Investigation of fungal contamination in indoor environments normally includes visual inspection and sampling. The samples to collect, the number, when and where to collect them and the methods to be used for sample analyses depends on the objectives or goal of the investigation. The samples that may be collected include air, dust or bulk samples. Swabs or clear cellophane tape can be used to sample for fungi from contaminated surfaces. The samples can be analyzed by either direct microscopy or by culture methods depending on the type of data required.

Testing for airborne mold spore concentration is achieved by impacting a known volume of air onto a surface coated with sticky material. As the air hits the sticky surface the spores and any other particulates in the air are trapped. In the laboratory the spores are identified under a microscope, categorised into various groups and counted. This method is excellent for estimating how contaminated the air is but it does not tell us what proportion of the counted spores are still viable. If an estimate of the proportion of viable mold spores is needed, then the air has also to be impacted onto some growth agar media. Viable mold spores would then grow on the media and appear as mold colonies, usually referred to as colony forming units (CFU). CFU is not a very accurate way of measuring the viable proportion of airborne mold spores. This is because a single colony can develop from one spore or a group of spores. Secondly, fast growing colonies tend to overgrow slow growing colonies. Also, the agar media used may not support the growth of all categories of viable spores present in the air.

Why selecting the right type of agar media is critical

There are several types of agar media used in a microbiology laboratory for culturing molds. These media may differ in their water activity, pH, nutrient content or composition. Molds differ in their growth requirements. Therefore, no single medium is suitable for each and every mold out there.

How would one select the media to use then?

It is easy to select the media to use if one is looking for a specific type of mold. However, in most mold investigation projects, one is interested in knowing the the kinds of viable molds present in the air and their concentrations. The problem of using a single type of media is that some molds may not grow well (or may not grow at all) in the selected media. Hence, although such molds may be the dominant contaminants in the air, they may end up being missed or underestimated. The solution, therefore, is to use more than one type of media or select one that is known to support a wide range of environmental molds. A good example is Malt Extract Agar (MEA). The problem with this media is that it also supports the growth of bacteria. If the environment sampled is contaminated with bacteria, the bacteria grow faster than molds and interfere with mold growth. This problem can be overcome by incorporating a suitable antibiotic or other suitable compounds (e.g., Rose Bengal) into MEA to suppress bacterial growth. Rose Bengal not only suppresses the growth of bacteria but also restricts the spread of fast growing molds thus making it easy for colony counting.

What about culturing of bulk samples?

The same applies to culturing of bulk samples such as pieces of building material or dust. Direct culturing of such material in a single type of media could give erroneous results. If a single media is to be used to culture these types of samples, it is recommended that a lab performs a direct microscopic examination of the samples before culturing. Direct microscopy allows identification of the dominant contaminant (at least to genus level) regardless of whether the mold is dead or cannot grow on media used.

Demonstrating the effect of media on mold growth

To demonstrate how results from a single media can be misleading, examine the 4 petridishes. Two bulk samples were cultured onto 2 different media (DG18 and MEA) after serial dilution. Sample 1 was cultured in petridishes marked “A”. Direct micrsocopic examination of sample 1, indicated it had Stachybotrys as the dominant mold and some slight growth of Penicillium. After incubation, Stachybotrys did not show up at all in DG18 but both Stachybotrys (cream colonies with dark centres) and Penicillium (blue colonies) appeared on MEA. The second sample had Stachybotrys only. After plating onto DG18 and MEA and incubation (see petridishes marked "B"), Stachybotrys appeared on MEA and but not on DG18. These observations clearly indicate how wrong conclusions can be made if the right type of media is not used either in air sampling or culturing of bulk samples.

References

Microorganisms in home and indoor work environments: diversity, health impacts, investigation and control. Flanning Brian, Samson, Robert A., and Miller, David J (Ed.), Taylor and Francis, 2001.

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Mold Sampling

The mold sampling method one chooses should be determined by the objective of the
investigation. One may sample air or surfaces for mold contamination. The standard
method for sampling air is to use a volumetric sampler e.g. RCS or Andersen N6 for
viable airborne spores and hyphal fragments and Air-O-Cell, VersaTrap, Allergenco and others such cassettes for total spore counts. Testing of surfaces may involve use of RODAC agar plates for smooth surfaces, and swabs and adhesive tape on all other surfaces. It is important to note that adhesive tapes may not work well on wet and porous surfaces. Bulk samples can also be taken and plated onto agar plates or analysed by direct microscopic examination. Dust samples can be collect from surfaces such as carpets, upholstered furniture and textiles.

Media For Mold Sampling

If one decides to collect viable air samples, the choice of media to use is very
important. Generally, malt extract agar (MEA) is used. It is a "broad spectrum" medium that supports the growth of a wide range of fungal species. However, antibiotics may have to be incorporated to surpress bacteria growth. Its main disadvantage is that fast growing molds tend to overgrow slow growers making it difficult to count colonies. To overcome this problem, DG18 and Rose Bengal can be used. These media have compounds added to them to slow down fast growing fungi and inhibit bacterial growth. If one is sampling a relatively dry environment, MEA+40% sucrose would be recommended for detecting xerophilic (dry loving) fungi.

Mold Identification

Currently, the only reliable means for routine identification of mold species is to
perform traditional mycological methods. This requires years of training and practice. Be sure to use a lab that has a qualified Mycologist on-board (preferably at PhD level). The lab should also be regularly participating in a recognised proficiency testing program such as the AIHA EMPAT program.

Performing Effective Mold Sampling

If you need to take mold samples, use properly trained personnel or to get yourself
trained. If you decide to undergo training, select a mold training course that provides skills and background information to enable you recognize indoor mold, develop effective sampling strategies, and interpret laboratory results.