Total Spore Count or Culture? The Right Method for Your Question

“Without culture there is no result.” – “You only count dead spores.” Anyone who sends air samples to a mould lab hears these lines sooner or later. Behind them are two laboratory methods that answer different questions and therefore produce different numbers: spores/m³ and CFU/m³.

This article puts the two side by side: the total spore count to DIN ISO 16000-20 and the culture-based method to DIN ISO 16000-17/-18. It explains how each works, where its limits lie, what the German UBA guideline and the ISO standards say – and which method fits which question.

In short: the total spore count counts every spore under the microscope, living or dead, and delivers spores/m³ per spore type within days. Culture grows viable spores on agar and delivers CFU/m³ after about a week, with identification down to species. The UBA guideline lists both as recognised rules of the trade and provides an assessment aid for each. Which one fits is decided by the question – not by the lab.

Two questions, two methods

Every air sample starts with a question. Either: how many mould spores are in this air, and of which types? Or: which viable species grow from it?

The first question is answered by the total spore count – also called particle sampling or spore-trap sampling. Spores are collected on a slide and counted under the microscope; the result is spores/m³. The second question is answered by culture, the classic viable air sampling: spores are collected on agar plates and incubated; the result is colony-forming units per cubic metre, CFU/m³.

The two numbers cannot be converted into each other. A colony can grow from a single spore or from an aggregate of spores, and a large share of the spores never forms a colony at all.

The UBA guideline puts it plainly: there is no sampling and detection method that suits every question. The investigation strategy has to match the objective, and the expert should set out in advance what conclusions the chosen method allows.

Total spore count: counting instead of growing

How the analysis works

A pump draws a defined volume of air through a sampling cassette. Inside, the air stream passes through a slit and hits a coated slide, and the spores stick to the adhesive layer – slit impaction to DIN ISO 16000-20. In the lab the trace is stained and read under the microscope.

Everything that is morphologically recognisable as a fungal spore is counted, plus hyphal fragments. The result is reported per spore type in spores/m³ and compared with an outdoor sample taken at about the same time.

Strengths

• The complete picture. Culturable and non-culturable spores are captured, living and dead alike. That matters for health: according to DIN EN ISO 16000-19, non-culturable spores do not differ from culturable ones in their allergenic and toxic effects.
• Moisture indicators that never show up on agar. The UBA guideline gives Stachybotrys chartarum as its example: often not detectable by culture, but detectable through the total spore count.
• Speed. The time-consuming incubation is not needed; the result is usually available within days.
• Clearance testing after biocide use. Killed spores are still allergenic. Only the total spore count shows whether they were actually removed after the biocide – the UBA guideline singles out exactly this case.

Limits

• No statement on viability. Living and dead spores look the same under the microscope.
• Identification only to genus or group. Many small, round spores cannot be told apart morphologically. Aspergillus and Penicillium are therefore reported as one spore type – the assessment aid in the UBA guideline (Table 10) works the same way. Species identification, say Aspergillus versicolor versus Aspergillus fumigatus, is not possible.
• Bacteria are not captured.
• Experience decides. Microscopic reading needs trained eyes and quality assurance; the UBA guideline points to proficiency tests for total spore counting.

Culture: what is alive and grows

How the analysis works

In viable air sampling the air is either directed straight onto agar plates (impaction, DIN ISO 16000-18) or collected on a filter that is then washed out and plated (filtration, DIN ISO 16000-16). DIN ISO 16000-17 specifies two media, DG18 agar and malt extract agar, incubated at 25 ± 3 °C.

Incubation usually takes about a week, with intermediate counts so that fast-growing colonies do not swamp the slow ones. At the end the colonies are counted and identified; the result is CFU/m³ per species or genus. For special questions, such as Aspergillus fumigatus, plates are additionally incubated at 36 °C or 45 °C.

Strengths

• Identification to species. A colony can be identified to species level – important where individual moisture indicators such as Aspergillus versicolor or clinically relevant species such as Aspergillus fumigatus are the point.
• Proof of viability. Only what grows is counted. Where the question is active growth, that is information in its own right.
• Broad track record. According to the UBA guideline, culture is the most widely used method; the assessment aid in Table 9 of the guideline is based on CFU/m³.

Limits

• Time. The result arrives after incubation at the earliest – for a clearance test with the containment still standing, a real cost factor.
• Viable spores only. Spores that dry out or die under sampling stress, or do not grow on the chosen medium, are missing from the result. The UBA guideline: depending on the spore spectrum, culture detects “markedly fewer moulds than are actually present”; some typical moisture indicators are generally poor growers.
• Selectivity of the medium. DG18 favours dry-tolerant species, malt extract others – hence two plates per sample. Whatever grows on neither remains invisible.
• Statistics. A robust count needs at least ten colonies per plate according to the UBA guideline; low concentrations of rare species are hard to quantify.

Which method for which question?

The overview matches typical questions to the methods; it does not replace an investigation strategy for the individual case.

Two rules apply to both methods. First: without an outdoor sample or a reference room no assessment is possible. Second: air samples are snapshots with a wide scatter – the UBA guideline recommends several measurements, for instance two volumes in duplicate.

What the guidelines say

UBA guideline (2017, updated 2024)

The guideline of the German Environment Agency lists both methods in its overview of recognised rules of the trade: the measurement of culturable moulds in air to DIN ISO 16000-16 to -18 and the total spore count to DIN ISO 16000-20. It justifies the total spore count by the fact that, as a rule, only part of the spores are culturable and that non-culturable spores have allergenic and toxic effects too; it is therefore “useful in many cases”. Each method has its own assessment aid – Table 9 for CFU/m³, Table 10 for spores/m³ – and for the check after remediation the guideline explicitly names one method or the other.

The guideline contains no obligation to combine both methods on air samples. It describes them as methods with different conclusions and leaves the choice to the question at hand. For material samples, by contrast, it explicitly recommends running microscopy and culture in parallel – more on sampling to the guideline in UBA mould guideline 2024: sampling.

DIN EN ISO 16000-19, DIN ISO 16000-20 and -17

The sampling strategy in DIN EN ISO 16000-19 states that non-culturable spores equal culturable ones in their allergenic and toxic effects, and that different methods are used “depending on the question”. A site inspection by a competent person comes before any measurement.

Part 20 describes the total spore count: impaction onto an adhesive surface, microscopic counting, no culture – the total spore count “including culturable and non-culturable spores”. Part 17 governs culture on DG18 and malt extract agar at 25 ± 3 °C.

VDI 4300 Part 10 and WTA 4-12

VDI 4300 Part 10 (2008) was the German measurement strategy for moulds and is still cited in older expert reports. The guideline has since been withdrawn; its content went into DIN EN ISO 16000-19 and DIN ISO 16000-20, and the UBA guideline refers to “the former VDI 4300 Part 10”. The current reference for strategy is DIN EN ISO 16000-19.

For verifying the success of a remediation, the German WTA guideline 4-12-21/D applies as well. The procedure is described in Clearance testing after mould remediation.

AirTrap XL and Sporecyte: the total spore count in practice

AirTrap XL is a spore-trap cassette for the total spore count: slit impaction onto a coated slide, the principle of DIN ISO 16000-20. Sampling runs with the Sporecyte pump at 15 l/min for 5 minutes, i.e. 75 litres per sample. More on What is AirTrap XL.

The slides are scanned at Apacor in Wokingham and analysed in the ISO/IEC 17025-accredited Sporecyte laboratory. Spore identification is AI-assisted; every result is checked by a specialist before the report is released. The report arrives within 48 hours of the samples reaching the lab: spores/m³ per genus, every indoor sample compared with the outdoor air, an elevation rating and images from the slide – more under What is Sporecyte and in Lab analysis.

Just as important is what the method does not do:

• No CFU, no statement on viability, no identification to species. Aspergillus and Penicillium are reported together.
• With 75 litres, a single counted spore corresponds arithmetically to around 13 spores/m³. Rare types with poor airborne dispersal therefore sit close to the detection limit. Since the UBA guideline treats even single spores of such types as a first indication, duplicate samples are worth taking.
• Without an outdoor sample there is no interpretation.
• The report supports the assessment by a competent person; it does not replace it. How to read the numbers is explained in Understanding your mould lab report.

If your question needs species identification or proof of viable spores, culture is the right method – in addition to or instead of the total spore count. Both samples can be taken at the same appointment at the same sampling point.

Frequently asked questions about total spore count and culture

“You only count dead spores” – so what does the result tell me?
All spores are counted, living and dead. For allergenic and irritant effects that is the relevant figure: according to the UBA guideline and DIN EN ISO 16000-19, non-culturable spores act the same way. For the question of a source in the room, what counts is the comparison with outdoor air and the spore spectrum, not viability. What the total spore count cannot do is say whether something is actively growing – for that you need culture or a material sample.

“Without culture there is no result” – is that true?
No. The UBA guideline lists the total spore count to DIN ISO 16000-20 as a recognised rule of the trade, gives it its own assessment aid and names it alongside culture as an option for the post-remediation check. What is true: without culture there is no species identification and no statement on viability. If that is what you need, you need culture.

Can I convert spores/m³ into CFU/m³?
No. Spores/m³ are usually markedly higher, because only part of the spores grow on agar and aggregates form just one colony. Assess each number within its own method – against the outdoor sample taken with the same method and with the matching assessment aid of the UBA guideline.

Why does the lab report Aspergillus and Penicillium together?
Their spores are small, round and cannot be reliably told apart under the microscope. The assessment aid in the UBA guideline works with the Penicillium/Aspergillus type for the same reason. If the species matters – Aspergillus versicolor as a moisture indicator, say, or Aspergillus fumigatus in a health question – culture is the way to go.

Which method is accepted in expert reports or in court?
Both, provided they are carried out to the standard, documented and analysed by a qualified lab. The UBA guideline recommends laboratories accredited to DIN EN ISO/IEC 17025 that take part in proficiency tests, and expects the expert to present an investigation strategy that makes clear what conclusions the chosen method allows. Whether a measurement is sufficient in a specific case is for experts and lawyers to judge.

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Written by the AirTrap team. Sources: UBA guideline on the prevention, detection and remediation of mould in buildings (2017, updated 2024), DIN EN ISO 16000-19, DIN ISO 16000-17, DIN ISO 16000-20, VDI 4300 Part 10 (withdrawn), WTA guideline 4-12-21/D. This article is professional guidance and does not replace the assessment of a competent person.