procedures are 

numbered and shown in the left column; additional 

details, precautions and comments are shown in the 

right column after corresponding numbers or letters in 

parentheses. General explanatory material, special 

notes, important cautions, and other miscellaneous 

information pertaining to a set of instruction~ appears 

in a headnote immediately before the start of the 

instructions. 
PREUMINARY TREATMENT 

Cleaning the sample 

MOST SAMPLES 

The amoant of sample processed for silts, shales, carbonates, cherts, welded tuffs, and 

other siliceous rocks: 30-40 grams; lignites and coals: 20 grams; Paleozoic coals: 50 

grams; peats and modern soils: approximately 60 grams. Clean surface of material 

thoroughly by one or more of these methods. 

1. Hold sample under running water and scrub with 

brush; blow with compressed air. 

2. Scrape sample with scalpel or knife, discarding 

all scrapings. 

3. Place crushed sample in 250-ml polyethylene 

beaker. Fill beaker with distilled water (tap water 

sometimes contaminates sample), stir, and allow to 

settle for approximately 10 minutes. Decant all water. 

PALEOZOIC COAL SAMPLES 

(1) After blowing with compressed air, sample may 

be placed on paper towel on hot plate (not over 100° F 

or 37.7° C) to dry. 

Clean by only one of the following methods: 

1. Outcrop samples of unweathered coal, mine 

samples, and diamond drill cores of coal may be 

washed with running water and a scrub brush or they 

may be placed in a beaker of water in an ultrasonic 

generator tank (40,000 Hz) for several minutes to 

remove surface contamination. 

2. Place weathered samples in a beaker. Fill 

beaker with water. Stir and allow to settle for 3 

minutes. 

(1) If samples are cleaned in an ultrasoni~ generator 

tank the water should be decanted and discarded. 

(2) Solid pieces settle rapidly and fines a":'e poured 

off. Repeat washing until water clears. Sample should 

be placed in an aluminum foil tray in hood until 

surface is dry. 

Breaking down the sample 

Special equipment: ceramic mortars and pestles 

MOST SAMPLES 

1. Break sample into pea size fragments using 

mortar and pestle. 

2 

(1) Mortar and pestle should be cleaned after each 

use. Use an abrasive cleaner; scrub with a stiff-bristle 

brush and rinse under running water. 
PALEOZOIC ·coAL SAMPLES 

1. Break along the banding with a chisel-pointed 

hammer into sheets or plates. Break sheets, by hand, 

into pieces approximately one-quarter of an inch in 

diameter. 

a. Broken outcrop or grab samples: run through a 

Jones sampler 1/ or riffle two times and take a 

suitably sized sample for maceration. 

b. Mine column and core samples: cut on a 

carborundum saw so that a complete ribbon or column 

sample of uniform size is obtained. 

(1) The use of mortar and pestle should be avoided if 

good megaspore recovery is expected. 

(a) Usually 40-50 grams. 

(b) Usually slightly less than one-fourth of an 

ordinary 2-inch core. 

DISAGGREGATION 

NOTE: Polycarbonate tubes are used in most of the following procedures. DO NOT use 

glacial acetic acid, acetone, undiluted nitric acid, methanol, ethanol, or xlyene in 

polycarbonate tubes. These reagents react with polycarbonate tubes and will destroy 

them. 

Removal of carbonates-HCl procedure 

1. To clean sample in 250-ml polyethylene or Pyrex 

beaker (Pyrex if heat will be used) add 10 pet aqueous 

hydrochloric acid (HCl) to slightly more than cover 

sample and stir. 

2. If a reaction occurs, add 100 ml of 10 pet HCl to 

beaker. Stir frequently. 

3. In order that disaggregated particles do not 

remain in HCl for too long, after 15 minutes decant 

into a 100-ml centrifuge tube, add distilled water to 

fill tube, centrifuge approximately 2 minutes at 2500 

rpm, and decant. Add 10 ml distilled water to tube, 

and agitate; fill tube with distilled water; centrifuge 

and decant. Add 100 ml 10 pet HCl to beaker and 

leave for 15 minutes longer. Repeat decanting into 

tube, washing and adding fresh 10 pet HCl to beaker 

until reaction ceases. 

4. After all reaction has ceased decant into the 

10Q-ml tube (leaving large particles in beaker); 

centrifuge and decant. 

(1) If no reaction (i.e. bubbling) occurs usil'9: 10 pet 

aqueous HCl, proceed to "Removal of silica and 

silicates, HF procedure" (p. 4 ). 

(2) If sediment threatens to foam out of beaker, add 

a few drops of water to slow reaction. A hot plate 

may be used to speed the reaction. Heat mu~t be used 

when dissolving dolomites. 

(4) Repeat this step, centrifuging and decanting, 

until all material in beaker (except large par~icles) is 

trans£ erred. 

!/ The use of commercial trade names is for descriptive purposes only and does not constitute endorse""lent of these 

products by the U.S. Geological Survey. 

3 
5. Add about 100 ml distilled water to material 

remaining in beaker. Allow larger particles to settle 

and decant into tube; centrifuge and decant. 

6. Add 10 ml distilled water to tube, mix, then fill 

tube with distilled water, centrifuge and decant. 

(5) Repeat this step 3 times to be sure material 

remaining in beaker is neutral. Check wit!:' pH paper 

to determine neutrality. 

(6) Repeat washing (usually 6-8 times) Wltilliquid is 

neutral. Check with pH paper to determin~ neutrality. 

Removal of silica and silicates-HF procedure 

NOTE: Precautions always to be observed when using hydrofluoric acid: Always use in 

fume hood with fan ON. Check hood with vaneometer. Vaneometer reading should be no 

less than 75 ft/min. Wear rubber gloves, apron, and face shield. The centrifuge should 

be placed directly in front of fume hood, or in hood, since HF fumes are fatal if enough are inhaled. --

MOST SAMPLES 

1. Place sample from mortar in 250-ml 

polyethylene beaker. Add a small amount of 10 pet 

HCl (to cover sample) to check for calcium 

carbonate. If a reaction occurs go back to "Removal 

of carbonates-HCl procedure" (p. 3 ). 

2. After "Removal of carbonates-HCl procedure", 

step 6, add a small amount of HF to the 100-ml tube; 

mix and decant into original 250-ml polyethylene 

beaker. Add more HF to rinse tube until all residue is 

transferred. 

3. Place beaker on platform rotator. 

4. If no reaction occurs with the addition of 10 pet 

HCl, allow sample to settle 5 minutes and decant the 

HCl. Add HF slowly (about 100 ml, depending upon 

size of sample) while stirring. If a violent reaction 

occurs and the sediment threatens to foam out of the 

beaker, squirt distilled water into the beaker (from a 

wash bottle) or place sample in cold water bath while 

adding HF. Place beaker on platform rotator. (This 

constant agitation will speed disaggregation of the 

sample.) 

4 

(1) About 100 ml of hydrofluoric acid (HF) is 

sufficient to dis aggregate the sample. If all Ca salts 

are not removed with 10 pet HCl before ac"riing HF to 

the sample, a precipitate will appear. Thir is ~~ally 

CaF 2 and is extremely insoluble (Ksp = 4 x 10- ). 

Thorough treatment with 10 pet HCl and careful 

washing to eliminate dissolved Ca salts before adding 

HF is very important. 

(3) If no rotator is available, using a polyethylene 

stirring rod, stir the sample at 5-minute intervals. 

(4) Continue agitation until the sample is 

disaggregated. The length of time that sair"Ole is left 

in HF will depend upon the volume of the sample and 

the lithology. Most samples will be disaggr?.gated 

within 1-2 hours. Since HF seems to have c corrosive 

action on pollen and spores it is best to stir constantly 

until the sample is disaggregated and wash 

immediately. (Some samples may have to ro.main in 

HF overnight or sometimes for days to disar~gregate. 

These samples, however, do not usually con~ain very 

good pollen or spores.) 
5. When the sample is disaggregated (fine sediment 

settled on bottom of beaker), add distilled water to fill 

beaker; allow to settle for one hour and carefully 

decant about half the liquid. 

6. Again add distilled water to fill beaker, let settle 

one hour, and decant about half of the liquid. 

7. After the second decantation, transfer residue to a 

1 00-ml polycarbonate centrifuge tube, leaving large, 

heavy particles in beaker. Fill tube with distilled 

water; centrifuge and decant. 

8. Add 5 ml distilled water to sediment in tube, stir; 

fill tube with distilled water. Centrifuge and decant. 

9. Optical Check. Using a dropper, place a drop of 

the washed sediment on a slide with a drop of distilled 

water and check with microscope to determine 

whether there are large organic aggregates that should 

be broken down. 

10. If maceration is fairly complete, but large 

aggregates of organic material remain, see 

"Oxidation: Organic residues-Schulze's Solution" 

(p. 8 ). 

11. If maceration is complete and palynomorphs 

seem clean, proceed to "Separation of organic residues 

by zinc bromide flotation" (p. 12). 

PALEOZOIC COAL SAMPLES 

(5) Alternate Method: When the sample is 

disaggregated, add distilled water to fill bea.lrer. 

Decant into 100-ml polycarbonate centrifuge tube; 

centrifuge and decant. Repeat decanting into tube, 

centrifuging and decanting, until all liquid is 

transferred from beaker to tube. NOTE: Lar~e, heavy 

particles on bottom of beaker should not be d~canted 

into tube. These remain in beaker for later 

treatment. After all floating material is trru,sferred 

to tube, centrifuged and decanted, agitate se~iment in 

tube with mixer, fill with distilled water, ceiJtrifuge, 

and decant. Repeat until the pH of the wate~ is 

neutral. 

(6) This step may be repeated several timer .. 

(7) Repeat until all material (except large, heavy 

particles) has been transferred to tube. 

(8) Repeat washing until the pH of the water is 

neutral. 

If silica is observed in water mounts following the procedures under "Release of 

palynomorphs .and humic compounds: Paleozoic coals-KOH procedure" step 4 (p. 8 ), 

perform the following procedures: 

1. Transfer residue to 250-ml polyethylene beaker. 

Add HF to cover sample and stir. 

2. After two to three minutes transfer sediment tc 

100-ml centrifuge tube. Add distilled water to fill 

tube; centrifuge and decant. 

3. Add water to fill tube; stir, centrifuge and 

decant. 

5 

(1) Observe all precautions noted on page 4-! HF 

has a corrosive action on spores and pollen, sc the 

treatment should be as short as possible. Twc to three 

minutes should be sufficient. 

(2) Centrifuge should be in or directly in fr,.,nt of 

hood because inhalation of HF fumes can be fatal. 

(3) Repeat washing until water is neutral. 
Treatment of coals, lignites, and organic residues 

OXIDATION 

NOTE: Oxidation procedures should always be carried out in hood. DO NOT BREATHF 

the gases given off during oxidation as they are toxic. Wear rubber gloves, apron, and 

face shield. CAUTION: The presence of pyrite in a sample may cause an explosion when 

Schulze's Solution is added. Always have a beaker of cold water available to dilute the 

mixture if necessary. 

Paleozoic coals-Schulze's Solution 

1. Place cleaned sample in 1-quart mason jar. 

2. Pour a very small amount of Schulze's Solution 

into jar (enough to wet sample). 

3. If no extreme reaction occurs it is safe to pour in 

200-250 ml Schulze's Solution. 

4. Stir sample at intervals with a glass rod. 

5. Optical Check: After a suitable interval 

(depending upon the speed of the reaction) withdraw a 

small amount of liquid with a dropper and put it into a 

15-ml Pyrex centrifuge tube to which about 10 ml of 

water has been added; centrifuge and decant. Place 

one drop of the washed sediment on a slide, add one 

drop of 5 pet KOH and cover with a cover glass. 

Check the degree of oxidation with microscope. 

6. When coal pieces are softened and when 

microscope check indicates that the oxidation process 

is complete add water to fill jar. 

7. After a settling period of 3 hours siphon water 

from sample. 

6 

(2) Preparation of Schulze's Solution: For low rank 

coals and organic residues mix one part rea~ent grade 

HNO~ (70 pet) with one part of a saturated' aqueous 

solution of KC103 (1:1). For high rank coal~ and very 

resistant organic residues mix two (2:1) or three (3:1) 

parts reagent grade HN03 with one part of a saturated 

aqueous solution of KC103• (The use of a l'~<lher 

concentration of HN03 on high rank coals can reduce 

time required to oxidize samples and may produce 

better results.) 

(3) In coals of high volatile A rank and higher, the 

reaction to 3:1 Schulze's Solution may be slight. (A 

slight reaction is indicated when a light yeJlow gas is 

released on first contact with Schulze's Sol ''tion and 

for the first minute.) Little or no reaction to Schulze's 

Solution usually indicates a long period of oxidation 

will be required. Better spore recovery mey be 

obtained through the use of a greater concentration of 

HN03• The use of fuming HN03 (90 pet), in place of 

reagent grade HN03 (70 pet), is recommenied. 

(5) If palynomorphs are released with the addition of 

5 pet KOH to the drop of sediment and if they are 

clean and sufficiently light in color, then tl'~ oxidation 

process is completed. 

(6) If sample is not completely oxidized l'Tlt coal 

pieces are slightly soft, the addition of hot water to 

the mason jar often will finish the oxidatio:r phase of 

the maceration. Softening of coal is deternined by 

resistance to pressure from the glass rod. 
8. Add water to fill jar. Let settle 3 hours and 

repeat siphoning process. 

9. Proceed to "Release of palynomorphs and humic 

compounds: Paleozoic Coals-KOH procedure" (p. 8 ). 

Other coals-Schulze's Solution (from Schulze, 1855) 

(8) This step must be repeated until the liquid is 

essentially neutral. It may be necessary to re:-:-eat the 

process as few as 6 times or as many as 10 or 12 times 

depending on the amount of sample and the 

concentration of HN03 used. 

Treatment of samples with Schulze's Solution should always be done in a fume hood. 

Have distilled water at hand. DO NOT BREATHE the gases given off during oxidation as 

they are toxic. 

1. Place cleaned sample in 250-ml polyethylene 

beaker. Cover sample with saturated aqueous solution 

of KC103 or NaCl03 (about 30 ml), and add an equal 

amount of HN03 for moderate treatment (1:1). 

2. After 5 minutes pipette off (use dropper with 

bulb) a few drops of mixture and put into 15-ml Pyrex 

centrifuge tube containing distilled water; centrifuge 

and decant. 

3. Using dropper, place a drop of sediment from 

centrifuge tube onto a slide and add one drop of 5 pet 

KOH (aqueous). Place cover glass over and check for 

degree of oxidation with microscope. 

4. When sediments are sufficiently oxidized (dark 

brown color given off when 5 pet KOH is added), 

oxidation may be stopped by adding distilled water to 

fill beaker. 

5. Pour material from beaker into 100-ml 

polycarbonate centrifuge tube which has been half 

filled with distilled water. Centrifuge and decant. 

6. Add 10 ml distilled water to tube, agitate; fill 

tube with distilled water; centrifuge and decant. 

7. Depending upon the type and condition of the 

sample after oxidation with Schulze's Solution, it may 

require treatment with a strong base such as 5 pet 

KOH, or with a milder base such as an organic 

detergent or an 80 pet acetone-water mixture. (See 

"Release of palynomorphs and humic compounds: 

Coals and organic residues-KOH procedure" (p. 9) or 

"Release of palynomorphs and humic compounds: 

Coals and organic residues-Organic detergent 

procedure" (p.JO).) 

7 

(1) If sediment threatens to foam out of be.~ker a 

squirt or two of water will slow the reaction. For 

more rapid oxidation use up to 90 ml HN03 (3:1). 

(2) If sample reacts vigorously (foaming yellowbrown gas given off), the reaction should be stopped 

after 10-15 minutes. If sample reacts little o:- not at 

all, it may be left overnight or as long as one week. If 

a sample must be left for more than a day, th~ spent 

Schulze's Solution should be decanted and a fro.sh 

solution added every day that the sample mus~ be left. 

(3) If fragments do not break down (no broV?n color 

given off) when 5 pet KOH is added, pipette cff test 

fractions at 15 minute intervals and check with 

microscope. 

(5) Repeat until all material is transferred from 

beaker to tube. It is necessary to dilute the f-:hulze's 

Solution by filling the centrifuge tube half full of 

water because HN03 will react with the poly·-::arbonate 

tube. 

(6) Repeat until water is clear and neutral. 

(7) Tschudy (1958) suggests the use of an 80 pet 

acetone-water mixture as a substitute for a s•rong 

base following Schulze's Solution for samples which 

contain degraded palynomorphs. Some samples may be 

treated by ZnBr2 flotation (see "Separation of organic 

residues by zinc oromide flotation" (p.12)) di1·~ctly 

after treatment with Schulze's Solution. 
Organic residues-Schulze's Solution 

After treatment with HF many samples will require oxidation with Schulze's Solution. 

NOTE: Follow same precautions as in "Other coals-Schulze's Solution" (p. 7 ). 

1. After the sample has been decanted into a 100-

ml tube and washed (leaving large heavy particles in 

beaker), add about 10 ml of saturated solution of 

KC103 or NaCl03 (aqueous) to sediment in tube. 

Agitate on mixer and transfer back to polyethylene 

beaker. Rinse remaining sediment from tube with 

about 10 ml of the saturated solution of KCl03 or 

NaCl03 and pour into beaker. 

2. Follow steps 2-7 under "Other coals-Schulze's 

Solution" (p. 7 ). 

RELEASE OF PALYNOMORPHS AND HUMIC COMPOUNDS 

Paleozoic coa}s-KOH proceclure 

CAUTION: Use KOH with great care because it will destroy pollen and spores if 

treatment is continued for too long a time; two to five minutes is usually sufficient. 

1. After the last siphoning of water (see 

"Oxidation: Paleozoic coals" step 8 (p. 7 )) add 10 pet 

KOH (aqueous) to the sample. 

2. Check for spores within 3 minutes after addition 

of the base. To make this check use the following 

method (water mount): Place a drop of the sediment 

on a slide with a drop of water. Place cover glass over 

this. Check with microscope to determine if 

palynomorphs are free and clear or if they will need 

more time in KOH. 

3. When spores are released and are clear (as seen 

with the microscope), add water to fill the jar. 

4. After 3 hours, siphon off water. Add water to 

fill jar. 

5. If silica is observed in water mounts (clear, 

angular, glass-like shards) see "HF Technique: 

Paleozoic coal samples" (p. 5 ). 

6. If no silica is present following the KOH treatment, 

screen the sample by washing through a 65-mesh 

screen. 

7. Decant the ( 65-m esh residue into a 1 00-ml 

polycarbonate centrifuge tube; centrifuge and decant. 

8 

(1) Amount of 10 pet KOH added depends on size of 

sample. A normal reaction to the base treatment is 

the release of a brown color in the liquid. 

(2) Usually 15 to 30 minutes treatment with a base 

is sufficient to free palynomorphs. Some sc-,mples may 

require no more than 3-5 minutes. Periods up to an 

hour are used, but if microfossils are not pr?.sent by 

that time, the sample is either not oxidized enough, is 

oxidized too much, or it is unfossiliferous. 

(4) This step is repeated until the liquid is neutral. 

It may be necessary to repeat the process 6 to 12 or 

more times, depending upon the size of the sample and 

the amount of humic matter released. 

{6) The large spores, megaspores, cuticle, and resin 

rodlets are retained on the screen, giving a>65-mesh 

residue which is stored in 2-oz poly-seal scr~w-cap 

vials in 95 pet methanol until ready to be e1-:amined. 

(See "Mounting in canada balsam: Translucent 

megaspores" {p.25).) 

{7) Continue decanting and centrifuging until all 

material is transferred to tubes. 
8. Add 10 ml water to tube, stir; fill tube with 

water; centrifuge and decant. 

9. Transfer residue to 15-ml Pyrex centrifuge tube; 

fill with water; centrifuge and decant. 

10. Proceed to "Mounting in canada balsam" step 1 

(p.Z4). 

Weathered Paleozoic coals-KOH procedure 

1. Place 1/4 of the riffled sample in a 250-ml Pyrex 

beaker and add 10 pet KOH (aqueous) to cover. If a 

brown color is released, wait 5 minutes. With a 

dropper, place one drop of the sediment on a slide and 

add one drop of water. Put a cover glass over this and 

check with microscope. 

Z. Check with microscope every five minutes to 

ascertain condition of spores. When spores are free 

and clear add water to fill beaker. 

3. Transfer material to 100-ml centrifuge tube, 

centrifuge and decant. Add water to residue; stir; 

centrifuge and decant. 

4. Screen sample by washing through a 65-mesh 

screen. 

5. Proceed to step 7 of "Release of palynomorphs 

and humic compounds: Paleozoic coals-KOH 

procedure" (p. 8 ). 

(8) Repeat washing until water is clear. 

(1) If no reaction occurs with the addition of KOH, 

it is necessary to oxidize the sample. (See "Other 

coals-Schulze's Solution" (p. 7 ).) If abundant spores 

and pollen grains are present after the KOH 

treatment, it is unnecessary to oxidize the sc-mple with 

Schulze's Solution. 

(Z) If spores and pollen are present but app"!ar thin, 

try washing sample in 10 pet KOH and halting the 

reaction almost immediately. If spores and rollen still 

appear thin, try a dilute solution of organic d~tergent 

in place of the 10 pet KOH. (See "Release of 

palynomorphs and humic compounds: Coals e-nd 

organic residues-Organic detergent proceduTe" 

(p. 10 ).) When palynomorphs are free, proceed to step 

3. 

(3) Repeat washing until water is clear and neutral. 

(4) Store >65-mesh residue as in step 6 of "Release 

of palynomorphs and humic compounds: Pale:ozoic 

coals-KOH procedure" (p. 8 ). 

Coals and organic residues-KOH procedure (from Kosanke, 1950) 

This treatment usually follows Schulze's Solution for coals and black organic residues. 

CAUTION: Use KOH with great care because it will destroy pollen and spores if 

treatment is continued for too long a time; two to five minutes is usually sufficient. 

1. After thorough washing as in step 7 of "Other 

coals-Schulze's Solution" (p. 7 ), add 5 pet KOH 

(aqueous) to 100-ml tube to cover sediment and stir. 

z. Fill tube with 5 pet KOH; stir, centrifuge and 

decant. 

3. Fill tube with distilled water; stir, centrifuge and 

decant. 

9 

(Z) KOH may be heated before adding to t:'l.e tube. 

This will speed the reaction. This step should take 

about three to five minutes. 

(3) Warm water, if used in this step, will S":leed the 

process. Repeat washing until liquid is clear and 

neutral. 
4. If there are large pieces of debris present in the 

sediment it may be screened through a 50-mesh 

stainless steel screen. 

5. If megaspores are present the>SO-mesh fraction 

should be stored in 2-oz poly-seal screw-cap vials in 95 

pet methanol until ready to be examined. (See 

"Mounting in canada balsam: Translucent megaspores" 

(p.25).) 

6. Proceed to "Separation of organic residues by 

zinc bromide flotation" (p.12 ). 

Coals and organic resid.ues-Organic detergent proced.ure 

(4) The>SO-mesh fraction should be checked 

microscopically for megaspores. The stainhss steel 

screen should be flamed after use to prevellt crosscontamination. 

In samples with oxidized or corroded palynomorphs, use the following procedure after 

treatment with Schulze's Solution. 

1. After thorough washing of residue and when 

liquid is neutral add about 20 ml warm organic 

detergent solution to sediment in 100-ml 

polycarbonate tube. Mix well. 

2. Fill tube with warm organic detergent solution; 

centrifuge and decant. 

3. Fill tube with warm distilled water; stir, 

centrifuge and decant. 

(1) Prepare organic detergent solution by adding 3 

grams of detergent (for example, Labtone) to 1000 ml 

distilled water (pH approximately 9); stir to mix 

thoroughly and warm on hot plate. 

(3) Repeat washing until water is clear and neutral. 

Coals and orianic residues-Sodium metasilicate procedure 

In some samples palynomorphs may have foreign material clinging to them after the 

foregoing preparations. The use of a sodium metasilicate ("metso") solution will, in mary 

cases, release this material from the grains. It is advisable to place a drop of the residt1.e 

on a slide, add one drop of metso, and check microscopically. It will be obvious under th~ 

microscope whether or not the metso is effective in cleaning the fossils. NOTE: 

Combined with short centrifuging this method will remove fine material from sediment. 

1. Add 5 ml of metso solution to residue in 15-ml 

Pyrex centrifuge tube. Mix. 

2. Fill tube with distilled water; stir, centrifuge, 

and decant. 

Peats and modern soils-KOH procedure 

1. Place dry sample in 600-ml Pyrex beaker. 

2. Add 5 pet KOH (aqueous) to slightly more than 

cover. 

3. Place on hot plate and bring to boil. Simmer 1-2 

minutes. 

10 

(1) Prepare metso solution by adding 2 grams of 

sodium metasilicate crystals to 500 ml of dbtilled 

water. 

(2) Repeat washing until decant is dear and neutral. 
4. Remove from hot plate, fill beaker with distilled 

water and stir. 

5. Transfer mixture from beaker to 10D-ml 

polycarbonate centrifuge tube; centrifuge and decant. 

6. After all material is transferred to the 

centrifuge tube, add distilled water to fill tube; 

centrifuge and decant. 

7. If residue is sandy, either (1) treat with HF (see 

"Removal of silica and silicates: HF procedurePaleozoic coal samples" (p. 5 )); or (2) proceed to 

"Separation of organic residues by zinc bromide 

flotation" (p.l2). 

8. Acetylate if necessary to remove cellulose content 

of palynomorphs. (See "Dissolution of celluloses by 

acetylation" below.) 

(5) If there are large clumps of debris present, 

residues should be washed through a 5D-mesb screen 

into the 10D-ml tube. Discard the >50 mesh residue. 

(6) Repeat washing until water is clear and r~utral. 

DISSOLUTION OF CELLULOSES BY ACETYLATION 

Precautions when mixing and using acetylation mixture: KEEP ACETYLATION 

MIXTURE AWAY FROM WATER. Wear rubber gloves, apron, and face shield. Mix 

reagent in hood. Examine Pyrex tubes (before using) for cracks or leaks, because leakage 

of the acetylation mixture into water will cause an explosion. 

1. Add glacial acetic acid (CH3COOH) to residues 

in 15-ml Pyrex centrifuge tube to fill; stir, centrifuge, 

and decant. 

2. Repeat glacial acetic acid wash. 

3. Add acetylation reagent to tube (approximately 5 

ml) and stir gently. Leave stirring rod in tube. 

4. Place tube carefully in gently boiling water bath 

for 3-1/2 to 4 minutes. Stir occasionally. 

5. Remove from water bath and add glacial acetic 

acid to fill tube. Stir, centrifuge, and decant. 

6. Repeat glacial acetic acid wash as in step 1. 

7. Add distilled water to fill tube; stir, centrifuge, and 

decant. 

11 

(1) Purpose of glacial acetic acid washes b to 

dehydrate residues. 

(3) Acetylation reagent: Nine parts acetic 

anhydride (CH3Co)2o and one part sulfuric acid 

(H2so4). Add acid to anhydride. Mix fresh just prior 

to use as reagent will take on water if left standing. 

(4) CAUTION: Do not allow water to boil high next 

to centrifuge tube lip; water produces a violent 

exothermic reaction with the reagent. 

(7) Repeat washing with distilled water Wltil there is 

no odor of acetic acid (6-8 washings). 
SEPARATION OF ORGANIC RESIDUES BY ZINC BROMIDE FLOTATION 

(from Funkhouser and Evitt, 1959) 

1. Remove as much water from sediment as 

possible by centrifuging and decanting. 

2. To the sediment in 100-ml centrifuge tube add 

ZnBr2 solution to cover sediment about 1/2 inch and 

stir thoroughly, using vortex mixer. It is necessary to 

mix sediment and ZnBr2 solution completely, because 

some sediments clump when the ZnBr2 solution is 

added and a clean and complete separation of the 

organic fractions cannot be achieved. 

3. Fold clear plastic tubing (thin-walled Tygon-see 

Funkhouser and Evitt, 1959, p. 372) and place in 50-ml 

polycarbonate tube which has been lubricated with 

glycerin to facilitate removal. 

4. Pour well-mixed sediment suspension into tubing, 

equalizing the amounts on each side. Rinse the lOQ-ml 

tube with more ZnBr2 solution and pour into tubing so 

that all sediment is transferred; after transfer rinse 

tube well with water. If the total amount of sediment 

in the tube measures more than 20 mm deep (as 

measured on the tube) the sample should be divided in 

half and floated in two tubes. 

5. Centrifuge 20 minutes at 2500 rpm. 

(International Equipment Co. centrifuge models 1 and 

2 will operate at such high speeds.) If safety limits do 

not permit this speed use a lower speed for a longer 

time to achieve the same results. 

12 

(1) To acidify sediment before adding ZnP•2 

solution, add 10 ml of 10 pet HCl (aqueous) to 

sediment in lOQ-ml centrifuge tube; stir thoroughly 

and fill tube with distilled water; centrifuge and 

decant. It is necessary to acidify the sedim~nt 

because the addition of ZnBr2 solution to sediment in 

which some water remains may cause a white 

precipitate (zinc hydroxide) to appear. 

(2) ZnBr2 solution: Prepare by adding one part 

distilled water (which includes a small amount of 10 

pet aqueous HCl) to two parts ZnBr~ (using 

hydrometer), making a specific graVIty of 

approximately 2.042. The solution must be acidified or 

a white precipitate (zinc hydroxide-Zn(OH)~) will 

appear when water is added to ZnBr2• 

Figure I.-Separation of sediment after centrifuging 

(step 5). 

(5) It is necessary to place tubes in such a manner 

that both sides of the tubing are in line with the 

direction of rotation. The tubing will fold over at the 

top and spill part of the contents if placed at right 

angles to direction of rotation. 
6. After centrifuging, remove tubing from the 

centrifuge tube; pinch tubing below the float with 

fingers or needle nose pliers and pour into the 100-ml 

tube which originally contained the sediment. 

7. Still pinching the tubing, rinse top out with 10 

pet HCl and pour this into the centrifuge tube, also. 

8. Add about 20 ml 10 pet HCl (to acidify wash 

water and avoid a precipitate of Zn(OH) 2) to float in 

100-ml tube. Fill remainder of tube with distilled 

water and stir thoroughly. Centrifuge for 3 minutes at 

2500 rpm and carefully decant. If interf.a.ce forms in 

tube, decant only to meniscus; add more water, stir, 

centrifuge 3 minutes, and decant. 

9. After two washes with distilled water the residue 

should be transferred to 15-ml Pyrex centrifuge tubes. 

10. Wash, centrifuge, and decant 6 times. 

11. If the organic residue now contains some mineral 

grains (derived from material adhering to the walls of 

the tubing in which separation was made) transfer 

sediment to a 15-ml polyethylene tube (with dropper). 

Rinse original tube with water and add to material in 

polyethylene tube. Fill polyethylene tube with water; 

stir, centrifuge, and decant. 

12. Add 5 ml HF to material in polyethylene tube (in 

hood). Stir and leave for 5 minutes. 

13. Fill tube with distilled water; centifuge, and 

decant. 

13 

(7) After transferring the float and rinsing the 

tubing with 10 pet HCl, the sediment and the 

remaining ZnBr4 solution may be set aside fer later 

checking to see tf palynomorphs remained with the 

heavy mineral portion of the residue. 

(9) If there are large pieces of debris pres~~t, screen 

the material by pouring into the 15-ml tubes through 

50-mesh stainless steel screens formed to fit into 

small funnels. All stainless steel screens sho•tld be 

washed and flamed after use. 

(13) Repeat washing 6 times. 
RECOVERY OF DINOFLAGELLATES 

If a sample is being prepared for dinoflagellates the disaggregation and treatment 

remain the same as for pollen and spores through "Separation of organic residues by zinc 

bromide flotation" step 10(p.13 ). From that point, treatment continues as follows: 

1. Using a filter assembly with a hand-operated 

vacuum pump (see fig. 2) place a 2Q-micron screen 

over the top of the assembly and secure with ring. 

Make sure that screen is not stretched tightly over 

opening. Screen should be depressed into a cone. Pour 

residue through screen, activate vacuum pump, and 

rinse with 10 pet methyl alcohol with wash bottle. 

2. Remove screen from filter assembly and place in 

clean beaker. Holding screen by one corner and using 

a wash bottle, rinse material from screen into beaker. 

3. Transfer material from beaker to original 15-ml 

centrifuge tube, centrifuge, and decant. 

(2) Fine residue (<ZO micron) may be discarded. 

However, if pollen and spores are also to be studied 

this residue should be poured into a 15-ml c~ntrifuge 

tube, centrifuged, and decanted. If palynomorphs 

require stain, proceed to "Staining" (p. 20). 

4. Put 2-3 ml of distilled water in tube, mix on (4) Repeat washing 4 times. 

vortex mixer, fill tube with distilled water, centrifuge, 

and decant. 

5. If palynomorphs require stain, proceed to 

"Staining" (p. 20 ) • 

Vacuum Pump 

Figure 2.-Filter assembly with vacuum pump. 

14 
FINAL CLEANING OF RESIDUES 

Settling procedure A: Gravity settling in Z tubes 

(removal of heavy debris) 

When the preparation is completed and the sediment is ready for slide preparation, some 

of the heavy particles may be removed by taking advantage of their differential settling 

rates. By saving all residues, one may experiment with these techniques without losing 

portions of the sample. 

1. Add 10 ml of distilled water to 15-ml tube 

containing sediment. Add a small amount of a wetting 

agent (such as Darvan #4) to prevent clumping; stir 

well, or place in ultrasonic generator tank for 30 

seconds. 

Z. Allow to settle about 30 seconds. 

3. Pour suspended material into a second tube, 

allowing only the heavy settled fragments to remain in 

the first tube. 

4. Using a dropper, place some of these fragments 

on a slide and check with microscope. 

5. Repeat steps 1-4, with appropriate settling 

interval, until residue is as free as possible of large 

fragments. 

(4) If palynomorphs are present in heavy se":tled 

residue, decrease settling time. If no palynoillorphs 

are present in the heavy settled residue, it may be 

discarded. 

(5) With each change in the settling interval, 

microscope checks must be made after transfer. 

Settling procedure ~: Gravity settling with Z tubes and dropper 

(removal of heavy debris) 

1. Repeat step 1 of "Settling procedure A" (above). 

z. Allow to settle 30 seconds. 

3. With dropper, draw material from the tube, 

leaving only heavy settled residue on the bottom. 

4. Check some of residue with microscope. 

5. Repeat steps 1-4, with appropriate settling 

interval, until residue is as free as possible of large 

fragments. 

15 

(Z) The settling period before either decanting or 

removal with a dropper should be either shortened or 

lengthened, depending upon the relative amounts of 

pollen and spores and larger fragments present. 

(Larger fragments settle more rapidly than p<'llen and 

spores.) 

(4) If palynomorphs are present in the heav; 

settled residue, decrease the settling time. Ir no palynomorphs are present in the residue, it 

may be discarded and the settling time increc~d. 

(5) With each change in the settling interval, 

microscope checks must be made. 
Settling procedure Q: Gravity settling with 4 tubes and dropper 

(removal of fine debris) 

This is basically the same technique as "Settling procedure ~" (removal of heavy debris). 

This may be used to remove fine debris from samples. However, the method requires a 

total of four clean, 15-ml Pyrex centrifuge tubes for each sample. (See fig. 3). 

1. Fill 15-ml tube containing residue with water; 

stir vigorously, place in holder, and allow to settle for 

10 minutes. 

Z. With dropper, remove top half of the water, and 

place in second 15-ml centrifuge tube. 

\I \./' \I 

A '/ '/ 

2 3 4 

r<() 

cl \/ '-/ \/ \/ L---~--~2~--3~--~4--~ 

(1) Time intervals may be decreased or increased 

until almost all fine debris has been discarded. 

\'1// \I 

B '/ 

3 4 

~ 

b ~ 

/ a 

Figure 3.-Settling procedure Q: Gravity settling with 4 tubes and dropper (removal of fine debri:-,). AI illustrate steps 1-6, respectively. 

16 
3. Fill first and second tubes with water; stir 

vigorously, and allow to settle for 10 minutes. 

4. Remove top half of water from second tube and 

place in third tube. Remove top half of water from 

first tube and place in second tube. 

5. Fill tubes with water; stir and allow to settle for 

10 minutes. 

6. With dropper, remove top half of water from 

third tube and place in fourth tube. Remove top half 

of water from second tube and place in third tube. 

Remove top half of water from first tube and place in 

second tube. 

7. Fill first tube with water; stir, and allow to 

settle for 10 minutes (or longer if fine portion 

contained palynomorphs). Tubes 2 and 3 are already 

filled and should not be stirred. 

8. Repeat steps 6 and 7. 

(6) Now the pipetted mixture in the fourth tube 

should be centrifuged and checked microscopically. In 

most cases it will contain nothing but fine debris and 

may be discarded. If palynomorphs are present in the 

fine portion, increase settling time. 

(8) Note: Repeat steps 6 and 7 until all fine debris 

has been discarded. 

Settling procedure ~: Gravity settling with alcohol washes 

(removal of fine debris) 

Through the use of alcohol washes, much of the debris in a sample will be even more 

finely divided. Use the following procedure. 

1. Add 2-3 ml 10 pet methanol to residue in 15-ml 

Pyrex centrifuge tube, stir; fill with 10 pet methanol; 

centrifuge and decant. 

2. Add 2-3 ml 25 pet methanol to residue in tube, 

stir; fill tube with 25 pet methanol; centrifuge and 

decant. 

3. Add 2-3 ml 50 pet methanol to residue in tube, 

stir; fill tube with 50 pet methanol; centrifuge and 

decant. 

4. Add 2-3 ml 75 pet methanol to residue in tube, 

stir; fill tube with 75 pet methanol; centrifuge and 

decant. 

5. Add 2-3 ml 95 pet methanol to residue in tube, 

stir; fill tube with 95 pet methanol; centrifuge and 

decant. 

6. Add 2-3 ml 75 pet methanol to residue in tube, 

stir; fill tube with 75 pet methanol; centrifuge and 

decant. 

7. Add 2-3 ml 50 pet methanol to residue in tube, 

stir; fill tube with 50 pet methanol; centrifuge and 

decant. 

8. Add 2-3 ml 25 pet methanol to residue in tube, 

stir; fill tube with 25 pet methanol; centrifuge and 

decant. 

17 
9. Add Z-3 ml10 pet methanol to residue in tube, 

stir; fill tube with 10 pet methanol; centrifuge and 

decant. 

10. Add Z-3 ml water to residue in tube, stir; fill 

tube with water; centrifuge and decant. 

11. Perform steps 1-8, "Settling procedure g" (p.16 ). 

Short centrifuging (removal of fine debris) 

Fine debris may be removed by centrifuging in 15-ml Pyrex tubes for short periods of 

time. Note: If a sample contains very small (10 micron) pollen or spores, short 

centrifuging may not be successful. Try "Settling procedure g" (p.16) instead. 

1. Place tubes in centrifuge and turn it on; when 

centrifuge has reached 1500 rpm turn off. 

z. Let centrifuge coast to a stop and remove tubes. 

3. Decant into labeled beakers (one for each 

sample). 

4. Add distilled water to tubes; stir, and repeat 

short centrifuging. 

5. Decant into beakers a second time. 

6. Check decanted portions microscopically. 

7. If no pollen or spores are present in the decanted 

portions this material may be discarded. 

8. If there are a number of pollen and spores 

present in the decanted fractions, transfer material 

from second set of tubes to first set and repeat steps 

1-6, increasing the centrifuging time. 

(1) A small clinical centrifuge is best for this 

purpose. 

(Z) DO NOT USE CENTRIFUGE BRAKE! 

(5) Repeat washing, short centrifuging, arri 

decanting until no float is seen in the tubes after 

centrifuging. 

(6) This can be simplified by transferring decanted 

material from beakers to clean 15-ml tubes, 

centrifuging about Z minutes and decanting. Repeat 

until all material in beakers is transferred. 

(8) If there is floating material after long~r 

centrifuging this material should be decante:<:l. and 

checked microscopically as in step 6. 

Swirling procedure: Separation of particles 

according to size and specific gravity 

(from Funkhouser and Evitt, 1959) 

The organic particles within a sample may be separated differentially according to size 

and specific gravity by swirling them in a watch glass using a technique similar to that 

used in gold panning. 

1. To the sediment in a 15-ml centrifuge tube, add 

about 10 ml distilled water. 

18 

(1) To prevent clumping of particles: Before 

swirling, add about 0.25 ml (small squirt from squeeze 

bottle) of dispersing agent (such as a saturated solution 

of Darvan :41:4 or a non-ionic detergent) to tb~ tube. 

Mix well. Place tube in ultrasonic generator tank. 

Agitate for 5 minutes. 
2. Place a 3-inch watch glass on a smooth, level, 

hard, light-colored surface. 

3. Place a small amount (5 ml) of the aqueous 

suspension in watch glass. Allow to settle for one 

minute. 

Figure 4.-Particles partly settled before 

swirling in watch glass. 

4. Holding the edges of the watch glass between the 

thumb and fingers, resting the heel of the hand on 

surface and keeping the rim of the glass level, move 

the glass smoothly in circles. Circles should be about 

4mm. 

Figure 5.-Particles segregated after swirling 

in watch glass. 

5. Tilt watch glass so the heavier particles in the 

center are strewn out for removal with a dropper. 

Figure 6.-Removal of heavier particles with a 

dropper. Watch glass tilted to strew 

particles out. 

6. Place finer particles remaining in suspension in 

15-ml tube; centrifuge, and check microscopically. 

7. Repeat swirling with particles in the second 

watch glass. Pipette off all material held in 

suspension and place in 15-ml tube. Centrifuge and 

check microscopically. 

19 

(2) A light-colored background is helpful when 

removing particles from the watch glass afte~ swirling. 

(3) If a suspension is very thick and dark, with 

heavy, opaque particles, put a smaller amount in the 

watch glass to swirl at one time. 

(4) Do not move glass rapidly or with a side to side 

motion as this will cause turbulence of the Sl' .. face of 

the liquid. 

(5) These particles may then be placed in another 

watch glass for even finer separation. The watch glass 

may be placed on the microscope stage and the 

sediment checked to determine success of tbe 

separation. 

(6) Short centrifuging of these finer partides may 

help further concentrate the fossils. (See "Short 

centrifuging "(p. 18 ).) Note: If there is a gr~at size 

difference among the pollen and spores present, the 

larger ones will be in the heavy fraction and the 

smaller ones in the fine fraction. 
8. Check remainder of met.terial in second watch 

glass microscopically. 

(8) These particles should be heavy fragrents and 

may be discarded. If there are a number of 

palynomorphs remaining in this fraction repeat steps 

1-5 until a clean separation is achieved. 

TREATMENT OF PALYNOMORPHS 

Staining 

After the various oxidation procedures outlined in this manual, many palynomorphs 

become translucent and require staining. Preparation of staining solutions: Triturate C.1 

gram Safranin 0 or Bismarck Brown Yin 100 ml ethanol (absolute ethyl alcohol) or 90 fCt 

methanol (methyl alcohol). Alternate method: Add 0.1 gram of Safranin 0 or Bismard· 

Brown Y to 100 ml 90 pet ethanol or methanol in storage bottle (ground glass seal) and 

place bottle in ultrasonic generator tank. Agitate for 15 minutes. 

1. Fill 15-ml Pyrex tube containing residue to be 

stained with 10 pet methanol. Stir, centrifuge, and 

decant. 

2. Fill tube with 25 pet methanol; stir, centrifuge, 

and decant. 

3. Fill tube with 50 pet methanol; stir, centrifuge, 

and decant. 

4. Add 5 ml 75 pet methanol to tube. Stir. Add 5 

ml Safranin 0 or Bismarck Brown Y staining solution 

to tube. Stir. 

5. After 15-min interval, using a dropper, ·remove 

small amount of residue, dilute it with a drop of water, 

and check microscopically to determine degree of 

staining of palynomorphs. 

6. When grains are sufficiently overstained, add 75 

pet methanol to fill tube; centrifuge, and decant. 

7. Add 2-3 ml 95 pet methanol to tube; stir and fill 

with 95 pet methanol. Centrifuge and decant. 

8. Proceed to step 6, "Mounting in Vinylite A Y AF" 

(p. 22) or step 5, "Mounting in canada balsam" (p. 24 ). 

20 

(1) It is necessary to begin methanol washes with 

dilute aqueous solutions and ever succeedin~ more 

concentrated solutions of methanol to prev,~nt 

clumping of sediments. If 95 pet methanol is added 

directly, most sediments will clump and are: almost 

impossible to disperse. 

(5) Some palynomorphs are more resistant to stain 

than others so microscope checks must be IJ"ade. 

(6) The grains must be overstained since further 

washing with methanol or ethanol is necessary before 

mounting and these reagents will destain 

palynomorphs. 
Bleaching 

Occasionally palynomorphs may be opaque even after oxidation procedures. Structural 

features are obscured, so it is necessary to bleach such grains in order to make 

morphologic determinations and photomicrographs. In many cases, palynomorphs may be 

opaque but corroded. Because these grains will sometimes disintegrate with the use of 

sodium hypochlorite, a milder bleach such as hydrogen peroxide (30 pet) is recommended. 

SODIUM HYPOCHLORITE PROCEDURE 

CAUTION: Sodium hypochlorite (ordinary household aqueous bleach-5.25 pet by weight) 

corrodes pollen and spores over prolonged periods and may cause changes in grain size. 

Use great care in controlling the length of time that samples are bleached. 

1. To residue in 15-ml Pyrex centrifuge tube add 5 

ml distilled water; stir and add 5 ml sodium 

hypochlorite. 

2. Add 2 drops of 10 pet HCl (aqueous) and stir. 

3. Place a drop of the suspension on a slide; place 

cover glass over drop and check with microscope. 

4. When grains are the desired color fill tube with 

distilled water; stir, centrifuge, and decant. 

5. Add 5 ml metso solution to tube, stir, and fill 

tube with distilled water. Centrifuge and decant. 

6. Add distilled water to tube; stir, centrifuge, and 

decant. 

HYDROGEN PEROXIDE PROCEDURE 

1. Add 5 ml 30 pet hydrogen peroxide to sediment 

in 15-ml Pyrex centrifuge tube. 

2. Dilute by adding 5 ml distilled water. Stir. 

3. Place a drop of the suspension on a slide, cover 

with cover glass, and check with microscope. 

4. When grains are the desired color fill tube with 

distilled water; stir, centrifuge, and decant. 

21 

(1) Further dilution of the sodium hypochlo~ite can 

be made if the mixture seems to be damaging the 

grains. The check with the microscope (step 3) will 

determine if this is necessary. 

(2) For some samples this bleaching process may 

take no more than a few seconds-in others, l.-3 hours 

or overnight. 

(3) A cover glass is necessary because fumes may 

harm the objective. Several checks may be n~cessary 

before the bleaching is completed. 

(4) Repeat washing 3 times. 

(5) For preparation of metso solution see "Sodium 

metasilicate procedure" step 1 (p.10). 

{6) Repeat washing until water is clear. 

(3) Use of a cover glass is necessary so tha~ fumes 

do not harm the objective. Several checks may be 

necessary before the bleaching is completed. 

(4) Repeat washing 6 times. 
SUDE PREPARATION 

Sampling of Residues 

Accuracy in sampling the total residue and in proportional representation of materials is 

essential to meaningful interpretation of the assemblage. If the residue in the vial is in a 

small amount of liquid a representative sample can be insured by agitating the 

sediment. This may be done by expelling air from the dropper bulb through the liquid 

several times before withdrawing the drop for mounting. Residues should be centrifuged 

before slide preparation until almost all liquid is removed. 

Mounting in Vinylite AYAF 

Some individuals prefer this mounting medium for palynomorphs because it is possible to 

mount palynomorphs in the same plane on the cover glass, and, thus, traversing slides i~ 

made simpler. It is not recommended that pollen and spores be stained during mountin~ 

through the use of A Y AF to which stain has been added. This method results in a 

background of much the same color as the palynomorphs, making photomicrography very 

difficult. Preparation of Vinylite AY AF (refractive index = 1.46}: Add 100 ml 95 pet 

methanol or ethanol to 2.0 grams A Y AF (prepare in a bottle which can be tightly 

sealed). Add 4 ml dibutyl phthalate (plasticiser). Let stand until AY AF pellets are 

dissolved (this will take 2.-3 days). Agitate bottle frequently to speed mixing. Note: [',., 

not use canada balsam for mounting A Y AF slides because A Y AF will become cloudy 

after a period of time. 

1. Add 2.-3 ml 10 pet methanol to residue in 15-ml 

Pyrex centrifuge tube, stir; fill with 10 pet methanol; 

centrifuge and decant. 

Z. Add 2.-3 ml 2.5 pet methanol to residue in tube, 

stir; fill tube with 2.5 pet methanol; centrifuge and 

decant. 

3. Add 2.-3 ml 50 pet methanol to residue in tube, 

stir; fill tube with 50 pet methanol; centrifuge and 

decant. 

4. Add 2.-3 ml 75 pet methanol to residue in tube, 

stir; fill tube with 75 pet methanol; centrifuge and 

decant. 

5. Add 2.-3 ml 95 pet methanol to residue in tube, 

stir; fill tube with 95 pet methanol; centrifuge and 

decant. 

6. Add 2.-3 ml absolute methanol to tube, stir; fill 

tube with absolute methanol; centrifuge and decant. 

2.2. 

(1) If residue has been through staining Frocess (see 

"Staining" p. ZO , steps 1-3 are omitted. 

(6) Excess water remaining in the preparation will 

cause the mixture on the cover glass to become white 

and cloudy. 
7. With dropper, transfer sediment to storage vial 

(see "Storage in Vinylite AY AF" p. Z8 ; rinse centrifuge 

tube with absolute methanol and add this to the vial. 

8. Add approximately Z ml A Y AF solution to 

storage vial, mix well, screw on cap, and label. Let 

stand 4-5 hours or overnight before making slides. 

9. Using a dropper, stir AY AF-sediment mixture by 

expelling air through the dropper. Place a drop of 

AY AF-sediment mixture on a clean cover glass. 

10. Add 3-4 drops A Y AF from the dropper bottle and 

mix with the dropper used to remove sediment and 

A Y AF from the storage vial. 

11. Place a second clean cover glass directly over 

the first. 

1Z. Hold the sandwich by its long edges and pull the 

two cover glasses apart laterally. 

13. Place cover glass 8repared-side up on slide and 

put on slide warmer (40 C). Allow to dry at least Z 

hours before mounting. 

14. Remove slide and cover glass from warmer, and 

put approximately 3-4 drops (amount depends on size 

of cover glass) of Histoclad mounting medium on the 

slide. Invert cover glass and, beginning with one end 

at the edge of the mounting medium, allow it to drop 

slowly into the medium. 

15. Allow slide to cure at room temperature for 4-5 

days. 

16. When slide is fully cured, clean any excess 

mounting medium from edges of cover glass with a 

razor blade. 

17. Slide is now ready for permanent labeling. 

Z3 

(7) An efficient method of removing alcohol from 

the sediment involves filling the storage vial ~ith the 

sediment and absolute methanol and centrifuging the 

vial (with cap on). (A smaller, empty vial, pla~ed in 

the bottom of the carrier, will prevent the top vial 

from slipping downward and wedging itself int':l the 

carrier.) Using a dropper with bulb, remove the 

methanol leaving only the sediment and a verr small 

amount of alcohol. 

(9) If residue is very concentrated the drop should be 

smaller than a drop from a sparse suspension. 

(10) Number of drops of AY AF solution depends on 

size of cover glass and concentration of residr<e: 

ZZx30 mm cover glass ....:. 3 drops of A Y AF; zz~~40 mm 

cover glass--4 drops of AYAF. If a suspension is quite 

sparse it is not necessary to place the drops of AY AF 

on the cover glass. Simply place 3-4 drops of the 

sparse suspension on the cover glass and proceed to 

step 11. (Droppers used in mounting with AY AF must 

be rinsed in absolute methanol before washing.) 

(11) The mixture will spread evenly to the ed7es of 

the cover glass. 

(13) Slides should be labeled temporarily witl a wax 

pencil, felt pen, india ink, or with a tape label. (Steps 

13-17 apply to both halves of the sandwich.) 

(14) Do not allow cover glass to fall immediately but 

let down gradually so that medium spreads without 

leaving air pockets. In order to elevate the cover glass 

on one end and allow it to drop slowly, a teasing needle 

or toothpick may be held under the end of the cover 

glass. The consistency of the Histoclad should be such 

that it flows evenly under the lowered cover glass. 

This procedure should take approximately 10 sa.conds if 

the Histoclad is of the proper cpnsistency. If the 

Histoclad is too thick there is a tendency for C';r 

bubbles to form. Use xylene or toluene to thir 

His to clad. 

(15) After 4-5 days the slide will be dry enou'!h to be 

used with care but curing time may vary from Z-3 

weeks. 
Mounting in canada balsam 

( 65-MESH RESIDUE 

1. Add 2.-3 ml 2.5 pet methanol to residue in 15-ml 

Pyrex centrifuge tube, stir. Fill tube with 2.5 pet 

methanol; centrifuge and decant. 

2.. Add 2.-3 ml 50 pet methanol to tube; stir. Fill 

with 50 pet methanol; centrifuge and decant. 

3. Add 2.-3 ml 70 pet methanol to tube; stir. Fill 

with 70 pet methanol; centrifuge and decant. 

4. Add 2.-3 ml 95 pet methanol to tube; stir. Fill 

with 95 pet methanol; centrifuge and decant. 

5. Add 2.-3 ml absolute methanol to tube; stir. Fill 

with absolute methanol; centrifuge and decant. 

6. Add 2.-3 ml of 50-50 mixture (by volume) of 

absolute methanol and xylene to tube; stir. Fill tube 

with 50-50 mixture; centrifuge and decant. 

7. With a clean, dry dropper, mix sediment by 

expelling air through dropper in sediment. 

8. Place 4-5 drops of canada balsam in clean, dry, 

porcelain evaporating dish on slide warmer. 

9. With dropper place 2.-3 drops of sediment in 

evaporating dish. 

10. Mix well by stirring with dropper. 

11. Using dropper place one drop of balsam-sediment 

mixture on clean, temporarily labeled slide on slide 

warmer. 

12.. Place cover glass directly on drop. 

13. Transfer remaining residue from centrifuge tube 

to poly-seal storage vial. Rinse tube with a 50-50 

ethanol-xylene mixture and put this in vial also. 

14. Leave slides on slide warmer or in warming oven 

until mount is set. 

15. After curing, remove slides from warmer and 

label. 

2.4 

(1) If residue has been stained (see "Staining" p.ZO) 

steps 1 and 2. are omitted. 

(8) Have slide warmer set at about 40°C. A 

disposable aluminum weighing dish may be used instead 

of porcelain. 

(9) Amount of sediment transferred to e'"~'aporating 

dish with balsam depends upon whether sediment is 

concentrated or sparse. (If sediment is cor~entrated 

use less.) 

(11) Amount of balsam-sediment mixture depends on 

size of cover glass. For a smaller cover glass (2.2. x 2.2. 

mm) use less mixture. 

(12.) Mixture will spread evenly to edges of cover 

glass. Practice will determine exactly how much is 

needed to spread exactly to edges of cover glass. 

(13) If a porcelain evaporating dish was used in slide 

preparation it must be rinsed, before washing, with a 

50-50 ethanol-xylene mixture to remove canada 

balsam. The dropper which was used to mix canada 

balsam and the sediment must also be rinse<\ with 50-

50 ethanol-xylene mixture. 

(14) Curing time commonly ranges from 1-2. weeks. 

(15) Canada balsam slides need not be sealed. 
TRANSLUCENT MEGASPORES 

1. Put >65-mesh residue in petri dish with water 

enough to float palynomorphs. 

2. With a flattened teasing needle, pick megaspores 

from residue and place in a second petri dish with 

sufficient 50 pet methanol to cover. 

3. When a representative number of megaspores 

have been picked, decant the 50 pet methanol from 

dish and add 95 pet methanol. 

4. Allow to settle and decant. 

5. Add 100 pet ethanol. Allow to settle and decant. 

6. Add 50-50 mixture of ethanol-xylene. 

7. Place small dots of canada balsam on a slide. 

8. With flattened teasing needle pick megaspores 

from ethanol-xylene mixture and place within the 

canada balsam dots. 

9. Allow to cure briefly on the warming plate 

(40°C). 

10. Add additional canada balsam (1-2 drops) 

between the megaspores and place cover glass directly 

over preparation. 

(7) Make one dot for each megaspore to be nounted. 

(9) Thirty to sixty seconds is commonly enough time. 

(10) The megaspores will remain in position when the 

cover glass is applied; thus, an orderly arrange:"llent of 

mounted megaspores is possible. 

Mounting in Glycerin Jelly 

Preparation of glycerin jelly: 2 grams Phenol, 66 grams glycerin liquid, 14 grams gelatin, 

colorless. Add gelatin to 38 ml distilled warm water. Then add glycerin. Heat very 

gently in hot water bath for 15-20 minutes (or until liquid starts to clear and white 

bubbly foam rises to top). Add phenol and heat and stir until dissolved. CAUTION: 

Avoid heating glycerin jelly for long periods at the boiling point. Gelatin will polymerize 

and the mixture will be useless. 

MOST SAMPLES 

1. Place 2 drops of hot glycerin jelly on a clean, 

warm slide (40°C) which has been temporarily labeled. 

2. Using a clean dropper remove a small amount of 

aqueous residue from 15-ml centrifuge tube and drop 

directly on jelly. 

3. Mix thoroughly with dropper tip. 

25 

(1) If too much glycerin jelly is used the mh~ture will 

spread out from all sides of the cover glass anri much 

of the preparation will be lost. If too little glycerin 

jelly is used, extensive air pockets will form d"tting the 

curing process. 

(3) Keep slide warm to reduce formation of bubbles. 
4. Place cover glass over mixture holding one edge 

up and allowing it to drop slowly until mixture spreads 

evenly to all edges of cover glass. 

(4) A teasing needle or a toothpick may be used to 

hold one end of the cover glass and allow it to drop 

slowly. 

5. Invert slide on the slide warmer on parallel strips (5) Allow to cure for 4-5 days. 

of wood designed to hold the slide about 4-5 mm from 

warmer so that the cover glass does not touch the 

warmer. The fossils will settle to the top of the cover 

glass. 

6. Clean edges of cover glass with water. 

7. Seal edges of cover glass with Glyptal clear (7) Slide is now ready for permanent lal ~ling. 

varnish No. 1202. Allow to dry 2-3 hours. 

DINOFLAGELLATES 

If palynomorphs have been stained they must be returned to water before mounting in 

glycerin jelly. Follow procedure below from step 1. If palynomorphs have not been 

stained begin procedure below at step 6. 

1. After staining is completed, add 7 5 pet methanol 

to fill centrifuge tube; centrifuge and decant. 

2. Add 2-3 ml 50 pet methanol to tube; stir. Fill 

with 50 pet methanol; centrifuge and decant. 

3. Add 2-3 ml 25 pet methanol to tube; stir. Fill 

with 25 pet methanol; centrifuge and decant. 

4. Add 2-3 ml 10 pet. methanol to tube; stir. Fill 

with distilled water; centrifuge and decant. 

5. Add 2-3 ml distilled water to tube; stir. Fill with 

distilled water; centrifuge and decant. 

6. Place a clean 76x25 mm (3x1 in.) glass slide on 

slide warmer (40°C). 

7. Place a clean 22x30 mm cover glass on slide. 

8. Pick up cover glass and put 2 drops of warm, 

dilute glycerin jelly directly on cover glass. 

9. Using a dropper, place one drop (or adequate 

amount) of residue on the dilute glycerin jelly and mix 

well, spreading mixture to edges of cover glass. 

10. Return cover glass to slide on warmer, prepared 

side up, and allow to dry for 30 minutes. 

11. Remove cover glass from slide and place 2 drops 

of warm glycerin jelly on slide. 

26 

(6) Slide should be temporarily labeled. 

(7) A small residue yield may necessitate using a 

smaller cover glass. 

(8) To prepare dilute glycerin jelly: Mi~ one part 

glycerin jelly with one part distilled water. 

(9) A toothpick, bent dissecting needle, or a small 

glass rod can be used to mix the residue ard jelly. 

(10) This process allows for evaporation of all water 

from glycerin jelly, and also aids palynomorphs to 

settle in one plane, against the cover glass-. 
1 2.. Turn cover glass over and place on the drops of 

glycerin jelly. The mixture should flow only to edges 

of cover glass. 

13. Slide should now be placed face down on the slide 

warmer on parallel strips of wood designed to hold the 

slide about 4-5 mm from the warmer so that the cover 

glass does not touch the warmer. The fossils will 

settle to the top of the cover glass. 

(12.) Number of drops of glycerin jelly used de:"lends 

on size of cover glass. If a mixture does not flow 

completely to edges, add warm glycerin jelly tc edges 

of cover glass where voids occur and the jelly will flow 

under the cover glass. 

Single Grain Mounts 

In the absence of any special equipment to isolate and extract individual specimens the 

following procedure has been used to obtain single grain mounts of pollen and spore 

specimens. 

1. Put a few drops of residue in a 15-ml centrifuge 

tube. If residue is in alcohol, wash with distilled water 

to remove alcohol. (See step 1, "Mounting in glycerin 

jelly: Dinoflagellates" (p.2.6).) 

2.. Heat a small hot plate to about 150°C. Place 

clean slide on hot plate. 

3. Extract enough hardened glycerin jelly from 

bottle to make a small smear on slide and let melt. 

4. Mix residue in tube with dropper. Extract a 

dilute representative sample from the tube with 

dropper and place one drop of residue on top of the 

melted glycerin jelly and mix immediately. 

5. Remove from hot plate and allow to cool. 

6. Place slide on microscope and locate specimen to 

be isolated. 

7. When specimen is located, turn slide over so that 

the glycerin jelly is facing downward and move the 

condensor of the microscope as close as possible to the 

slide without touching the jelly. 

8. Using ink and a quill pen, place a very small dot 

of ink on the top of the upside-down slide where the 

point of light shines through the slide. 

9. Place the slide on the stereoscope, highest 

power. Cut away excess glycerin and spores on the 

slide, leaving the single specimen mounted in a bit of 

glycerin jelly. 

10. To mount a cover glass over the specimen place 

the slide on a warmer. While melting, put a minute 

amount of melted glycerin jelly on a cover glass and 

place it directly over the ink dot, thus sealing the 

single grain on the slide. 

2.7 

(5) Place a petri dish over slide to keep it from 

collecting dust. 

(7) In this manner a fine point of light is obtained 

and encircles the specimen. 

(8) This allows you to mark the exact spot w'lere the 

specimen lies on the slide. 

(9) To cut away the excess use a new razor b1ade. 
11. To completely seal the grain, melt some 

carnauba wax on the warmer; place the slide on 

warmer until warm (enough to melt wax but not warm 

enough to melt glycerin jelly); place a drop of the wax 

at the edge of the cover glass, allowing it to flow 

under the cover glass. 

1 Z. Immediately remove the slide from the warmer 

and allow it to lie flat until cooled and set. 

(11) It takes only a few seconds for the slide to reach 

the temperature at which the wax will flow under the 

cover glass. 

(1 Z) If the slide is left on the warmer too long it will 

become too hot and destroy the specimen. Time 

needed for the slide to cool and set is at l~ast 15 

minutes. 

STORAGE OF UNMOUNTED MATERIAL 

Residues may be stored in any size bottle. The fossil preparations of the U.S. Geological 

Survey are stored either in Z-dram or Z-oz flint glass vials with poly-seal screw caps. 

Storage in water 

When mounting in glycerin jelly the residues are in water and are ready for storage when 

slide-making is completed. 

1. Using a dropper, remove residues from 15-ml 

centrifuge tube and transfer to storage bottle. 

Z. Rinse tube with enough distilled water to reclaim 

all the residue. 

3. Add 3 ml 25 pet methanol to prevent fungal 

growth; add Z-3 drops glycerin, and screw on the cap. 

(Z) When completed, the storage bottle should be 

approximately half full. 

Storage in Vinyli te A Y AF 

Residues which are already in A Y AF (in storage bottles) are ready for storage when 

slide-making is completed. (See "Slide preparation: Mounting in Vinylite AY AF" step 8 

(p.23).) In time, alcohol may evaporate, leaving the residues imbedded in AYAF. Slide~ 

may be prepared later by adding 95 pet alcohol to dissolve the A Y AF. 

Storage in ethanol and xylene 

1. After mounting in canada balsam (see p. Z4) 

transfer (with dropper) the remaining residue from the 

15-ml centrifuge tube to Z-dram flint glass vial with 

polyethylene screw cap. Rinse tube with 50-50 

mixture of absolute methanol and xylene and add this 

to the vial. 

Z. Screw on cap and store. 

Z8 

(1) Vial should be one-half to two-thirds full after 

transfer. 
REFERENCES 

Funkhouser, J. W., and Evitt, Wm. R., 1959, 

Preparation techniques for acid-insoluble 

microfossils: Micropaleontology, v. 5, no. 3, p. 

369-375. 

Kosanke, R. M., 1950, Pennsylvanian spores of Dlinois 

and their use in correlation: Illinois State 

Geological Survey Bulletin, no. 74, p. 8-11. 

29 

Schulze, Franz, 1855, Uber das Vorkommen 

wohlerhaltener Zellulose in Braunkohle 1:"1d 

Steinkohle: Berlin, Berliner Koniglichen 

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