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How to improve spore trap results

Spore trap data should be reliable if results are to be used in assessing indoor spore types and concentrations and ultimately to evaluate the validity of exposure potential or remedial success.  Spore trap results are primarily used in indoor mold investigations to:

  • assess the airborne spore concentrations and the potential for human mold exposure
  • justify remedial recommendations and implementation
  • evaluate quality control and post-remedial assessments of mold remediation projects.
spore trap results
Non-viable Air Sampling

In an earlier article, Understanding Spore Trap Results, we discussed the factors that influence the reliability of spore trap results. These factors include:

  • The amount of air sampled
  • The non-uniform distribution of spores in the air
  • Related to the non-uniform distribution of spores in the air, is the distance from the source to the air sampling point. For actively growing mold, the spore concentration is higher closer to the moldy structures/materials
  • Changes in fungal spore types and concentrations within the day. The release of spores from moldy materials is not continuous and is dependent on ambient conditions such as temperature, air movement and relative humidity (rh). Ambient conditions also influence the types of spores released. For example, at very high humidity  fewer spores of Aspergillus/Penicillium are released than at low rh
  • The experience of the analyst.

How to improve spore trap results

Bearing in mind the factors that influence the reliability of spore trap results, how can we then improve the spore trap results? Below are some suggestions on how we could minimize the effect of the above factors. Professional judgement is required as some of the some of the suggestions may not be applicable in every situation. Since most of the suggestions involve increasing the number of samples collected, it is important to evaluate whether the added cost and sampling time are worth it.

Amount of air sampled

By increasing the amount of air sampled we can improve the spore trap results. As was discussed in article, Understanding Spore Trap Results, by collecting 75L of air we’re sampling only 7.5% of 1000L, the ideal amount of air that should be sampled. Unfortunately increasing the amount of air especially in a dusty environment, would lender the spore traps difficult if not impossible to analyze. We therefore recommend collecting at least 150L or more whenever the environmental conditions allow. If sampling a dust free environment, collecting more than 150L would be recommended. If using the 15L per minute pumps, it would take slightly more than an hour to collect a sample which, to some extent, takes care of the changes of spore concentration with time.

Non-uniform distribution of spores in the air

Since the spores are not uniformly distributed in the air, the only way to minimize the effect of this factor is to take more than 1 sample at different locations within the same room. For example if collecting air samples in a room, you could take a sample from each corner of the room and one in the middle, that means a total of 5 samples. If dealing with huge open spaces, the number of samples to collect would be more. Also, if dealing with very tiny rooms, you could collect only 2 samples.

The spatial and temporal variability of spore concentration in the air

The spatial (relating to space) and temporal (relating to time) variability of spore concentration in the air can be minimized by taking samples at different locations and at different times within the day. For examples we can decide to take some samples at same locations early in the morning, at noon and in the evening.

Conclusion

The important thing is first to define the purpose for spore trap air sampling. Then design a sampling strategy that is practical and that would give you data good enough for your defined purpose.

References

Michael Oberle, Markus Reichmuth, Reto Laffer, Cornelia Ottiger, Hans Fankhauser and Thomas Bregenzer (2015). Non-Seasonal Variation of Airborne Aspergillus Spore Concentration in a Hospital Building. Int. J. Environ. Res. Public Health 2015, 12, 13730-13738.

Robertson, L.D and Robert Brandys (2010). A multi-laboratory comparative study of spore trap analyses. Mycologia January/February 2011 vol. 103 no. 1 226-231.

Hyvärinen A; Vahteristo M; Meklin T; Jantunen M; Nevalainen A; Moschandreas D (2001). Temporal and spatial variation of fungal concentrations in indoor air. Aerosol Science and Technology 35, 688 – 695.

Understanding Spore Trap Analyses and Results

When enumerating airborne fungal spores, most labs, for practical reasons, analyze a percentage of the sample trace (often 15-25%) instead of 100%. A few labs claim that they analyze 100% of the sample trace. Obviously analyzing 100% of the sample trace sounds better than analyzing say 15 or 25%. In fact, some people argue that analyzing less than 100% of the sample trace is incorrect, inaccurate and gives skewed results. However, it is important to note that the accuracy of spore traps is affected by so many factors such that the contribution to inaccuracy/accuracy by the percentage of the sample trace analyzed in the lab is highly likely insignificant if at least 15% of the sample is analyzed. Even if 100% of the sample trace is analyzed, the results of spore traps would still be skewed in one way or another. Perhaps the most important of these factors is the sample volume collected. The volume of air collected for analysis is in most cases 75L or 150L. However, the final spore count is reported as the number of spores per cubic meter of air. That means ideally we should collect and analyze 1000L of air because by collecting 75 or 150L of air, the samples are only 7.5% and 15% respectively of what should have been analyzed. Unfortunately the spore traps (Air-O-Cell, Allergenco, etc.,) are not designed to collect large volumes of air especially in a dusty environment. In a dusty environment spore traps get overloaded with dust making it difficult for the analyst to see the spores.

The other factor that skews results is the fact that fungal spores are not uniformly distributed in the air.  That means if the spores are not uniformly distributed in the air, even the position and the height at which we place the sampler would skew the results.

Another factor that skew results is the fact that the spore concentration in a building varies not only with the season but also with the day and time of the day. In one study it was found that the spores of Aspergillus fumigatus can increase temporary to a significantly high concentration and then decline to “normal” level within 30 min only. This means the 5 or 10 minutes samples that are collected using spore traps are only a measure of spore concentrations at the time of sampling.

Spore trap- notice the non-unform distribution of Asp/Pen spores
Asp/Pen- notice the non-uniform distribution of spores

Is Analyzing 25% of Spore Trap Sample Trace Incorrect and Inaccurate?

The simple answer to this question is NO. First, spore traps are not meant to determine the actual spore count in the air. They are meant to give an idea of the types of spores present in the air and the concentrations of these spores relative to each other. Analyzing spore traps by direct microscopy is a complex process and there are limitations. Depending on the experience of the analyst there are counting and identification errors. Because of eye fatigue, counting errors are likely to be higher for a sample trace analyzed at 100% than one analyzed at 25%.

At MBL we use the ASTM Standard “Standard Test Method for Categorization and Quantification of Airborne Fungal Structures in an Inertial Impaction Sample by Optical Microscopy (Designation: D 7391 – 17 e1)“. This is the only standard currently available for spore trap analysis. The standard does not specify the minimum percentage of the sample trace to count and for good reasons. Practically, not every sample can be analyzed at 100%. The density of particulates (spores and other particulates) on the sample trace and the size of particulates determine how easy and fast the sample can be analyzed. Analyzing 100% of a highly loaded sample is not only difficult but could take one to several hours and will have little or no added value to the results because counting errors will be made during the analysis.

When Is It Possible to Analyze 100% of Sample Trace

The only time it is practically possible to analyze 100% of sample trace is when you have relatively few scattered spores and other particulates on the slide. With heavily loaded samples it’s very difficult for the analyst to keep track of what they have and haven’t counted under the microscope. As mentioned earlier, the analyst is likely to be fatigued and count many spores twice or miss counting some of them altogether. Compare the 2 images below which are showing spores and other particulates in just a single field of view of a microscope. Again, even for samples with low particle density, does analyzing 100% of sample trace add any value to the results? We would like to hear from you…

Spore trap- mixture of spores
Heavily loaded sample with tiny spores- Difficult to count 100%
Spore trap- Pithomyces spores
Big scattered spores- Easy to count 100%

Fungal Spore Identification Course

Master fungal spore identification by taking the Fungal Spore Identification Course! Upon completion participants will have the necessary skills to sample for airborne fungal spores, analysis samples, accurately count and identify a variety of spore types, and calculate airborne spore. concentrations.

What Do Your Mold Lab Test Results Mean?

Testing air quality for mold using a professional mold testing kit
Testing air quality for mold

Interpreting lab test results for indoor mold is currently not standardized. Therefore, it would not be surprising if you give 10 different people same copy of lab test results for interpretation and you get 10 different opinions. However, if you tell the 10 people what you wanted to find out from those results and also give them visual assessment results and the history of the building, you are likely to get most of the people arriving at the same conclusion. In other words, lab test results are meant to answer a question or prove/disprove a hypothesis that was formulated prior to collection of samples. This is only possible if you have a well defined objective for collecting mold samples for lab testing. The 3 common objectives for collecting mold samples are:

  1. To determine if building materials or contents are colonized by mold. Mold may or may not be readily visible with unaided eyes depending on the stage of growth and the color of the mold against that of the material it’s growing on.
  2. To determine whether the indoor environment has “normal and typical” types and amounts of airborne mold spores. Mold spores are found everywhere including indoors. Mold becomes a problem when it grows indoors. A number of molds that grow indoors, for example, some species of Stachybotrys and Aspergillus are known not only to cause allergy but also produce highly toxic byproducts (mycotoxins). Such molds when present in high numbers pose a health risk to the occupants.
  3. To determine if mold removal was effective. This is sometimes referred to as “Clearance testing”. Mold removal or remediation is meant to bring the mold levels to normal condition.

Once the sampling objective has been defined, the next thing is to decide on the most appropriate types of samples to collect. The types of samples you need to collect is determined by the type of data that would answer a question or prove/disprove your hypothesis.

Types of Mold Samples

Air Samples.

Air samples can be collected in 2 different ways. By impacting air on some growth media (culture air samples) or impacting air on inert media (non-culture air samples). The choice of either method depends on the objective of the investigation. If you are interested in determining whether specific species of mold are present in the air then you should collect culture air samples. If you want to determine how contaminated the air is, then non-culture air samples are the most appropriate. You use both sampling methods if you are interested in determining the species present and also the level of contamination.

Lab test results for air samples are usually qualitative and quantitative. Without standards and guidelines, interpretation of results of mold air samples relies on comparisons of indoor vs. outdoor results.  There are three primary sources of mold spores found in indoor environment. These are outdoor air carried in through doorways and windows; spores carried in on people, pets, or items brought into the building; and mold that grow and produce spores indoors as a result of excess moisture.

Surface Samples

Tape Slide for Sampling Visible Mold
Tape Slide for Sampling Visible Mold

Surface samples include bulk (pieces of building material and dust), swab and tape-lift samples. The primary purpose for collecting surface samples is to determine whether building materials and content are colonized and by what type of mold. With the exception of dust samples we do not recommend quantifying mold on surfaces of building materials. Quantification data obtained from these type of samples is usually not a good representative of the actual contamination level in the building and in most cases this data is not accurate and can therefore be very misleading. And even if the data was accurate, it cannot be used to determine risk of mold exposure and therefore has no practical use in determining health risk.

So, What Do Your Mold Lab Test Results Mean?

Mold lab test results should never be interpreted on their own. Your own visual assessment during the inspection and the building history should be given more weight. Some labs offer help with results interpretation but for them to provide meaningful interpretation you should provide them with building assessment data and what you were trying to determine through lab testing.

Determining if building materials or contents are colonized by mold

Tape-lift samples are the most appropriate for determining if building materials or contents are colonized by mold. In cases where scotch tape cannot be used to collect a sample, e.g., on wet surface, dry swabs can be used to collect the samples. Bulk samples may also be used. Presence of both mycelia and spores is an indication of mold growth.

Determining whether the indoor environment has “normal and typical” types and amounts of airborne mold spores

Air samples are the most appropriate to determine whether the indoor environment has “normal and typical” types and amounts of airborne mold spores.  “Normal and typical” types and amounts of airborne mold spores would be defined as the average amount and types of mold spores found in buildings with no history of water damage or ventilation problems. A building with normal and typical amounts of mold spores may have several spore types at levels that are lower than those found outdoors. Even in buildings with no history of water damage, spores of Penicillium/Aspergillus may be prevalent exceeding the absolute levels and relative percentages of these spores outdoors. Moisture-indicator molds such as Chaetomium, Stachybotrys, and Ulocladium species should be absent.

When the relative airborne spore concentration is greater indoors than outdoors, it indicates that the source of spores is not outdoor air alone, but also an indoor source, such as mold growth associated with a leaky roof, leaky foundation, plumbing leak, or any significant moisture source that is sustained over a long period.

Determining if mold removal was effective

To determine if mold removal was effective, visual inspection of the surfaces that have been remediated is key in assessing the effectiveness of the remediation effort. The remediated surfaces should show no evidence of present or past mold growth. The visual assessment is backed up with laboratory testing of surface and air samples. Presence of a few airborne indicator mold spores after mold removal does not mean remediation was not effective.

Some professionals believe air sample lab test results are not reliable and “clearance testing” performed after mold removal should just be visual. However, air testing is an important additional tool to ensure the building occupants are not exposed to elevated levels of airborne fungal spores and spores were not spread everywhere during the remediation process.

Interpreting Numerical Data of Viable Airborne Mould Samples

Viable Air sample taken on RCS agar strip
Viable Air sample taken on RCS agar strip

Another article on airborne mould samples provides guidelines for interpreting numerical data of viable air samples. To see the guidelines for non-viable air samples click Guidelines for Interpreting Non-viable Air Samples. The guidelines may be used to decide whether further investigations are required after initial investigations.

As was indicated in the last Issue, numerical laboratory results cannot be used as the primary determinant of whether there is a mould problem but should always be used to supplement visual inspection data and other available information such as the building history.

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 of 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 Viable Airborne Mould Samples

For a comprehensive assessment of the sample results, all the 3 steps require to be followed where applicable.

  1. Consider the concentration of Cladosporium and other genera which may reach high concentrations in the outside environment (sterile mycelia, yeasts, Alternaria, Botrytis).
    • If the concentration (CFU/m3) of one genus in the indoor air is lower than 0.7 to 1.0 times the concentration in the outside air, i.e., Ic ≤ Oc x 0.7 (+0.3), then indoor source is unlikely (background level).
    • If the concentration (CFU/m3) of one genus in the indoor air is lower than 1.5 ± 0.5 times the concentration in the outside air, i.e., Ic ≤ Oc x 1.5 (± 0,5), then indoor source is possible and further investigations are required.
    • If the concentration (CFU/m3) of one genus in the indoor air is more than 2 times the concentration in the outside air, i.e., Ic > Oc x 2, then indoor source is probable and immediate further investigations are required.
  • Consider the total concentrations for those species that are unlikely to occur in high concentrations in outside environment.
  • If the difference in concentration between indoor air and outside air is lower than or equal to 150 CFU/m3, i.e., Ic – Oc ≤ 150, then indoor source is unlikely (background level).
  • If the difference in concentration between indoor air and the outside air is greater than 150 CFU/m3 but lower than or equal to 500 CFU/m3, i.e., Ic – Oc > 150 ≤ 500, then indoor source is likely and further investigations are required.
  • If the difference in concentration between indoor air and the outside air is more than 500 CFU/m3, i.e., Ic – Oc > 500, then indoor source is likely and immediate further investigations are required.
  • Consider the concentration of one species that is unlikely to occur in the outside environment.
    • If the difference in concentration between indoor air and the outside air is lower than or equal to 50 CFU/m3, i.e., Ic – Oc ≤ 50, then indoor source is unlikely (background level).
  • If the difference in concentration between indoor air and the outside air is greater than 50 CFU/m3 but lower than or equal to 100 CFU/m3, i.e., Ic – Oc >50 ≤ 100, then indoor source is possible and further investigations are required.
  • If the difference in concentration between indoor air and the outside is more than 100 CFU/m3, i.e., Ic – Oc > 100, then indoor source is likely and immediate further investigations are required.

  Health Canada Guidelines

Viable air sample taken with an Andersen sampler
Viable air sample taken with an Andersen sampler

Health Canada, 1993 technical guide defines the “normal” indoor air mycoflora as that which is qualitatively similar and quantitatively lower than that of outside air. Based on a 3-year average, 40 CFU/m3 for Cladosporium, Alternaria, and non-sporulating basidiomycetes is considered as normal.

Further investigation may be necessary if more than 50 CFU/m3 of only one species other than Cladosporium or Alternaria is present. Concentrations of up to 150 CFU/m3 are acceptable if there is a mixture of species reflective of the outside air spores.

Higher counts may suggest dirty air filters or other problems. Concentrations of up to 500 CFU/m3 are also acceptable in summer if the species present are primarily Cladosporium or other tree and leaf fungi (phylloplane). Values higher than 500 CFU/m3 may be indicative of failure of the filters or contamination in the building.

To get hands-on experience on the application of these guidelines register for our Mold online training program today!

References

  1. Health Canada. Indoor air quality in office buildings: a technical guide. A report of the Federal & Provincial Advisory Committee on Environmental and Occupational Health. 55 pp. (1993). Umweltbundesamt 2002.
  2. Leitfaden zur Vorbeugung, Untersuchung, Bewertung und Sanierung von Schimmelpilzwachstum in Innenräumen. Erstellt durch die Innenraumlufthygienekommission des Umweltbundesamtes.

To see the guidelines for non-viable air samples click Guidelines for Interpreting Non-viable Air Samples

Airborne Fungal Spores: Non-viable and viable Air Sampling Methods

Non-viable air sampling for fungal spores
Non-viable air sampling for fungal spores

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?

Viable Air Sampling using QuickTake 30
Viable Air Sampling using QuickTake 30

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.

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