Problems attributed to molds in indoor air are usually associated with on- site proliferation of the molds in the affected building. Only occasionally are mold conidia from an outdoor source suspected as being potential sources of symptoms. Molds proliferating within a building usually do so in discrete locations where moisture and substrate conditions are conducive. Typical sites of indoor mold proliferation are damp cellulosic materials (e.g. wallboard paper, wallpaper, carpet backing, damp papers); debris in ventilation ducts, in carpets, or in mattresses or upholstered furniture; poorly maintained humidifiers; insulation on which organic film has accumulated; constantly humid painted, caulked or plastic surfaces (e.g., windowsills, shower stalls, cold air return vents); and potted plant soils. The ultimate goals of diagnostic indoor air mold studies are:

1) to determine if mold propagules, particularly those bearing irritating or immunosensitizing chemical components, are being produced and dispersed within the building to an extent which may account for (or predict) symptomatology; and,

2) if a connection between molds and symptoms is likely, to find and eliminate sites of mold amplification within the building.

These goals are usually addressed through one or both of the following strategies:

1) Air sampling (or sometimes dust sampling) to determine the level and types of viable fungal propagules within building trouble spots, and to suggest the presence of significant indoor mold growth sites ("amplifiers"); and

2) Physical search of likely problem areas to detect conspicuous mold amplifiers.

Further details of these two strategies are outlined on the page:

A. Sampling for Fungal Bioaerosols

Major types of mycological air sampling techniques

Air sampling for fungal structures is, at the most fundamental level, divided into techniques based on the culture of live propagules, and techniques based on the trapping and visualization of living or dead materials. This article will primarily deal with the former kind of sampling methodology. The reason is that many situations of suspected indoor air contamination involve toxigenic, allergenic fungal genera with small, nondescript conidia, such as Penicillium and Aspergillus. These are often difficult to assess accurately with particle-trapping devices such as Rotorod samplers and spore traps, where culturing cannot be done and analysis of samples tends to be biased toward the identification of larger, distinctively shaped, and/or dark-pigmented structures. Common fungi possessing such large or dark propagules, such as Cladosporium, Alternaria, Pithomyces, and Bipolaris, are often relatively innocuous fungi predominantly coming from outdoor sources. Other conspicuous allergens such as basidiospores of the bracket fungus Ganoderma applanatum or Ustilaginaceous smut teliospores may also be counted with unparalleled accuracy, but these are once again from outdoor sources and have little relevance to the major questions assessed by our working group. It should be noted, however, that Kozak et al. (1980) found a Rotorod sampler to be very useful for visualizing non-viable Stachybotrys, Ulocladium and Alternaria conidia emanating from contaminated carpets in homes. Although these authors actually located and identified the problem molds by a combination of inquiries about water damage, site inspection, and direct sampling from suspect surfaces, they felt that the Rotorod could be an important component of a detailed evaluation. They recommended its use in combination with an Andersen sampler for viable propagules, plus rigorous site examination, history-taking, and direct sampling. Given the often poor viability of Stachybotrys propagules, and the resulting uncertainty of using viable sampling techniques for detecting this organism, the recommendation should be strongly considered whenever a hidden Stachybotrys source may be present in a building.

The air sampling techniques elucidating viable propagules can be dichotomized into two categories: those relying on gravity to effect sedimentation of the mold propagules onto growth medium, and those based on pumping a measured amount of air onto or through a propagule collecting device. The former category is typified by the sedimentation plate, while the latter contains a variety of volumetric air sampling machines. The former will be dealt with first.

Settle (sedimentation) plates

There are a large number of publications substantiating the fact that settle plates elucidate a biased sample of the viable airborne mold propagules (e.g., Sayer et al., 1969). The reasons for this are twofold. Firstly, propagules have differential settling rates according to their weight and aerodynamic form. Settle plates are particularly efficient at detecting large conidia in indoor air, while the proportion of conidia belonging to important small-spored genera such as Aspergillus and Penicillium is underestimated. Secondly, whereas pumping samplers cause some air disturbance, settle plates are still. Some disturbance is usually necessary in air sampling to resuspend settled conidia which would ordinary become airborne under conditions of normal human activity in the rooms being investigated (Buttner and Stetzenbach, 1993). Actual normal human activity substantially improves fungal isolation even where pump samplers are used. It is important to note, though, that higher air turbulence levels, as may be found in outdoor sampling, may tend to keep smaller particles in suspension and decrease their settling onto sample medium surfaces.

Despite these limitations, settle plates are still widely used, at least in preliminary studies, in remote or impoverished areas, or as an adjunct to physical searching for amplifiers. As semiquantitative samplers, when adequately exposed (a commonly used protocol is for one hour at tabletop level under conditions of ordinary room activity (Verhoeff et al., 1992)), they can readily be used to discern the likely presence of significant indoor mold amplifiers (except in special cases, e.g., Stachybotrys amplifiers mainly consisting of non-viable materials). A problematic indoor mold amplifier, if it consists of small spored fungi such as Aspergillus or Penicillium, generally produces a sufficiently large quantity of airborne propagules that these species show up as a significant proportion of the isolates occurring on an adequately exposed settle plate. The gravitational bias against these smaller conidia is partially compensated for by the high numbers of conidia produced by any medically significant amplifier (subject to normal disturbance).

Some specific sampling deficiencies have been attributed to settle plates by researchers investigating parameters not relevant to the detection of significant indoor amplifiers. The strength of this technique lies in the fact that epidemiologically important toxigenic or allergenic fungi, unlike invasively pathogenic fungi, must be present in large quantities. The quantitative species distribution of fungi growing in any habitat tends to have an inverse exponential distribution, in which there are very large numbers of individuals representing a few predominant taxa, and very small numbers of individuals representing each of an indefinitely large number of minor taxa (Good, 1953; Gochenaur, 1984). Hence, to point out that settle plates tend to grow fewer taxa than impacted air plates (e.g., see Sayer et al., 1969) or that they detect members of a particular fungal group in fewer sites than impacted air samplers do (e.g., see Hyvarinen et al, 1993) is seldom if ever of practical significance. With adequate exposure times, especially under conditions of typically low air turbulence indoors, only members of the asymptotic "tail" of minor taxa are strongly likely to be missed in any given habitat. The best general definition of an adequate exposure time is that necessary to obtain an adequate representation of the smallest spore type of practical interest. In indoor studies, this spore type is often the Aspergillus/Penicillium conidium; the present author finds that the majority of 1-hour, indoor settle plates he receives from putatively mold-affected buildings have members of these taxa as predominant species.

Published comparative studies between volumetric and gravitational techniques often have inadequacies. Sayer et al., 1969, compared 15 min. samples taken by means of a vacuum sampler drawing 28.3 L/min of air with gravity plates exposed an entirely inadequate, and only desultorily parallel 15 min. Solomon (1975), in a better designed study, found that 30 min settle plate samples showed very little correlation with 1 - 10 min Andersen volumetric samples, and that numerically predominant, small-spored taxa were sometimes missed or very poorly represented. Verhoeff et al. (1990; 1992), however, found a strong correlation between Andersen samples and duplicate 60 min settle plate samples. The earlier of these two studies showed that the number of species isolated on settle plates on conventional high water activity medium (malt extract agar) was not significantly different from that obtained with four major types of volumetric air samplers. The later study showed that settle plates isolated significantly fewer species than the Andersen sampler, but did not comment on whether these species were relatively abundant or uncommon. No study to date has deviated from the prevailing focus on abundance and commented on the extent to which different types of samples allowed the recognition of synecological patterns signifying the presence and types of indoor fungal amplifiers. Such patterns (e.g., Penicillium brevicompactum + Aspergillus versicolor, usually signifying moist but not currently saturated wall covering paper) can be seen even in a relatively light deposition of smaller spored types on a settle plate (or in a light outgrowth of low viability spore types in either gravitational or volumetric sampling), and are sufficient to direct further on-site investigation. Either a heavy or a light deposition of such a pattern indicates the likely presence of a larger or smaller, closer or more distant, exposed or more concealed, but in any case undesirable mold proliferation site. Notwithstanding the serviceability of longer settle plate exposures in detecting these patterns, however, settle plates are best used in combination with a thorough initial site inspection to detect any macroscopically visible mold growth. (Because of low-viability propagule types like Stachybotrys, the same caution holds for volumetric samples).

Volumetric air sampling should be regarded as superior and used whenever it can be made available.

In Canada, the species predominant in indoor mold amplifiers ordinarily form a small or insignificant proportion of spora in outdoor air samples (except near large compost sites such as municipal leaf dumps or where abundant dust from stored crops or wood is encountered). Common examples of fungi strongly associated with indoor proliferation are Aspergillus versicolor, A. fumigatus, A. niger, members of Penicillium subgenus Penicillium (with a few exceptions), and black-spored Scopulariopsis species. Receipt of settle plates predominantly colonized by significant numbers of such fungi is an excellent indicator of potentially problematic indoor mold amplification. Accompanying outdoor air control plates, exposed sufficiently far away from the building studied to avoid outflow of building bioaerosols, are strongly recommended: they are characteristically negative for these fungi.

False negative or ambiguous 1-hour settle plates may be obtained from buildings with very restricted mold amplifiers, or with very still air in undisturbed rooms, or with amplifiers of poorly culturable species (e.g., Stachybotrys chartarum) or with amplifiers 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 from indoor plants, and possibly Chaetomium). The health effect of the species with low aerial dispersal has been suggested to be insignificant (Kapyla, 1985), but since intermittent or cumulative airborne dispersal of desiccated material may occur, some wet-spored species may be quite significant. Stachybotrys is an example of a wet-spored fungus for which significant airborne dissemination and health effects are well substantiated. Also, noxious volatiles may be produced by some wet-spored fungi. Little is known, however, about the health effects of the volatiles of wet-spored indoor fungi; many such species are not odoriferous to ordinary olfaction.

With settle plates, as with any other culturing of airborne molds, the most informative level of interpretation usually requires that the analyst be able to distinguish among the predominant species and species-groups of the genus Penicillium, Aspergillus and other relatively complex fungal groups. Also, if outdoor air controls are inadequate, he or she must be sufficiently familiar with the local ecology of molds and yeasts to detect deviations from their ordinary seasonal frequency in outdoor air. In Toronto, for example, a high number of Penicillium subgenus Aspergilloides colonies on a household settle plate in winter is an excellent indicator of mold proliferation indoors; the same finding in September might be nondiagnostic. Such interpretation of uncontrolled samples requires a mycologist with some aerobiological baseline data.

Since meaningful analysis of settle plates is based primarily on recognizing the synecological assemblage of isolates consistent with the presence of indoor mold amplifiers, and only secondarily concerned with the actual numbers of colonies detected, the problem of establishing acceptable and unacceptable numbers of colonies in indoor samples cannot be addressed. Any actions taken against indoor mold proliferation must therefore be triggered by factors other than the demonstration of a threshold count. Locating and examining any mold amplifiers not detected in preliminary inspection is a logical follow-up step once settle plates have revealed that these amplifiers are present. The substrate nature of the amplifier can usually be read from the settle plate. For example, Stachybotrys indicates very moist cellulose, often in previously flooded or soaked material, not uncommonly in sheltered areas behind wallpaper or wallboard paper growing in contact with the glue. Eurotium suggests, among other things, carpets with accumulations of dry skin scales and dust; Aspergillus versicolor suggests humid wallboard or other humid cellulose, including cellulosic dust within ducts; and so on.

In practice, common indications for characterization and remediation of the discovered amplifiers are: 1) occurrence of symptoms consistent with adverse reaction to indoor molds; and/or, 2) building management or administrative concerns that the amplifiers might cause symptoms in future, or might indicate or exacerbate degradation of materials, or might cause offense due to noxious odors or to the cosmetic, esthetic, psychological or political disadvantages of harbouring conspicuous decay. Once established mold amplifiers have been demonstrated, managers usually find themselves under strong pressure to clean them up.

Vacuum/culture (Pump) samplers

Pump samplers for viable propagules can be broken down into: 1) samplers impacting a stream of air onto a fungal medium surface; 2) samplers trapping propagules from an airstream in a viscous fluid which can then be plated on growth medium; or, 3) samplers trapping propagules on a membrane filter which can be eluted onto growth medium. In category #1 are slit samplers such as the New Brunswick slit-to-agar sampler, sieve impactors such as the Andersen and SAS samplers, and centrifugal impactors such as the RCS sampler (details of sampling with these devices are outlined by Muilenberg, 1989; see Glossary in Davies et al., 1995, for information about Andersen, RCS and slit samplers; information on a newer version of the RCS sampler has been published by Benbough 1993; see Verhoeff et al., 1990 for a chart showing the air intake rates, usual sampling times, and particle size biases of volumetric and non-volumetric sampling devices and techniques). In category #2 are liquid impingers and modifications of slit samplers to deposit propagules in easily melted glycerol/gelatin gels (Blomquist et al. 1984). In category #3 are various assemblages of pumps and filter cassettes drawing measured quantities of air through membrane filters impervious to fungal conidia.

A considerable literature exists comparing the efficacy of these samplers. Indeed, until recently, such studies have greatly predominated over other kinds of studies that might have predictive value in the analysis of indoor mold problems, e.g., studies of the biological effects of exposure to mold conidia, or studies of the composition of fungal communities associated with indoor proliferation and related symptoms. Even though much useful information has been gathered by the analysis of sampling devices, and accurate sampling is important, an overemphasis on this essentially nonbiological topic is deleterious. At present, even if propagule concentrations in room atmospheres were known with absolute accuracy, we would be little further ahead in understanding the association (if any) between these numbers and symptoms, and would not be assisted in the location or remediation of mold proliferation sites. In any case, correlative statistics should allow any moderately imperfect but reasonably consistent sampler to yield numbers which could be meaningfully gauged against symptoms, toxin levels, and a variety of related topics. These numbers must be broken down by fungal group, not given as total CFU, since the totality of spora includes varying proportions of potentially irritating and relatively benign particles.

Several recent studies have been performed comparing the sampling efficiencies of different pump samplers. Most of these studies have embodied some uncontrolled variables: for example, some fail to standardize the sampling durations and volumes, and many generate data by sampling in unpredictably non-homogeneous room air. They must therefore be interpreted with considerable caution, and those interested in this topic are advised to do a more detailed review than can be accomplished here. A few recent studies are worthy of mention, but the conclusions mentioned below should not necessarily be taken at face value. Buttner and Stetzenbach (1993) analysed the efficiencies of Andersen 6-stage, SAS, Burkard (suction slit impactor for direct examination of particles) and settle plates in a controlled room with known concentrations of Penicillium chrysogenum conidia. The Andersen sampler gave the most accurate and consistent results. Verhoeff et al. (1990) did comparative field trials in houses with the slit-to-agar, single-stage (N6) Andersen, RCS and SAS samplers. The slit sampler and the Andersen were concluded to be the most precise. Smid et al. (1989) similarly compared the single-stage Andersen, slit, RCS and SAS samplers. Once again the Andersen and slit samplers were reported to give the best results, with RCS reasonably comparable; SAS underestimated CFU counts by about 50%. These authors concluded that the RCS sampler remained useful because of its convenience of use and acceptable accuracy. A new version of the RCS sampler has recently been favourably evaluated (Benbough 1993).

In heavily contaminated environments, e.g. barns, Andersen samplers may suffer from overexposure, with multiple propagules being counted as one after impaction via the same sieve hole, with subsequent colony overgrowth. Correction factors have been published for moderately overexposed plates, but are inadequate for heavily overexposed plates. Diminishing the sampling time is a possible response, but has the disadvantage that spatial/temporal discontinuities in airborne mold propagule distribution may skew results. For example, a 30 second exposure may fortuitously sample a current of relatively clean air from a window draft not generally representative of a contaminated room; or, likewise, a short exposure may sample the peak of a burst of conidia from a disturbed mold amplifier or reservoir, and may show numbers well above those typically found in the room. For this reason, devices trapping propagules in liquid may be more accurate in heavily contaminated environments. With such samplers, sampling times can be longer without overwhelming the analytic capabilities of the system. Thorne et al. (1992) found that both impinger samples and eluted Nuclepore filters were more accurate than Andersen samples in barns housing swine. Blomquist et al. (1984) modified a slit sampler to deposit spores on agar or glycerol/gelatin gels, and then homogenized or liquefied these gels and plated them out in a classic dilution series. When glycerol/gelatin gels were used, results were comparable to those obtained using eluted Nuclepore filters.

Impingers have not been widely accepted in ordinary indoor mold sampling work. Most potentially problematic airborne molds have highly water- repellent conidia which, in contact with aqueous media, tend to adhere to surface films and hydrophobic surfaces, and to clump together in minute air pockets. Trapping of such hydrophobic particles in impingers is not efficient (Muilenberg, 1989). My own experience confirms this: a comparative study of Andersen and impinger samples from a hospital under renovation showed that impingers underestimated CFU by 90% or more (unpublished data). Thorne et al. (1992) reported considerably better results using impingers designed to impact the air stream on the liquid surface, rather than bubbling the air through the liquid. In barns housing swine, this technique, which would be expected to minimize re-entrainment loss of hydrophobic particles in impinger exhaust, allowed isolation of significantly higher numbers of CFU per cubic metre of air than did Andersen sampling. Unfortunately, the authors did not mention whether the fungi isolated by the impingers were predominantly hydrophobic or hydrophilic types.

In conclusion, for public buildings, various slit, sieve and centrifugal samplers should give reasonably comparable results. Absolute propagule count is not a realistic sole criterion for building remediation, since large counts from outdoor air are possible at some times of year, particularly in buildings with openable windows or with air filters that do not exclude smaller fungal conidia. The most efficient use of samplers, arguably, should be to detect conidial shedding by indoor mold amplifiers. Although such shedding may very well result in high CFU counts, and high counts in general will be more significant than low counts, certain factors may cause a truly problematic amplifier to yield low to moderate counts: for example, sampling at a distance from the amplifier (Room 211, flooded last year, has grossly molded carpet backings and wall cavities, but your only sample on that floor was from the opposite end of the corridor), misleading air distribution patterns (the basement door had been closed for several hours before you sampled the living room and bedrooms of 221 Grove St., but is often open and issuing mold conidia during times when the family experiences problems), and low conidial viability (your two colonies of Stachybotrys are also representatives of the 120 non-viable but toxic conidia which were in the litre of air you sampled). Pasanen et al. (1989) have found that viable spore counts were sometimes less than 25% of the total spores detected by scanning electron microscopy in farm and urban homes. Other difficulties are outlined by Miller (1992). Such information strongly argues on behalf of using air sampling as a detector of amplifiers and a semiquantitative indicator of approximate bioaerosol density rather than as an absolute arbiter of indoor air standards.

The relevance of spora counts is greatest where there is a diffuse and difficult-to-access amplifier present in a building. Two recurrent examples are mold growth in ventilation ducts in buildings with self-contained air recirculation systems, and molds apparently associated with a multiplicity of lightly and sporadically contaminated books in a library. In these cases, the idea of finding a discrete amplifier and eliminating it, the practical solution for the great majority of indoor mold problems, may be problematic. Although heavily contaminated ducts or books must clearly be cleaned up or otherwise dealt with (as must ducts or books with confirmed Stachybotrys colonization), the possibility of lightly contaminated environments is evident. The traditional question of determining an air contamination level requiring action is relevant in these instances.

Clearly it simplifies matters to restrict the analysis only to those fungi associated with the amplifiers, and to exclude fungi known to be associated with any incident outdoor air. (It is unlikely that indoor and outdoor types will have an additive effect as their toxin chemistry and antigenicity will be largely distinct; the possibility of additive glucan effects needs further investigation.) The difficulty is to know what factor to correlate numbers of indoor-generated mold propagules with in order to assign health significance to the findings. Essential dose/response information needed to correlate numbers of fungal propagules of particular chemical composition to health effects in humans is absent for all molds, and, as tolerance to molds appears to vary biologically among individuals, and appears to relate at least partially to the vagaries of allergic sensitization, acceptable dose information would doubtless be arduous to acquire even if ethical tests could be devised. Surrogate tests such as tests for the responses in vitro of human cells (e.g., alveolar macrophages) are in their infancy, and animals lack the ability to corroborate or disconfirm the persistent, subjective symptoms commonly reported in cases of indoor mold proliferation. The need for objective measures of adverse responses to mold inhalation is great, and devising such measures would be an important step in coming up with scientific correlates between spore counts and the need for remediation of buildings.

In the absence of any direct indicators of mold bioaerosol numbers exceeding human tolerance levels, a reasonable indicator of potentially significant problems would seem to be the coincidence of: 1) symptoms attributed to building air quality and compatible with mold exposure (non-specific upper respiratory or "flu-like" symptoms, mucous membrane irritation, exacerbation of asthma, wheeze, shortness of breath, etc., with remission within hours of leaving building and recurrence upon re-entry into building) and 2) levels of toxigenic or allergenic species originating within the building significantly exceeding levels detected in comparable buildings where, after adequate study, significant indoor mold amplifiers are not thought to exist. Some typical values for normal Canadian public buildings were given by Nathanson (1993) and were revised by Davies et al. (1995).

This paper will not discuss direct air or dust sampling methods for fungal biochemicals. Those detecting general fungal materials such as chitin, glucan and ergosterol lack the ability to discriminate between fungal elements from indoor and outdoor sources. Hence they will tend to give unambiguously interpretable single-case results (as opposed to multi-case statistical trends) only in cases where there is an extreme indoor buildup, or in cases where indoor accumulation of outdoor fungal material is otherwise known to be insignificant. Tests detecting specific toxins or volatiles may be very useful, but in their specificity are beyond the scope of this article. See Miller (1992) for a summary of some limitations of sampling for volatiles.

Dust, carpet, surface swab or contact plate samples may serve in place of air samples, but may elucidate many normally settled elements (e.g. Mucorales; also Fusarium other than predominant species on local agricultural crops) which in many cases are not significantly present in the air. On the other hand, dust samples have the advantage of containing a relatively long-term record of the history of fungal deposition within a building, and may thus relieve investigators of problems posed by the bioaerosol variability of different air currents seen in short-duration air samples. Further investigation is needed to give criteria for the interpretation of dust sample results, but results to date show some promise. Swabs may play a useful role in the sampling of patches of mold growth which have been detected visually, but they are inferior to surface scrapings since they tend to select spores and leave conidiophores/pycnidia/ascomata behind.

Media used in sampling

The media used in sampling fungal air and dust spora are diverse. They fall into several distinct categories: generally permissive media of high water activity, designed to allow growth and in-situ identification of a wide range of fungi (e.g., Sabouraud, 2% Malt Extract Agar, V-8 agar), generally permissive media with components restricting colony diameter ("restrictive media"), minimizing colony overgrowth and allowing in-situ identification of at least some fungi (e.g., Rose Bengal agar, various high-water-activity media containing Dichloran, Littman oxgall agar), media of low water activity, with or without factors restrictive of colony diameter, for isolation of moderately osmotolerant to xerophilic fungi (e.g., Dichloran 18% glycerol agar, Czapek's + 40% sucrose agar, 2% Malt Extract Agar + 10% salt ["malt and salt agar"]), and media selective for particular groups of fungi ("selective media", e.g., Sabouraud/cycloheximide medium for the majority of human pathogens, Onygenales, Herpotrichiellaceae, and Ophiostomatales; media with benomyl for Basidiomycetes, Zygomycetes, Endomycetes, Pleospora/Cochliobolus anamorphs, and Microascaceae). At least two apparently irreconcilable dichotomies must be addressed by the person trying to select a single medium for an indoor fungal study: firstly, that no one medium will optimize growth both of the significant fungi adapted to high substrate water activity (e.g., Stachybotrys) and the significant fungi requiring lowered water activity (e.g., Eurotium, Wallemia); and secondly, that the best media for identifying organisms in situ are also the most problematical for colony overgrowth and formation of spurious satellite colonies in shipping and handling.

Currently the two most widely used media for general sampling are malt extract agar (MEA) and dichloran 18% glycerol agar (DG18). The former was recommended by the American Conference of Governmental Industrial Hygienists (Burge et al., 1987), while the latter has been shown to be useful in a variety of recent studies (e.g., Verhoeff et al., 1990). The limitations of MEA are that it allows extensive colony overgrowth and supports osmophilic fungi poorly; DG18 supports osmophiles well, and facilitates growth of the moderately osmotolerant fungi which form the majority of indoor species of concern (Penicillium, Aspergillus), but causes poor growth in moderately osmointolerant fungi such as Scopulariopsis (Reenen-Hoekstra et al., 1991) and may support Stachybotrys and other highly osmointolerant species poorly or not at all (Samson, pers. comm). An near-ideal sampling protocol might include both media. "Malt and salt" agar may be a good alternative for DG18: long used for isolation of osmotolerant fungi, it also allows growth of Stachybotrys chartarum (Miller, pers. comm.). The colonies of this fungus are restricted but are readily seen.

In general, workers who, for practical reasons, prefer to use a single medium must be mindful of the types of fungi they will be excluding from their data.

Because most indoor environments contain a variety of osmophilic Aspergillus species, DG18 often tends to isolate the greatest number of species in comparison trials (e.g., Verhoeff et al., 1990); if a single medium must be chosen it may be optimal, but it should not be used alone except in combination with thorough visual and microscopic visual search to detect excluded fungal types, especially Stachybotrys. Such physical searching is recommended for Stachybotrys in any case, since it may be predominantly represented in the environment by non-viable conidia. Despite this, it is not uncommonly obtained in air samples, and any investigator wishing to maximize the likelihood of detecting significant Stachybotrys amplifiers is obliged to consider the use of air sampling with an appropriate high water activity medium.

The present author uses Littman oxgall agar extensively, primarily because of its tendency to repress sporulation and prevent satellite colony formation and colony overgrowth during shipping and handling in transit from test sites to the laboratory. It grows Stachybotrys well, has been observed to grow Eurotium (Aspergillus glaucus and allies) in high numbers in at least some cases, grows Wallemia occasionally (but probably not in a good representation of its true population predominance), and does not grow Aspergillus restrictus and allies. In its only formal comparison test as an indoor mold sampling medium, it showed significantly fewer colonies than three other media, including Sabouraud agar, at the sixth day of incubation (Morring et al., 1983). The authors conceded, however, that this time period was too short for a full evaluation. Littman (1948) showed that the eponymous medium outperformed Sabouraud agar over longer incubation periods. Littman oxgall agar, however convenient it may be for shipping, requires much labour, since the majority of colony types must be subcultured for identification. Further, they must be subcultured soon after plates are received since, as nonsporulating colonies, they may become non-viable within 2 - 3 weeks. This medium would therefore be a suboptimal choice for anyone doing his/her own sampling and mycology, or receiving plates or strips within a day or two of exposure.

Rose bengal agar or its dichloran-supplemented variant are also restrictive of overgrowth and, while delaying or repressing sporulation to a lesser extent than Littman oxgall agar, may be relatively robust in shipping while permitting a relatively high level of in situ identification. It must be borne in mind that rose bengal generates high-energy oxygen species on exposure to light, and illuminated medium may become lethal to fungi. Dichloran rose bengal agar has grown significantly fewer colonies than other media in at least one study (Verhoeff et al., 1990), although this effect was not observed in others (e.g., Smid et al, 1989). Unfortunately Verhoeff et al. (1990) did not record the time period allowed for incubation. Restrictive media in general slow colony growth rates, and for a fair biological (as opposed to purely practical) trial, such media should be observed only after sufficient incubation to ensure maximal colony outgrowth. An in-house trial showed that Littman oxgall agar gave colony numbers equivalent to those obtained with MEA in hospital renovation air samples incubated 14 days (unpublished).

The fact that all existing fungal sampling media have recognized shortcomings is a further blow against the former aerobiological ideal of using a perfected, standardized sampling device with a perfected, standardized growth medium to evaluate potential fungal aerosol problems with reference to standard guidelines for acceptable numbers of CFU. This ideal, which was always predicated on the reduction of all members of the three major terrestrial fungal phyla to a single, alchemical mass substance, clearly must yield to the reality of biological diversity. As more becomes known about the actual hazardous substances associated with airborne fungal materials, methods indicating the occurrence or likely occurrence of these substances will be developed. In the meantime, the investigator engaged in detecting potentially significant amplifiers must simply ensure that an adequate diversity of techniques is used to cover the diversity of possible amplifiers.

B. Direct detection of amplifiers.

Procedures for the direct detection of mold amplifiers may be used either after an air sample has predicted the presence of amplifiers, or as a preliminary survey. Common places where significant amplifiers can be visually identified are in water-damaged walls on or under wallpaper or wallboard paper (whether painted over or not), on the backings of water- damaged carpets, on HVAC coils, pans, vanes and so on, on damp papers (e.g., after flood, including floods created by firefighting operations), within walk-in refrigerators and incubators, and in any moist organic materials, including any moist object composed of cellulose. If insulation is exposed, it may be visibly discolored with mold, as may the inner or outer surface of its covering paper.

Amplifiers may be visible on windowsills (Kapyla, 1985) as well as shower stalls and washroom fixtures. These windowsill and washroom amplifiers, if small and not involving cellulosic material (i.e., molds are growing only on paint, ceramic, grouting or plastic) are seldom problematic. More extended amplifiers in these situations may be problematic, and even hypersensitivity pneumonitis, which normally requires long-term heavy exposure to develop, has on rare occasion been linked to heavy growth of fungi in shower or other washroom amplifiers.

The most convenient way to investigate these amplifiers is to impress them with cellulose-acetate (e.g., Scotch (TM) brand) tape, either transparent or frosted varieties, and then to examine these pieces of tape directly under a microscope after adding a drop or two of water or water/detergent solution. The sticky surface of the tape often displays the mold materials in spectacular detail, as well as revealing such items of interest as fungivorous mites, mite faecal pellets, and sometimes other arthropods such as booklice and small millipedes. To transport the tape strips from the site to the lab, the most convenient technique is to tape them down flat onto the inside of a sturdy, clean plastic bag (does not need to be sterile), label the bag, and then transport. In the laboratory, the tape strips can be peeled off the plastic, cut into convenient lengths as necessary, and put onto slides for examination. Transparent tape can be taped directly onto a slide; frosted tape must be taped onto a coverslip, then the coverslip must be inverted and placed tape-side-down on a slide to allow stable examination of the mold material attached to the tape's underside.

Amplifiers which are not immediately visually evident on site may also be elucidated by microscopic sampling. Common practices are the transparent tape sampling of duct interiors, direct microscopy of duct insulation, slide examination of humidifier basin materials, and examination of small fibre samples cut or pulled away from filters or carpet backings in which mold growth is suspected. Further direct detection of amplifiers may be performed by culturing, e.g., plating out of humidifier fluids and plating out of scrapings or swabs from recognized or suspected mold growth on walls or other surfaces. Beware of mite infestations in your fungal cultures!

If an amplifier is detected and there is some doubt about whether it disseminates a significant number of propagules into the environment, air sampling with a nearby Andersen sampler or equivalent, in the presence of normally vigorous room activity, should determine its relative influence. The predominant species produced within the amplifier must, however, be known so that appropriate culturing techniques may be used in the air sample. Also, the material must be known to be viable. Air sampling is not appropriate for quantitative evaluation of Stachybotrys or certain other fungi poorly culturable from airborne propagules. Bear in mind that irritating toxigenic fungi may remain irritating in a dead condition, and allergenicity also persists even when the materials stimulating the allergic reactions are dead.

Verhoeff, AP. et al. 1992. Presence of viable mold propagules in air in relation to house damp and outdoor air. Allergy 47: 83-91.