Which Sampling Method Should One Use for Airborne Fungal Spores?

Concerns about health issues, especially allergic reactions from inhaling fungal spores, has made air sampling an important component of indoor mold investigation.

Air can either be sampled onto some growth media for culture analysis (a.k.a viable or culturable samples) or on a sticky surface or a filter membrane for direct microscopic examination (non-viable or non-culturable samples).

Often, it is debated as to whether one should take non-viable samples, viable samples or a combination of the two. Either method can be used without the other or both can be used together (at the same time) depending on the objectives of the investigation. For marijuana grow operations, the Calgary Health Regions requires use of both methods for fungal air testing.

Both methods have their advantages and disadvantages that need to be taken into account when deciding which method to use. Before we discuss when one should use non-viable or viable sampling, let us understand what the terms non-viable, viable and spore trap mean.

Non-viable Air Samples

“Non-viable air samples” refer to samples that are taken on some sticky media or on a filter membrane or tape and subsequently examined directly under a microscope for enumeration and identification of mould spores and hyphal fragments without culturing. In other words, the samples are taken for analyses by direct microscopic examination (DME).

Results are presented as a listing of various categories of moulds and the corresponding number of spores or hyphal fragments per cubic meter of air (Spores/m³). This term is technically inaccurate since it seems imply that the spores collected using this method are dead. However, both viable and non-viable propagules are collected but are indistinguishable under the microscope and hence both are enumerated.

The major advantage of non-viable sampling is that the observation of spores under the microscope is not dependent on the viability of spores or not. The other advantage is that since the samples do not require culturing, results can be obtained the same day the samples were collected. One of the disadvantages of this method is that majority of spores can only be identified to group level (genus) and some are recorded as unidentified spores.

Viable Air Samples

“Viable air samples” refer to samples that are taken on some growth media and subsequently incubated for mould propagules (spores and/or hyphal fragments) to germinate and form colonies. The resulting colonies are then enumerated and/or transferred to other media for identification to genus or species. Results are presented as a listing of the recovered moulds and their corresponding number of colony forming units per cubic meter of air (CFU/m³).

That is, the analysis of viable air samples involves culturing. The term is also technically inaccurate because some (sometimes most) of the propagules impacted on the growth media may not germinate not because they are not viable but because of the selectivity of the growth media used, competition from fast growing moulds or that some moulds can only grow on living hosts.

The major advantage of viable sampling is that the moulds can be identified to individual (species) level. The disadvantage of this method is that it cannot detect dead spores yet these spores can still cause allergic reactions.

Spore traps

“Spore traps” is commonly used to refer to non-viable air samples. However, whether sampling is done for culture analysis with an RCS, Andersen or for DME with Air-O-Cell or other similar cassettes it involves spore trapping. “Spore traps” is therefore applicable to both viable and non-viable samples.

When should one use non-viable, viable or both sampling methods?

The easiest way to decide on this is first to define the objectives of air sampling, decide on data required from sample analysis and the questions these data are meant to answer. The objective might be broad or very specific. It’s important to know that some spores are better identified and quantified via non-viable samples while others require viable samples.

When to use non-viable sampling

If the objective of air sampling was to have an idea of how contaminated the air is, then the data required would be total fungal spore counts. Non-viable samples would then be the best to take because counting includes both those propagules that can grow on laboratory media and those which cannot grow either because they are dead or would not grow on the selected media.

Non-viable sampling may also be selected when the objective of air sampling is to determine the total counts for airborne spores prior to and after remediation to assess the effectiveness of remediation. In this case viable air samples would not be necessary.

When to use viable sampling

If the objective of air sampling was to determine whether the air contains a specific species of mould e.g., Aspergillus fumigatus, then viable sampling is required since non-viable analysis would not distinguish A. fumigatus from other Aspergillus species and not even from Penicillium species and related genera.

For detecting a specific species, a selective media that would support the growth of the mould of interest would also be selected. If identification to species was required for a broad range of moulds, then media that support growth of a wide range of moulds should be selected.

When to use both non-viable and viable sampling

If the objective of air sampling was to determine the total airborne mould concentration and at the same time determine the proportion of viable propagules, their composition and the species then both sampling methods should be used. This would possibly be the case in hospitals where concern is not only the total concentration of airborne mould but also the viable species present.

Conclusion

Viable and non-viable air samples are complementary since each have limitations that can be overcome by using the other. However, one may use either method on it’s own depending on the objectives of air sampling, the data required and the questions these data are intended to answer. You can learn more about our mold testing services here.

The guidelines may be used to decide whether further investigations are required after initial investigations. However, numerical laboratory results cannot be used as the primary determinant of whether there is a mould problem but should always be used together with visual inspection data and other available information such as the building history. See our course How To Interpret Mold Results.

Generally, indoor airborne mould concentrations are compared with those of outdoor. The presence of one or more species of mould indoors, but not outdoors, suggests presence of a growth source in the building. This comparison may not be possible during winter since outside concentrations may be below the detection limits as mould growth is slowed by the cold weather. The same principles for comparing outside with indoor can be used to compare the test rooms with the control rooms.

These guidelines are mainly summarized from those developed by the German Federal Environmental Agency (Umweltbundesamt, 2002) and the Health Canada (Indoor Air Quality in Office Buildings: A Technical Guide, 1993).

Guidelines for Interpretation of Numerical Data of Non-viable Mould Air Samples

For a comprehensive assessment of the sample results, all the six steps require to be followed where applicable.

1. Consider the concentrations of spore types which may reach high concentrations in the outside environment (for example Ascospores, Alternaria/Ulocladium, Basidiospores, Cladosporium).
   • If the total concentration of these spore types in the indoor air is lower than or equal to 1 to 1.4 times the outside concentration, i.e., I_c ≤ O_c x 1 (+0.4), then indoor source is unlikely (background level).
   • If the total concentration of these spore types in the indoor air is lower than or equal to 1.6 (± 0.4) times the outside concentration, i.e., I_c ≤ O_c x 1.6 (± 0.4), then indoor source is possible and further investigations are required.
   • If the total concentration of these spore types in the indoor air is more than twice the concentration in outside air, i.e., I_c > O_c x 2, then indoor source is likely and immediate further investigations are required.

1. Consider the concentration of Penicillium/Aspergillus spore types.
   • If the difference in concentration between indoor air and outside air is lower than or equal to 300 spores/m3, i.e., I_c – O_c ≤ 300, then indoor source is unlikely (background level).
   • If the difference in concentration between indoor air and outside air is greater than 300 spores/m3 but lower than or equal to 800 spores/m3 i.e., I_c – O_c > 300 ≤ 800, then indoor source is possible and further investigations are required.
   • If the difference in concentration between indoor air and outside air is more than 800 spores/m3, i.e., I_c – O_c> 800, then indoor source is likely and further investigations are required immediately.

     

2. Consider the concentration of Chaetomium spp.

•  If the concentration in indoor air is lower or equal to the concentration in outside air, i.e., I_c ≤ O_c, then indoor source is unlikely (background level).

• If the difference in concentration between indoor air and outside air is lower than or equal to 5 spores, i.e., I_c – O_c ≤ 5, then indoor source is possible and further investigations are required.
• If the difference in concentration between indoor air and outside air exceeds 5 spores, i.e., I_c – O_c > 5, then indoor source is likely and further investigations are required immediately.
• Consider the concentration of Stachybotrysspp.
  • If the concentration in indoor air is lower or equal to the concentration in outside air, i.e., I_c ≤ O_c, then indoor source is unlikely (background level).
  • If the difference in concentration between indoor air and outside air is not more than 2 spores, i.e., I_c – O_c ≤ 2, then indoor source is possible and further investigations are required.
  • If the difference in concentration between indoor air and outside air exceeds 2 spores, i.e., I_c – O_c > 2, then indoor source is likely and further investigations are required immediately.
• Consider the concentration of various other unidentified fungal spores that do not belong to the basidiospore or ascospore types.
  • If the difference in concentration between indoor air and outside air is not more than 400 spores/m³, i.e., I_c – O_c ≤ 400, then indoor source is unlikely (background level).
  • If the difference in concentration between indoor air and outside air is greater than 400 spores/m³ but not more than 800 spores/m³, i.e., I_c – O_c > 400 ≤ 800, then indoor source is possible and further investigations are required.
  • If the difference in concentration between indoor air and outside air is exceeds 800 spores/m³, i.e., I_c – O_c > 800, then indoor source is likely and further investigations are required immediately.
• Consider the concentration of hyphal fragments.
  • If the difference in concentration between indoor air and outside air does not exceed 150 fragments/m³, i.e., I_c – O_c ≤ 150, then indoor source is unlikely (background level).
  • If the difference in concentration between indoor air and outside air is greater than 150 fragments/m³ but not more than 300 fragments/m³, i.e., I_c – O_c > 150 ≤ 300, then indoor source is possible and further investigations are required.
  • If the difference in concentration between indoor air and outside air exceeds 300 fragments/m³, i.e., I_c – O_c > 300, then indoor source is probable and further investigations are required immediately.

Guidelines for Interpretation of Numerical Data of Viable Airborne Mould Samples

These will be covered in Part II of this article. Or feel free to visit our homepage to view more mould and bacteria testing information, resources and services.

References

Health Canada (1993). Indoor air quality in office buildings: a technical guide. A report of the Federal Provincial Advisory Committee on Environmental and Occupational Health. 55 pp.

Umweltbundesamt (2002). Leitfaden zur Vorbeugung, Untersuchung, Bewertung und Sanierung von Schimmelpilzwachstum in Innenräumen. Erstellt durch die Innenraumlufthygienekommission des Umweltbundesamtes.