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 mold sampling 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. It’s therefore important to select mold sampling agar media wisely.
How would one select mold sampling media to use then?
It is easy to select the mold sampling 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 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 mold sampling 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 to some extent. 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 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.
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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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A number of methods can be used to test air for mold or other microbial contamination. One of the oldest methods of testing air for microbial contamination is the settle plates method. Though the method is semi-quantitative, it is still considered a useful method. In industries such as food, pharmaceutical and cosmetics the method is used to assess the likely number of microorganisms depositing onto the product or surface in a given time. The method involves opening and exposing petri dishes containing agar medium suitable for growth of microorganisms of interest. If one is interested in testing for mold, agar plates containing malt extract agar (MEA) supplemented with some antibiotics to suppress bacterial growth would be used. The agar plates are left open at table-top level at selected points in the room for half-hour to 4 hours. This allows mold spores and fragments to settle onto agar media by gravity. Mold test kits (involving growth media) are settle plates.
Settle Plates Results
The number of microorganisms deposited onto the agar surface of the plate over the period of exposure is determined by incubation of the agar plates at 25ºC for 5- 7 days and counting colonies that develop. The results can be expressed as number of colony forming units (CFUs) per unit time. The counted colonies can then be further characterised to genera or species. Higher numbers of CFUs and/or presence of potential pathogenic or toxigenic molds such Aspergillus fumigatus and Stachybotyrs chartarum are indicators of a problem.
Disadvantages of Settle Plates
Settle plate method is an extremely useful method for assessing air contamination by microorganisms. It is easy to conduct and very cost effective. However, only viable microorganisms would be detected by this method and hence it may give a false impression that the air is “clean” if most of the airborne microorgainisms are dead. False negatives may also be obtained from buildings with:
- very restricted mold growths.
- very still air in undisturbed rooms.
- species of poorly culturable molds (e.g., Stachybotrys chartarum).
- molds consisting of species with poor airborne dissemination (e.g., Aureobasidium on windowsills, Cladosporium on painted cold air vents, Fusarium and many other wet-spored fungi).