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Scientists cannot prove that a prehistoric substance was used as medicine simply because it looks medicinal in an excavation. They build a testable chain of evidence: establish the material’s archaeological or historical context, recreate a plausible sample, expose selected microorganisms to it under controlled laboratory conditions, measure growth against positive and negative controls, and then test whether the result survives chemical, toxicological and host-relevant follow-up. A clear zone on an agar plate can show antibacterial activity under those exact conditions. It cannot by itself prove deliberate prehistoric treatment, effectiveness inside a human body, an appropriate dose or safety.
A 2026 peer-reviewed study of reconstructed birch tar illustrates the method. Some samples inhibited Staphylococcus aureus, while none measurably inhibited Escherichia coli. The result is evidence of organism-specific activity in an exploratory assay—not proof that Neanderthals used birch tar as an antibiotic or that anyone should use it as a remedy.
The evidence chain scientists try to build
Testing an ancient material is an experimental reconstruction, not a time machine. Researchers must connect an excavated object, a historical recipe or an ethnographic parallel to a reproducible laboratory sample, then separate what the experiment demonstrates from what it merely makes plausible.
1. Form a historical and archaeological hypothesis
The starting point is context. Investigators ask where the material was found, how old the context is, what it was made from, and whether there is evidence for its production or use. A substance associated with wounds, tools or burial deposits may prompt different hypotheses from one found in food-processing equipment.
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For birch tar, archaeological finds establish tar as a hafting adhesive in Middle Palaeolithic contexts. Historical and ethnographic accounts also record medicinal uses of birch-derived products in some communities. Those records justify asking whether tar could inhibit bacteria, but they do not demonstrate that prehistoric people intended to treat infections. The medicinal interpretation remains a hypothesis to test.
2. Reconstruct a plausible sample
Ancient material is often altered by burial, contamination, evaporation or chemical transformation. A modern substitute must therefore match the proposed raw ingredients and production conditions as closely as practical. Researchers document species, mineral composition, heating or extraction, containers, temperatures and contamination controls so that another laboratory can reproduce the sample.
In the 2026 birch-tar study, investigators used bark from Betula pendula and Betula pubescens. They produced tar by three routes: tin distillation, a raised clay structure and a condensation method. The procedures were chosen as experimental reconstructions relevant to prehistoric production, while the tin process also has historical documentation. Comparing methods matters because heat, oxygen exposure, vessel material and condensation can change the chemicals that reach a test culture.
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A material that affects one bacterial group may fail against another. Good experiments identify the organisms precisely and include contrasting cell types when the question concerns broad antibacterial action. The birch-tar researchers tested S. aureus ATCC 25923, a Gram-positive bacterium, and E. coli ATCC 25922, a Gram-negative bacterium. The strain numbers make the work reproducible rather than leaving “staphylococcus” or “coliforms” undefined.
The choice is still a limited screen. Two laboratory strains do not represent every pathogen, resistant isolate, fungus or virus that could have affected people in the past. A negative result against one organism narrows the claim; it does not show that the material has no biological activity whatsoever.
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4. Use a controlled growth assay
One common first screen is agar disc diffusion, related to the Kirby–Bauer method. Bacteria are spread across an agar plate to create a uniform lawn. A measured amount of the candidate substance is placed on a sterile paper disc, and the plate is incubated. If growth is reduced around the disc, the clear ring is measured in millimetres.
For the birch-tar experiment, each sterile filter-paper disc was 6 mm in diameter and received 20 μL of tar solution. The discs were placed on agar inoculated with the two test bacteria. Gentamicin was the positive control: an established antibiotic should produce a clear zone if the culture and incubation conditions are functioning. Dimethyl sulfoxide (DMSO), the solvent control, was expected not to inhibit growth. Experiments were performed in duplicate.
5. Measure against controls, not against an impression
The diameter of the clear zone is a measurement of visible growth reduction in that plate, not a direct measure of clinical potency. A larger ring can result from stronger inhibition, but it can also reflect how readily a chemical diffuses through agar. A poorly diffusing compound may look weak even when it affects cells on contact; a highly diffusing compound may produce a broad ring at a concentration that would not be achievable in tissue.
Controls establish whether the result is interpretable. A working gentamicin zone shows that the test bacteria and assay supported inhibition under the reported conditions. No zone around DMSO indicates that the solvent, rather than the tar, did not account for the effect. Replication helps reveal variability, although duplicate plates are still a small sample and do not replace a larger statistical design.
6. Confirm, identify and test relevance
A screening result is the beginning of validation. Researchers can repeat the experiment with independent preparations, use broth-growth or absorbance measurements, determine concentration dependence, and test whether the effect is bactericidal or merely slows growth. Chemical analysis can identify the active constituents and show whether different production methods create different mixtures.
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To approach a medical question, testing must also move beyond bare agar. Host-mimicking media, protein-rich fluids, wound-like conditions and models that approximate infection environments can reveal whether activity survives in the presence of salts, proteins and human cells. Toxicity testing is essential before any claim about treatment is made.
The 2026 birch-tar study, step by step
What was reconstructed
The researchers made birch tar from two birch species and three production processes. This design allows a limited comparison of raw material and manufacturing route instead of treating “birch tar” as one chemically uniform substance. The samples were experimental reconstructions, not portions of tar recovered unchanged from a Neanderthal site.
What was tested
The assay used S. aureus ATCC 25923 and E. coli ATCC 25922. The team applied 20 μL of each tar solution to 6 mm sterile discs on bacteria-seeded agar, with gentamicin as the positive control and DMSO as the negative control. Each condition was run in duplicate.
What the plates showed
| Observation | Reported result | What it supports |
|---|---|---|
| Largest birch-tar zone | 10.5 ± 0.7 mm for one raised-structure sample against S. aureus | That reconstructed sample reduced visible growth of S. aureus under the assay conditions. |
| Other active samples | Several samples produced zones of 7.0–7.5 mm against S. aureus; one sample showed no activity | Activity varied with the reconstruction and was not consistent across all samples. |
| E. coli response | No measurable inhibition beyond the 6 mm disc for any of the six samples | No detectable activity against this strain in this assay; it does not establish inactivity against every Gram-negative organism. |
| Positive control | Gentamicin zones of 22–23 mm against both bacteria | The assay’s antibiotic control worked under the reported conditions. |
| Negative control | DMSO produced no inhibition | The solvent did not explain the clear zones. |
The contrast is important. The largest tar ring, 10.5 ± 0.7 mm, was less extensive than the 22–23 mm gentamicin control and occurred only with one reconstruction against one organism. Reporting the organism, sample type and control in the same sentence prevents the number from being mistaken for a general antibiotic potency measurement.
What the result does and does not establish
The defensible conclusion is narrow: some experimentally produced birch-tar samples inhibited S. aureus in an agar diffusion test, with effects varying by sample. The same six samples did not measurably inhibit E. coli in that assay.
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- It does not demonstrate broad-spectrum antibacterial activity.
- It does not establish an effective dose, treatment schedule or ability to clear an infection in a person.
- It does not prove that Neanderthals deliberately applied birch tar to wounds.
- It does not establish that the reconstructed material is safe on skin, in a wound or inside the body.
The authors describe the work as exploratory and call for tests that isolate the variables affecting activity, improve measurement and characterize the chemistry. Birch-tar extracts can contain compounds associated with toxic or mutagenic concerns, including combustion-related chemicals. A laboratory inhibition ring is therefore not a recommendation to use tar medicinally.
Why one assay is not enough
Diffusion is not the same as dose
Disc diffusion measures the combined effect of a substance’s activity, concentration and movement through agar. Complex archaeological materials may contain poorly soluble compounds, volatile components or particles that remain close to the disc. Researchers should therefore report the preparation and solvent and, where possible, confirm the result with liquid-culture growth measurements or another quantitative method.
Biology depends on the test organism
Gram-positive and Gram-negative bacteria differ in cell-envelope structure, which can change susceptibility. A result for S. aureus cannot be generalized to E. coli, fungi, parasites or viruses. Testing additional strains, including clinically relevant resistant isolates, can show whether an effect is narrow or reproducible.
Replication and statistics matter
Duplicate experiments provide an initial check but leave substantial uncertainty about variation between preparations, plates and days. Stronger evidence uses independent batches, more replicates, blinded measurement where feasible, prespecified concentrations and statistical analysis that reports uncertainty rather than only the largest zone.
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Historical medicinal clay demonstrates why methods must be adapted to the substance. Samian Earth is not interchangeable with birch tar, and evidence for one cannot be transferred to the other.
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A 2015 Journal of Archaeological Science study combined scrutiny of ancient texts with field and laboratory investigation of Samos bentonitic clays. The researchers examined the clay’s identity and mineralogy and tested candidate compositions against selected bacterial cultures. They reported that a few hundred parts per million of boron or boric acid in the clays could significantly reduce bacterial colonies in their experiment.
The result links a chemical component to an observed laboratory effect; it does not prove that every historical dose contained the same amount, that users understood boron’s role, or that the clay was safe or effective for all illnesses. Later work on archaeological medicinal earths likewise combines chemical and mineralogical characterization with bioactivity testing. In clay studies, particle size, mineral phases, trace elements and preparation can all matter; in tar studies, plant species and thermal process may dominate.
| Comparison point | Birch tar | Medicinal clay |
|---|---|---|
| Material evidence | Middle Palaeolithic tar associated with hafting, plus historical and ethnographic medicinal parallels | Ancient textual references and Samos bentonitic clays investigated in the 2015 study |
| Reconstruction or characterization | Betula pendula and Betula pubescens; three production methods | Mineralogical and chemical examination of candidate clays and their components |
| Biological readout | Disc-diffusion inhibition zones against two named bacterial strains | Reduction of bacterial colonies in the reported experiment |
| Key reported finding | Some activity against S. aureus; none against E. coli beyond the disc | A few hundred ppm of boron or boric acid associated with significant colony reduction |
| Interpretive limit | Does not prove prehistoric medicinal intent, clinical efficacy or safety | Does not make all historical clays equivalent or establish clinical treatment value |
How to judge a claim about an ancient antibiotic
When reading a paper, museum label or popular claim, check each link in the evidence chain:
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- Reconstruction fidelity: Does the experiment reproduce plausible ingredients, heating, extraction and storage? Are contamination and solvent controls described?
- Microbial scope: Are organisms identified to strain, and does testing include more than one bacterial type?
- Assay design: Is the method disc diffusion, liquid growth, colony counting or another measurement? What positive and negative controls were used?
- Replication: Were independent preparations and multiple replicates tested, or was the result obtained once?
- Strength and specificity: Is the effect concentration-dependent? Does it reproduce across batches and organisms? Are uncertainty and measurement limits reported?
- Chemical explanation: Have active compounds or minerals been identified, and do they vary with production method?
- Safety and translation: Has toxicity been assessed in relevant cells or models? Has the material been tested in host-mimicking conditions rather than only on bare agar?
- Historical inference: Does archaeological evidence show use for medicine, or is medicinal use merely plausible from later texts or ethnography?
How a careful conclusion should be worded
Scientific language should preserve the boundary between observation and interpretation. “The reconstructed sample inhibited S. aureus in an agar diffusion assay” describes an observation. “The material was an antibiotic used by Neanderthals” adds claims about prehistoric intent, human effectiveness and terminology that the experiment cannot establish.
Tjaark Siemssen and coauthors state the limitation directly in their 2026 PLOS ONE article: “Yet, further research going beyond the exploratory character of this study is necessary, that isolates the parameters that affect the antibiotic activity of birch tar produced using different methods, and by checking results with the cultivation method or using absorbance measurements, the accuracy of the results could be further improved.” That is the appropriate endpoint for a preliminary finding: a reproducible lead for better experiments, not a recovered prescription.
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