Acetolysis and pollen slide preparation, explained
What each step of pollen preparation actually does, from alkali and hydrofluoric acid to Erdtman's acetolysis and the choice of mounting medium, and when a lab should skip a step on purpose.
Acetolysis is a laboratory treatment, introduced by the Swedish palynologist Gunnar Erdtman, that heats pollen in a freshly made mixture of acetic anhydride and concentrated sulphuric acid (classically 9 parts to 1). It dissolves cellulose and the living contents of the grain and leaves the outer wall, the exine, clean and easy to read under the microscope.
Acetolysis is one step in a longer sequence. A typical preparation of lake or peat sediment adds a known number of marker spores, removes carbonates with hydrochloric acid, humic material with hot alkali, silicate minerals with hydrofluoric acid or a dense liquid, and then acetolyses what is left before mounting the residue on a slide.
This guide explains why each step exists, what it removes, what it does to the grains, and which sample types skip which steps. It is not a bench protocol: several reagents involved are seriously hazardous and belong only in a properly equipped laboratory with training and a risk assessment.
Why pollen needs to be concentrated
A gram of lake mud contains far more mineral grains, plant fragments and humic material than pollen. Put it straight on a slide and the grains are buried and too sparse to count efficiently.
Preparation works because the pollen wall is unusual. The exine is made of sporopollenin, one of the most chemically resistant materials in nature. Bennett and Willis (2001), in their standard chapter on pollen in lake sediments, describe the outer walls as “extremely resistant to chemical and physical attack”.
That resistance lets a lab attack almost everything else in the sample with acids, alkalis and heat, and keep what survives. Each step targets one kind of unwanted material. The aim is a residue rich in pollen and spores, with the grains in a condition that can be compared with reference slides.
The standard sequence at a glance
Protocols differ between labs and textbooks (Faegri & Iversen; Moore, Webb & Collinson), but the core sequence for Quaternary sediments looks like this. Not every sample needs every step.
| Step | What it removes or does | Typical reagent or tool | Main risk |
|---|---|---|---|
| 1. Measure and spike | Fixes a known sample volume or mass and adds marker spores so concentrations can be calculated | Volumetric sampler; Lycopodium tablets | Low |
| 2. Carbonate removal | Dissolves calcium carbonate (shells, marl, chalk) and the tablet matrix | Dilute hydrochloric acid (HCl) | Corrosive; CO₂ frothing |
| 3. Humic removal | Breaks down humic acids and disaggregates organic sediment | Hot dilute potassium or sodium hydroxide (KOH/NaOH) | Caustic |
| 4. Sieving | Removes coarse debris and, with a fine mesh, clay-size particles | Coarse mesh; fine mesh around 7–10 µm on some protocols | Loss of very small grains if mesh is too coarse |
| 5. Silicate removal | Dissolves quartz and clay minerals, or separates pollen from them by density | Hydrofluoric acid (HF), or dense liquid such as sodium polytungstate | HF: very high |
| 6. Acetolysis | Dissolves cellulose and cell contents; cleans and slightly darkens the exine | Acetic anhydride + concentrated sulphuric acid, heated | High: corrosive, reacts with water releasing heat |
| 7. Dehydrate and mount | Transfers the residue into the mounting medium | Ethanol / tert-butyl alcohol (for silicone oil) or water (for glycerine jelly) | Low to moderate |
Between steps the sample is centrifuged, the liquid is poured off, and the residue is washed. Washing matters as much as the reagents: carried-over acid or water can ruin the next step. A glacial acetic acid rinse before acetolysis, for example, removes water that would otherwise react with the acetic anhydride.
Acetolysis: what Erdtman's method does and doesn't do
Erdtman described the method in its revised form in 1960, and it became the default treatment in pollen morphology and much of Quaternary palynology. You will find the short definition in our palynology glossary under “acetolysis”.
What happens chemically
The mixture of acetic anhydride with a little concentrated sulphuric acid acetylates and breaks down cellulose and other polysaccharides. Heated briefly in a water bath, it strips away cell contents, the cellulose-rich inner wall (intine) and much fine plant debris. Sporopollenin is not dissolved, so the exine survives and its sculpture and apertures stand out.
What it does well
- Cleans the residue. Cellulose fragments that would clutter the slide are removed.
- Clarifies wall features. Apertures, pores and ornament become easier to see because nothing inside the grain obscures them.
- Standardises appearance. Fossil grains have lost their contents anyway; acetolysing reference pollen makes modern and fossil grains directly comparable.
What it does to the grains
Acetolysis is not neutral. Hesse and Waha (1989) put it bluntly: acetolysis “destroys all pollen material with the exception of sporopollenin”. They documented “total breakdown or gross modification of thin exine structures” in some genera, and argued that serious morphological work should look at both acetolysed and non-acetolysed pollen.
Size changes too. Reitsma (1969), measuring recent hazel (Corylus avellana) and oak (Quercus robur) pollen, found that the size of acetolysed grains was affected by the treatment before acetolysis and by the duration of acetolysis. That matters whenever size is used to separate taxa, for example cereal-type from wild grass pollen.
Removing minerals: HF and alternatives
Many sediments are mostly silt and clay. Those silicate minerals do not dissolve in hydrochloric acid or alkali, so something else is needed.
Why HF is used in palynology
Hydrofluoric acid dissolves silicate minerals while leaving sporopollenin untouched. In mineral-rich lake sediments and most sedimentary rocks it is the most effective way to get a concentrated residue. After HF, a hot HCl wash is commonly used to remove fluoride precipitates that would otherwise cloud the slide.
The trade-off is safety and waste. HF is exceptionally hazardous (see Lab safety), and it needs dedicated fume hoods, plastic labware, trained staff and controlled disposal.
Alternatives to HF
- Dense-liquid flotation. Pollen and spores are lighter than mineral grains. Spinning the residue in a liquid of suitable density floats the organic fraction off. Munsterman and Kerstholt (1996) introduced sodium polytungstate (SPT) as a non-toxic alternative to bromoform for this separation.
- Tested, but not identical. Leipe, Kobe and Müller (2019) compared SPT and lithium heteropolytungstate with HF on five lake and peat samples. Results partly disagreed statistically with the HF preparations; the differences looked sample-specific rather than taxon-specific. The alternative is workable, but it should be validated on the material in question.
- Non-acid disaggregation of rocks. Riding and Kyffin-Hughes (2006) tested sodium hexametaphosphate and hydrogen peroxide on rocks from the Ordovician to the Paleogene. These methods “generally proved to be as effective as the mineral acid procedure”, though results on Paleozoic samples were more variable and one Ordovician sample failed to break down.
- Fine sieving. Washing through a mesh around 7–10 µm removes much of the clay without chemistry, at the cost of possibly losing very small grains.
Adding markers: Lycopodium tablets for concentrations
A pollen count on its own gives percentages: how much of the assemblage is oak, grass or pine. Percentages cannot tell you whether oak became more abundant or everything else declined. For that you need concentrations (grains per cm³ or per gram) and, with a dated core, accumulation rates.
The standard solution is to add a known number of exotic marker grains at the start. Stockmarr (1971) introduced tablets containing a known number of Lycopodium (clubmoss) spores for this purpose. Each production batch has its own stated mean number of spores per tablet, and the tablets dissolve during the acid steps. The analyst counts marker spores alongside the pollen.
The calculation, with an illustrative example
Pollen concentration = (pollen grains counted × marker spores added) ÷ (marker spores counted × sample volume)
| Input (hypothetical numbers) | Value |
|---|---|
| Sample volume | 1 cm³ |
| Marker spores added (one tablet, batch mean assumed for this example) | 20,000 |
| Pollen grains counted | 400 |
| Marker spores counted on the same traverses | 100 |
| Pollen concentration | 400 × 20,000 ÷ (100 × 1) = 80,000 grains/cm³ |
| If the sediment accumulated at 0.1 cm per year (from dating) | 80,000 × 0.1 = 8,000 grains/cm²/year (pollen accumulation rate) |
The precision of the result depends on how many marker spores you count, not just how many pollen grains. Maher (1981) worked through the statistics of marker-grain concentration estimates in detail; the practical upshot is that counting very few markers gives wide error margins. Always use the batch value supplied with your tablets, never the round figure used above.
Mounting and storing slides
The final residue is stirred into a mounting medium, a drop goes on a slide and a coverslip on top. The choice of medium affects what you can see and how stable the grains stay.
| Glycerine jelly | Silicone oil | |
|---|---|---|
| Handling | Melted, sets as a gel; simple, water-based | Stays liquid; residue must first be dehydrated (e.g. tert-butyl alcohol) |
| Viewing | Grains fixed in place | Grains can be rolled by nudging the coverslip to see other views |
| Grain size | Grains can swell, and the medium affects their state of expansion | Often preferred when size is diagnostic, though solvent residues can affect size |
| Staining | Easy to add stain (e.g. basic fuchsin or safranin) | Staining must be done before dehydration |
| Storage | Coverslip usually sealed; slides kept flat | Coverslip must be sealed (edges ringed) or the oil creeps; kept flat |
Andersen (1960) introduced silicone oil as a mounting medium precisely because “the qualities of the embedding media influence” the state of expansion of pollen exines, which matters for fine identifications. His later work (1978) found that residual solvent from the silicone oil preparation could itself affect the size of hazel pollen. There is no perfect medium: record which one you used and compare like with like.
Whatever the medium, label slides permanently with sample code, depth, preparation date and method, and keep the unmounted residue in a small vial with a preservative if your lab's protocol allows. Residues let you make new slides later without reprocessing the sediment.
Which steps for which sample?
The same word, “preparation”, covers very different workflows. The table below shows why, summarising the published method each field relies on.
| Sample type | Steps usually used | Usually skipped | Why | Reference method |
|---|---|---|---|---|
| Lake sediment / peat (Quaternary) | Markers, HCl, KOH, sieving, HF or dense liquid, acetolysis | Strong oxidation | Mixed mineral and organic matrix; concentrations and clean exines needed for counting | Faegri & Iversen (1989); Moore et al. (1991); Bennett & Willis (2001) |
| Sedimentary rock (pre-Quaternary, incl. energy industry) | Crushing, HCl, HF, heavy-liquid separation, sieving | Often acetolysis | Mineral matrix dominates; the aim is to free palynomorphs from rock, and some palynomorphs can be damaged by harsh treatments | Riding & Kyffin-Hughes (2006, 2007) for acid and non-acid options |
| Honey | Dissolve in water, centrifuge, mount sediment | HF, KOH; acetolysis optional | Almost no mineral matrix; the harmonised method works on the untreated sediment | von der Ohe et al. (2004) |
| Air (volumetric trap tapes) | Tape segment mounted directly on a slide, usually stained | All chemical steps | Grains are already on a clean surface and counts must be fast and comparable across a monitoring network | Galán et al. (2014) |
| Forensic trace samples | Adapted from soil/sediment methods, scaled to tiny samples, with strict contamination controls | Depends on the item and on other analyses planned | Samples are small and irreplaceable, and other experts (e.g. DNA) may need the same material | Wiltshire (2016); Mildenhall, Wiltshire & Bryant (2006) |
If you work with honey, our guide to honey pollen analysis explains the harmonised method step by step, including why some labs add acetolysis and why reference slides must then be prepared the same way.
Lab safety
This section is not a safety protocol. It explains why these steps are restricted to properly equipped laboratories. Always follow your institution's risk assessment, the supplier's safety data sheet and in-person training.
- Hydrofluoric acid (HF). The US Centers for Disease Control and Prevention warns that skin contact “may not cause immediate pain or visible skin damage”, that swallowing even a small amount can affect major organs and may be fatal, and that calcium gluconate products are used in treatment. Labs using HF keep HF-specific first aid on hand and train staff before they touch it.
- Acetic anhydride. The NIOSH Pocket Guide lists eyes, skin and respiratory system as targets, with effects including skin burns and airway irritation, and a ceiling exposure limit of 5 ppm. It reacts with water, releasing heat and acetic acid, which is one reason residues are rinsed in glacial acetic acid before and after acetolysis.
- Concentrated sulphuric acid and hot alkalis. Both are corrosive; the acetolysis mixture is heated, adding a splash and fume hazard.
- Waste. Acid and fluoride wastes need neutralisation and disposal under local rules, not the sink.
When not to acetolyse
Acetolysis is a default, not a rule. There are good reasons to leave it out:
- You need the grain's contents or intine. Studies of pollen development, wall structure or function need non-acetolysed material, as Hesse and Waha (1989) argued.
- You want DNA. Acetolysis destroys everything except sporopollenin, so DNA-based identification (metabarcoding of honey, air or sediment samples, or ancient DNA from pollen) needs untreated material or a separate subsample.
- The standard method doesn't use it. Honey analysis under the harmonised method and routine airborne pollen monitoring work without it. Adding acetolysis changes the appearance and size of grains relative to the method's reference data.
- The sample is small and irreplaceable. In forensic casework, every treatment consumes material. Processing choices are made with the whole case in mind.
- The palynomorphs are fragile. Some thin-walled pollen types and some non-pollen palynomorphs are damaged or lost.
Whatever you choose, report it. A methods section that states the preparation, the mounting medium and the marker batch lets readers compare your counts and sizes with others.
Further reading
The two classic lab manuals are still the best place to see complete protocols with their reasoning: Faegri & Iversen's Textbook of Pollen Analysis and Moore, Webb & Collinson's Pollen Analysis, both listed on our books page. For the next stage, reading what is on the slide, continue with our guide to identifying pollen grains under the microscope.
FAQ
- What is acetolysis mixture?
- It is a freshly prepared mixture of acetic anhydride and concentrated sulphuric acid, classically in a 9:1 ratio, as described by Erdtman. It is heated briefly with the sample to dissolve cellulose and cell contents while leaving the sporopollenin exine intact. It must be made and used in a fume hood by trained staff.
- Why is HF used in palynology?
- Hydrofluoric acid dissolves silicate minerals such as quartz and clay, which make up most of many sediments and rocks, while leaving sporopollenin untouched. That concentrates pollen and spores. Because HF is extremely hazardous, some labs use dense-liquid flotation (for example sodium polytungstate) or non-acid methods instead, after validating them.
- Can you prepare pollen slides without acetolysis?
- Yes. Honey is analysed on the untreated sediment under the harmonised method, air-trap tapes are mounted directly, and DNA or developmental studies need non-acetolysed pollen. Without acetolysis grains keep their contents and look different, so compare them with reference material prepared the same way.
- Does acetolysis change pollen size?
- Yes. Reitsma (1969) found that the size of acetolysed hazel and oak pollen depended on the treatment before acetolysis and on how long acetolysis lasted. The mounting medium also affects size. When size is used for identification, standardise preparation and mounting and use reference data prepared the same way.
- Why add Lycopodium spores to a pollen sample?
- They are exotic markers added in a known number at the start. Counting them alongside the pollen lets you calculate how many pollen grains were in the original sample per cm³ or per gram, not only percentages. With a dated sequence, concentrations become pollen accumulation rates.
Sources
- Erdtman G. (1960). The acetolysis method: a revised description. Svensk Botanisk Tidskrift 54: 561–564.
- Hesse M., Waha M. (1989). A new look at the acetolysis method. Plant Systematics and Evolution 163: 147–152. doi:10.1007/BF00936510
- Reitsma Tj. (1969). Size modification of recent pollen grains under different treatments. Review of Palaeobotany and Palynology 9: 175–202. doi:10.1016/0034-6667(69)90003-7 (open PDF)
- Stockmarr J. (1971). Tablets with spores used in absolute pollen analysis. Pollen et Spores 13: 615–621.
- Maher L.J. (1981). Statistics for microfossil concentration measurements employing samples spiked with marker grains. Review of Palaeobotany and Palynology 32: 153–191. doi:10.1016/0034-6667(81)90002-6
- Andersen S.T. (1960). Silicone oil as a mounting medium for pollen grains. Danmarks Geologiske Undersøgelse IV. Række 4: 1–24. doi:10.34194/raekke4.v4.7007
- Andersen S.T. (1978). On the size of Corylus avellana L. pollen mounted in silicone oil. Grana 17: 5–13. doi:10.1080/00173137809428847
- Munsterman D., Kerstholt S. (1996). Sodium polytungstate, a new non-toxic alternative to bromoform in heavy liquid separation. Review of Palaeobotany and Palynology 91: 417–422. doi:10.1016/0034-6667(95)00093-3
- Leipe C., Kobe F., Müller S. (2019). Testing the performance of sodium polytungstate and lithium heteropolytungstate as non-toxic dense media for pollen extraction from lake and peat sediment samples. Quaternary International 516: 207–214. doi:10.1016/j.quaint.2018.01.029
- Riding J.B., Kyffin-Hughes J.E. (2006). Further testing of a non-acid palynological preparation procedure. Palynology 30: 69–87. doi:10.1080/01916122.2006.9989619
- Riding J.B., Kyffin-Hughes J.E., Owens B. (2007). An effective palynological preparation procedure using hydrogen peroxide. Palynology 31: 19–36. doi:10.1080/01916122.2007.9989632
- Bennett K.D., Willis K.J. (2001). Pollen. In Smol J.P. et al. (eds) Tracking Environmental Change Using Lake Sediments, vol. 3: 5–32. Springer. doi:10.1007/0-306-47668-1_2
- Faegri K., Iversen J. (1989). Textbook of Pollen Analysis, 4th ed. (Faegri K., Kaland P.E., Krzywinski K., eds). Chichester: Wiley.
- Moore P.D., Webb J.A., Collinson M.E. (1991). Pollen Analysis, 2nd ed. Oxford: Blackwell Scientific.
- von der Ohe W. et al. (2004). Harmonized methods of melissopalynology. Apidologie 35: S18–S25. doi:10.1051/apido:2004050
- Galán C. et al. (2014). Pollen monitoring: minimum requirements and reproducibility of analysis. Aerobiologia 30: 385–395. doi:10.1007/s10453-014-9335-5
- Wiltshire P.E.J. (2016). Protocols for forensic palynology. Palynology 40: 4–24. doi:10.1080/01916122.2015.1091138
- Mildenhall D.C., Wiltshire P.E.J., Bryant V.M. (2006). Forensic palynology: why do it and how it works. Forensic Science International 163: 163–172. doi:10.1016/j.forsciint.2006.07.012
- CDC. Hydrogen fluoride: chemical emergencies fact sheet. cdc.gov
- NIOSH Pocket Guide to Chemical Hazards: acetic anhydride. cdc.gov/niosh