How Many Air Samples To Take for Mold Investigations | Zygomycetes
By Dr. Harriet Burge, EMLab P&K Chief Aerobiologist and Director of Scientific Advisory Board
The obvious answer is "as many as possible." As we all know, airborne spore populations are extremely variable both in space and time, and no single sample that doesn't include a majority of the air in a space is truly representative. Obviously, in most incident investigations we cannot collect enough samples to get a representative picture of the air spora. Instead, we rely on visual observation and carefully constructed protocols to test specific hypotheses. Even when we do this, single samples are rarely going to provide significant data. So, what is the bottom line for collecting air samples?
Sampling to Document Human Exposure
Incident investigations are rarely designed to document human exposure to an aerosol, although some investigators (and many of their clients) believe they are. At a minimum, documenting human exposure would require sampling in all spaces the person occupies, and during all activities that the person does in those spaces. For example, if you wanted to document Aspergillus fumigatus exposure to a transplant patient in his/her hospital bed you would have to collect samples when the ventilation system is on and off, whenever a nurse, physician, helper, maintenance person, or visitor comes into the room, and certainly whenever the windows are opened (which they should not be!).
In order to interpret data from this protocol, specific recovery guidelines are set, and the number of samples necessary to meet these guidelines is calculated. For example, in the case of Aspergillus fumigatus spores in a transplant surgical suite the guideline might be less than 1 spore per cubic meter. Obviously, each sample set will have to include enough air to allow detection of this low number, and many samples (possibly hundreds) will have to be collected.
Epidemiological studies rely on a few samples collected in many locations to develop relationships between sampling (exposure) data and health outcomes. While single samples per site are often used for these kinds of studies, the errors introduced because of variability over space and time for each site is enormous, and significant relationships are probably masked.
In order to interpret epidemiological data, extensive analysis protocols are designed and calculations made to determine the number of sites to be sampled (rather than the number of samples/site). In some cases, pilot studies will be done to determine variability within sites. Rarely, replicate samples are collected at each site.
Clearance sampling in the case of mold investigations is generally done to document that remediation efforts have been successful, not to prove that exposure is not continuing. If human exposure is of concern then the guidelines for exposure event sampling need to be followed. This is a point of contention in many studies where building occupants want to be assured that the environment is now "safe" to reoccupy. In most mold cases, these assurances cannot be made. One can only document that the mold contamination for which the remediation was initiated has been successfully removed.
The big problem with designing clearance sampling protocols is that you are trying to prove the negative case, which is never possible. Instead, we work toward increasing the probability that the former aerosol is no longer there. The more samples that are collected, the higher the probability that our data is representative. In clearance situations, you can often take a 10 minute spore trap without overloading the sample. Note that a single 10 minute Air-O-Cell® sample represents slightly more than 5 cubic foot of air. You can easily calculate the percentage of the air in the space being studied represented by this one sample. If air samples are to be used for clearance sampling, I would suggest that at least 5% of the air in the space be sampled. You can help make each sample more representative by thoroughly mixing the air in the space before sampling, being sure that the process of mixing the air doesn't compromise the efficiency of your sampler. Even with thorough mixing, at least 2% of the air should be sampled. For a 1000 cu. ft. room you would need 4 samples to achieve 2%. If you have collected 4 samples, you can calculate the median and range, and compare these to your own pre-determined guidelines. Note that without predetermining your guidelines, interpretation will not be possible. One approach to guideline development is to sample before and during remediation, then decide the percentage change you want to see in the clearance samples. This process is best done for individual spore types.
Sampling to Document Aerosolization from Sources
One important and effective strategy that can minimize the number of samples to be collected is to document the potential for aerosolization from suspected sources. This involves visually identifying potential sources or reservoirs, sampling near the source with no activity, then creating representative activity and sampling again. Thus, you can sample near an electrical outlet (after using a smoke tube to determine that air is flowing out of the outlet), then bump a chair into the wall (or use an equivalent disturbance) and sample again. Here the minimum number of samples per suspected reservoir is two, and I always collect 4 (2 before activity and 2 after).
Similarly, you can evaluate the contribution of a suspected source to the ambient aerosol in the room by collecting duplicate room air samples with no disturbance, then collect the 4 source samples. Then you can compare taxa in the air to those that increase in the source samples following activity.
Data interpretation guidelines again have to be predetermined before sampling. One possibility is to decide that the range of the before and after source samples should not overlap. For the second strategy, you would look for unusual spores that are present in both the source and the air sample.
Sampling for Comparison to the Outdoor Air Spora
A common method for assessing the indoor environment is to collect indoor and outdoor samples for comparison. The actual data comparison may be done using indoor/outdoor ratios, or (better) the MoldSCORE. Many people use single samples to make these determinations. I think this is a mistake. Ideally I would collect 2 outdoor samples before entering the space, then 2 for each room of concern, then 2 more outdoor samples on leaving. The absolute minimum I would use would be the 2 outdoor samples and 2 for each room, omitting the final outdoor samples. The reasons for this should be obvious by now. No one particulate sample is characteristic of any environment unless it includes a representative sample of the air in that environment. This is because the particles are never randomly distributed in the space.
The Bottom Line
No single particulate sample is representative of any environment, especially when the particles are bioaerosols. Because of the nature of the sources, distributions of bioaerosols are never random, and change dramatically in space and time. Most of the comments above refer to spatial variation, and the fact that time is also a variable must always be considered. However, the fact that a single visit to a space is often the only chance we have to collect samples, concentrating on spatial variability seems appropriate. Sampling and analysis are relatively inexpensive compared to your time, and good sampling protocols will allow you to save time later in interpretation, and gain the goodwill of your clients when you can readily defend your protocols.
By Murali Putty, EMLab P&K Analyst
Zygomycetes are one of the four major groups of fungi, the others being the Oomycetes, the Ascomycetes, and the Basidiomycetes. Zygomycetes are common, fast growing fungi, often overgrowing and inhibiting other fungi nearby. They are one of the most ecologically diverse groups of fungi, functioning as saprophytes on substrates such as fruit, soil, and dung, as harmless inhabitants of arthropod guts, as plant mutualists forming ectomycorrhizae, and as pathogens of animals, plants, amoebae, and especially other fungi. They can be also found in flowers, stored grains, mushrooms and insects. Approximately 600 species of zygomycetes are known.
Rhizopus and Mucor are two of the most common zygomycetes that are frequently identified from soil and house dust as well as in both outdoor and indoor air samples. The other commonly seen zygomycetes include Absidia, Syncephalastrum, Circinella, Mortierella, Mycotypha, Cunninghamella, and Choanephora.
Zygomycetes are distinguished from other classes of fungi by the production of sexual spores known as zygospores and by the production of asexual spores called sporangiospores. The sporangiospores are usually produced in a sporangium. Each sporangium may contain thousands of sporangiospores.
Sporangiospores are the most abundant spores produced and are primarily disseminated by wind. Animals, insects, and rain can also contribute to dissemination. The zygospores are formed within a zygosporangium. A single zygospore is formed per zygosporangium. The zygospores are large and are resistant to environmentally adverse conditions, therefore they are not widely disseminated. Zygomycetes are generally coenocytic (a mycelium that is multinucleate and lacks regular septation, i.e. no cross walls). This is an important characteristic for identification during direct microscopic examinations where mycelium from many different fungi may be present.
A number of species of zygomycetes are used in Asian food fermentations, such as Rhizopus oligosporus in the Indonesian food tempeh, and Actinomucor elegans in Chinese cheese or sufu.
Some species cause storage rots of fruits (particularly strawberries by Rhizopus stolonifer), some cause diseases in plants (e.g., Choanephora cucurbitarum flower rot of cucurbits). They are capable of growth on a variety of other substrates including soil, grains, vegetables, and wallboard paper when sufficient water is present. Some species can cause life-threatening opportunistic infections of diabetic, immuno-suppressed, and immuno-compromised patients. Some zygomycetes are isolated from domesticated animals in tropical and subtropical regions of the world, including the U.S. gulf states.
The presence of zygomycetes can be easily identified in tape lifts. Rhizopus for example, are distinctive, readily identifiable, if rhizoids and all sporulating structures are clearly visible. Zygomycete spores are clear round spores. They will grow rapidly on most fungal media. Some species of zygomycetes, like Rhizopus species, can cover culture plates within 24 to 48 hours.
1. O'Donnell, K L (1979). Zygomycetes in Culture. University of Georgia, USA.
2. EMLab P&K: Fungal Library
3. Clinical Microbiology Reviews: Zygomycetes in Human Disease
4. Tree of Life: Zygomycota
5. Wikipedia: Zygomycetes
This article was originally published on July 2008.