Dust sample, quantitative fungal culture [C120]

Published: July 8th, 2009

Revised: October 4th, 2009

The analysis of vacuum collected dust is widely used to estimate microbiological exposures in human population health studies. In this context, dust analysis has repeatedly proven to be a useful proxy of exposure, because it serves as a reservoir for particles to which people are exposed in indoor environments. Dust itself also serves as a long-term integrative air sample, capturing transient airborne spore bursts, and retaining representative particles amid the growing bulk of textile fibres, hairs and skin scales that comprise the predominant mass-fraction of house dust.

Dust analysis is a useful adjunct to the investigation of indoor environments for fungal amplification sources. Although the total levels of culturable fungi in dust are not necessarily reflective of the condition of a building, careful evaluation of the species composition of the fungal cohort of dust can often lead a skilled investigator to meaningful revelations that otherwise would be missed. Although there are no guideline documents on the interpretation of dust sampling data, excellent baseline information for the expected fungal population of dust fungi is given in Dr. Scott’s PhD thesis, Studies on indoor fungi (2001).

Dust is usually obtained by vacuum collection from a predetermine area of flooring. Vacuum dust collectors range from very sophisticated devices, such as those used in research studies, to relatively simple “interceptor” filters that can be affixed to the sucking end of a household vacuum cleaner. The Dustream® collector, pictured at right, is low-cost, widely accepted method for vacuum dust collection that does not require specialized equipment. The red adapter at the base allows to be attached to hoses of a range of sizes. It can use used for culture-based tests, as well as endotoxin, glucan, and allergen assays such as mould and dust mite. Note that samples obtained from household vacuum cleaner bags are almost useless for biological analyses, since the typically growth-promotive environment within the collection bag, over time and depending upon the amount of moisture present and the frequency of cleaning and maintenance, causes changes in the composition and abundance of the captured fungal population.

Field collection method

1. Don a pair of clean, disposable nitrile gloves. Use painter’s tape to outline a 1 m² area of flooring. If a contiguous space cannot be located to accommodate the full area, define as many sub-areas as necessary to demarcate a total floor area of 1 m².
2. Remove the Dustream® nylon mesh collection thimble from its polypropylene storage bag, and place the filter thimble into the collector shell. Affix the collector shell to the extension wand of the vacuum cleaner, using the red adapter ring, if necessary.
3. Switch the vacuum cleaner on, and proceed to vacuum the entire surface within the demarcated area. To ensure thorough coverage, make 7 repeated passes over each stroke prior to proceeding to the adjacent stroke. Covering the entire area in this manner should take approximately 10 min. In the event that a sufficiently large amount of dust is present that the vacuum stalls or overheats due to back-pressure, record the actual area vacuumed to the nearest stroke in which 7 passes were completed.
4. Remove the nylon mesh collection thimble from the collector shell, and place the thimble into a clean vial or polypropylene bag. Be sure to label the container with the sample number, job number, date and time of collection, and the surface area obtained. Use an indelible marker to ensure that the label data is not easily wiped off of the container. Wipe any residual fine dust from the collector shell using a 70% isopropyl alcohol wipe prior to beginning another sample collection.

Service options

Results reporting

The results of this test will be reported quantitatively, as Colony Forming Units per milligram of dust (CFU/mg), and broken down by species, species-group, or genus, depending on the taxon observed. For clients desiring to know the mass of total and/ or fine (< 150 um) dust recovered, we would be happy to supply these data for a small additional fee.

Bulk/ swab/ TefTex wipe, semiquantitative fungal culture [C111]

Published: July 8th, 2009

Revised: October 4th, 2009

Bulk samples

In the laboratory, a semiquantitative fungal culture of a bulk sample is generally carried out by aseptically taking tiny shavings from the specimen and sprinkling them over the surface of a Petri dish filled with growth medium. Alternatively, a dacron swab may be used in the laboratory to recover any loose debris from the surface of a bulk sample, and then rolled on the surface of a growth medium. A third method for semiquantitative testing of bulk samples involves washing all or a portion of the surface of the material with a sterile buffer solution, and transferring an aliquot of the resulting suspension to a Petri dish. Which particular procedure is indicated is a subjective decision and it depends to a great extent on the size, composition and overall appearance of the sample. The advantage to starting with a bulk sample is that it permits the laboratory to determine which approach is most suitable, possibly including several parallel approaches to achieve the most accurate result.

Swab samples

Despite their ease of handling, swab samples are generally poorly suited as field methods for sample collection. Wherever possible, it is always best to collect a bulk sample, and reserve the use of swab methods to the laboratory is this is deemed an appropriate approach to processing the specimen. Despite this proviso, swabs are used with great frequency in field investigations, and when taken correctly and in appropriate circumstances, they can provide useful information in the assessment of an interior condition. The swabs we find most suitable for fungal sampling have a woven Dacron tip, and lack a gel or liquid medium in their transport tube (in other words, they are “dry” swabs). This is important because exposure of collected spores and other cells to any moisture may result in cell germination. If the surrounding medium in which these cells germinate is unsuitable for growth, the nascent fungus may die. By contrast, moist transport media are a necessity for bacterial sampling, since many types of bacteria die quickly in the absence of moisture.

Field sample collection

1. Don a pair of nitrile gloves and remove the swab from the blister pack or tube, taking care to handle it only by the distal end or lid.
2. The area to be tested should be an area roughly between 1-100 cm². Gently rub the swab tip in a back and forth motion on the surface being tested, rotating the swab slightly as the sampling proceeds to cover the entire sampling area. Note that the precise area sampled is only important if relative comparisons are likely to be needed between swab samples.
3. Insert the swab into the transport tube and secure the lid. Be sure to write the sample collection data on the attached label, including the job number, sample number, sampling date, surface type sampled and approximate area swabbed.
4. Multiple swabs may be placed in a clean, new reclosable zippered plastic bag, and stored in a dark, cool place until receipt by the laboratory. There is no need to ship swab samples with an ice pack or other means of temperature control. We have found that simply packing the swabs in a padded envelope provides sufficient temperature protection in most cases.

TefTex wipe samples

Certain sampling settings, such as irregular surfaces, are poorly suited to either bulk or swab sampling. Wipe samples may be more suited to these situations, since much more force can be applied to the sampling surface, and the flexibility of the wipe allows it to better conform to the contours of the sampling surface. We use TefTex wipes for biological sampling. These wipes are made from virgin teflon twill fabric. They are individually packed, sterile and pyrogen (endotoxin) free, and have an effective sampling surface of approximately 10 cm². Wipe samples may be collected in a manner similar to the above swab sampling procedure. Once the wipe has been sampled, it should be folded in half such that the sampled surface is in the interior of the fold, and replaced in the labeled transport pouch.

Service options

Results reporting

Results of semiquantitative bulk samples, swabs and TefTex wipe samples are given as 1+, 2+, 3+, etc., where the numeral preceding the plus sign indicates the order of magnitude of each taxon observed in the sample.

Bulk sample, quantitative fungal culture [C110]

Published: July 8th, 2009

Revised: October 4th, 2009

Solid or so-called “bulk” materials, such as wood, carpet, and other insoluble, solid items, can be evaluated by quantitative fungal culture to determine their culturable fungal content. All or a portion of the specimen is weighed, placed in a buffer solution and vigorously agitated to suspend adherent spores or vegetative hyphae. Aliquots of suspension are plated, the plates are incubated, and resulting colonies are identified and counted.

Although this methodology may seem straightforward, (indeed, in the laboratory it is a relatively simple procedure), the test is extremely difficult to standardize. This is because the surface area to volume ratios and densities of materials vary considerably, and most fungal colonization is likely to be restricted to the surface of the material. Although similarly sized coupons of comparable materials could be reasonably compared using this test, the method does not provide meaningful, information in an absolute sense on the fungal content a sample. Still, there remain a few indications for this test. For example, this approach may be informative where there is an interest to compare a set of identical or nearly identical objects based on the concentration of culturable fungi recovered. Such an item might be an industrial part, an herbal supplement or a biomedical device, where items from the same or different lots are compared. This test is generally not useful in indoor environmental investigations.

Field collection method

There is no specific field collection method for this test, per se. Items to be tested should be placed individually in a labeled tube or envelope to which a label containing all relevant sample information has been affixed. The test is semi-destructive, since depending on the size of the specimen, we may need to dissect portions of it in the laboratory to obtain a suitably sized coupon for analysis.

Service options

Results reporting

Results of this test are reported in Colony Forming Units per milligram of material (CFU/mg), and broken out according to species, species-group or genus observed (the identification level depends on the taxon in question).

Opportunistic fungal pathogen screen [C104] NEW!

Published: July 8th, 2009

Revised: May 10th, 2010

Serious infections caused by filamentous or dimorphic fungi are rare in immunologically normal people and normally only caused by one of several pathogenic species. In people with varying degrees of immune dysfunction, however, serious fungal infections are common, and may be caused by a much larger set of fungi many of which are not pathogenic under normal circumstances. These fungi are sometimes called “opportunistic pathogens”, in reference to their proclivity to invade tissues given the opportunity presented by immune compromise. While not typically a problem in the broad community, opportunistic fungal pathogens can be important agents of serious and even fatal disease in health care institutions.

Culture-based methods are the current gold-standard for the detection of opportunistic fungal pathogens, and 1 CFU/m³ has been advised as an action limit (Mayhall 2003, p 706). This test provides a rapid, 48 hr turn-around, culture-dependent evaluation of the airborne presence of the most commonly occurring agents of opportunistic fungal disease.

Taxa included

• Absidia species (including A. corymbifera)
• Acremonium species
• Aspergillus species complexes (including A. flavus, A. fumigatus, A. lentulus, Neosartorya species, Emericella/ Aspergillus nidulans, A. niger, A. terreus, and A. ustus)
• Cunninghamella species (including C. bertholletiae)
• Dematiaceous fungi with opportunistic potential (including Curvularia species and relatives, Cladophialophora species and others)
• Exophiala species
• Fusarium species complexes (including F. solani, F. dimerum, F verticillioides, and F. oxysporum)
• Mucor species (including M. circinelloides and M. indicus)
• Paecilomyces species (including P. lilacinus and P. variotii species complexes)
• Phaeoacremonium species
• Phialemonium species
• Rhizomucor species
• Rhizopus species (excluding R. stolonifer)
• Scedosporium species (including S. apiospermum and S. prolificans species complexes)
• Trichoderma species (including T. citrinoviride/ longibrachiatum complex)
• Trichosporon species

Field sample collection

1. This test requires the use of an instrument that permits a high sampling flow rate. Instruments including the RCS High Flow, the Andersen N6 and the SAS sampler are suitable, however validation studies have so far only been completed on the Andersen N6. The test requires the collection of a total of 1 cubic metre of air on tandemly collected samples which together are considered as a single sample for the purposes of evaluating the airborne fungal concentration against the target action level of 1 CFU/m³. The medium used for this test should be modified Sabouraud’s Glucose agar amended with chloramphenicol. This modified agar is a proprietary Sporometrics medium with which we are pleased to provide our clients interested in using this test.
2. Prior to use, the sampling head and exterior of the device should be disinfected with 70% isopropyl alcohol and allowed to dry. This treatment will help to prevent carry-over contamination from previous usages. As well, a clearly-defined sampling plan should be constructed before beginning sampling.
3. Verify that the expiration date on the sampling medium is current, and that there is no evidence of damage to the surface of the collection medium (e.g. drying or cracking, colour changes, small glistening, pasty or fuzzy dots that might be microbial colonies, or free water on the medium surface)
4. Insert the sampling medium into the air sampler following manufacturer’s recommendations. Wear latex or nitrile gloves during this process to prevent handling contamination.
5. Turn the sampler or sampling pump on to initiate air flow. A maximum air volume of 200 L on a single sample should be collected in as many tandem samples are required to sample an overall volume of 1,000 L of air. All other aspects of this test are similar to IAQ analysis, fungal culture [C100] >
6. After the sampling period has concluded, carefully remove the sampling medium from the device, taking care not to touch the surface of the medium. For Petri plate media, replace the lid and secure it around the edge with tape or Parafilm®. Ensure that you use a water-impermeable marker to label the strip, since the strip cannot be identified if the markings become accidentally wetted. Preprinted self-adhesive stickers work well for sample labeling purposes. Be sure to note the order in which samples were collected, and the sample sets that comprise a single collection.
7. Place samples in a reclosable clean plastic bag and keep in darkness until receipt at the laboratory. Do not store or ship samples on freezer packs, since this leads to condensation on the agar surface which can lead to the formation of spurious satellite fungal colonies, artifactually inflating the count. Samples need to be protected from abrupt temperature changes during shipment, so it is a good idea to keep them in an insulated box during sampling. The bag containing sample media may be wrapped in bubble wrap or paper and packed into a box or sturdy envelope for shipping. Samples must be received at the laboratory no later than 24 hr post sampling. Please ensure that you use a reliable courier service to meet these requirements. In the past we have experienced numerous problems with Purolator in this regard, and we recommend our clients to use FEDEX > for the shipment of time critical samples.

Service options

Results reporting

Results of this test are reported quantitatively per taxon identified in Colony Forming Units per cubic metre of air (CFU/m³) within 24 hr of sample receipt (weekends included).

IAQ analysis, quantitative fungal culture [C100]

Published: July 8th, 2009

Revised: May 10th, 2010

Culture-dependent air sampling remains a mainstay of fungal air sampling. Culture-based methods involve the collection of airborne particles by impaction or centrifugation on the surface of a growth medium. The airborne burden of culturable fungi is then extrapolated based on the colony counts obtained after incubation of the sampled media. Culturable air sampling results are usually expressed in Colony Forming Units per cubic metre air (CFU/m³). Although these techniques cannot be used to evaluate human exposure, they are often useful in detecting the presence of indoor fungal contamination. Unlike spore trap sampling, because counts are derived from actively growing colonies, culture-based air sampling affords an ability to identify recovered colonies to a very low level, often to the species level. Culture-based sampling methods do however have several notable limitations, as outlined below.

Methodological limitations

1. Sampling intervals must remain brief to minimize osmotic changes in sampling media during collection. In contrast to more integrative sampling techniques possible in spore trap sampling, the use of brief sampling periods increases the influence of transient airborne spore bursts on sample results.
2. Standard growth media are selective in their nature, and may not equally support the growth of all fungi present in the air at a given time. Many commonly occurring airborne fungi have specific nutritional requirements that cannot be satisfied by a single general purpose growth medium. Others may be entirely “non-culturable” (e.g. powdery mildew fungi in the genus Erysiphe and others). The growth media typically recommended for culture-based air sampling are intended to optimize recovery of the most relevant subset of the problem indoor fungi.
3. A large proportion of the fungal content of air is non-culturable and thus cannot be detected by this sampling method.
4. Long incubation times are often necessary to obtain sufficient growth and sporulation to permit accurate identification (e.g. 7-21 d). Attempts to expedite this process by shortening the incubation time or increasing the incubation temperature tend to bias the outcome by selecting for rapidly-growing fungi or thermophilic species, respectively.

Culture-based sampling is a useful investigative tool to help determine the presence of indoor fungal growth. Culture-dependent sampling is the preferred air sampling technique when low level fungal identifications are required (e.g. to the species-level). This method is also useful where high levels of non-fungal background particulate may be present that would interfere with spore trap testing. Several commercially available sampling instruments can be used for culture-based air sampling, including the RCS Standard and RCS High Flow, Anderson N6 and 2-stage samplers and the Surface Air Sampler (SAS).

Field collection method

1. Prior to use, the sampling head and exterior of the device should be disinfected with 70% isopropyl alcohol and allowed to dry. This treatment will help to prevent carry-over contamination from previous usages. As well, a clearly-defined sampling plan should be constructed before beginning sampling.
2. Verify that the expiration date on the sampling medium is current, and that there is no evidence of damage to the surface of the collection medium (e.g. drying or cracking, colour changes, small glistening, pasty or fuzzy dots that might be microbial colonies, or free water on the medium surface)

RCS YM strip (above), containing a proprietary Rose Bengal Agar

3. Insert the sampling medium into the air sampler following manufacturer’s recommendations. Wear latex or nitrile gloves during this process to prevent handling contamination.
4. Turn the sampler or sampling pump on to initiate air flow. Flow rates of most units are predefined by the manufacturer of the device. These tend to be in the range of 25-60 L/min. The duration of sampling is usually the only user-controlled variable. During summer months, sample duration should be reduced avoid given the normally high outdoor levels of phylloplane (plant leaf surface) fungi. A final sampling volume of 80-100 L is usually sufficient for most summertime purposes. In the winter, the sampling interval may be doubled unless there is a basis to expect high airborne fungal levels. It is generally not recommended at any time to sample air volumes larger than 200 L on a single sample.
5. After the sampling period has concluded, carefully remove the sampling medium from the device, taking care not to touch the surface of the medium. For Petri plate media, replace the lid and secure it around the edge with tape or Parafilm®. In the case of a sampling strip (e.g. RCS), replace the strip in the transport sheath in the same orientation it was removed (e.g. the surface of the growth medium should project into the “dome” inside the sheath — think of it a bit like an ice cube tray). Replacing the strip into the sheath upside down will invalidate the result and the sample cannot be analyzed. Seal the edges of the strip with tape to prevent drying. In all cases, label the sample sheath with the job number, sample number and collection date. Ensure that you use a water-impermeable marker to label the strip, since the strip cannot be identified if the markings become accidentally wetted. Preprinted self-adhesive stickers work well for sample labeling purposes.
6. Place samples in a reclosable clean plastic bag and keep in darkness until receipt at the laboratory. Do not store or ship samples on freezer packs, since this leads to condensation on the agar surface which can lead to the formation of spurious satellite fungal colonies, artifactually inflating the count. Samples need to be protected from abrupt temperature changes during shipment, so it is a good idea to keep them in an insulated box during sampling. The bag containing sample media may be wrapped in bubble wrap or paper and packed into a box or sturdy envelope for shipping. Samples must be received at the laboratory no later than 24 hr post sampling. Please ensure that you use a reliable courier service to meet these requirements. In the past we have experienced numerous problems with Purolator in this regard, and we recommend our clients to use FEDEX > for the shipment of time critical samples.

Service options

Results reporting

Results of this test are reported quantitatively per taxon identified in Colony Forming Units per cubic metre of air (CFU/m³). Of the microfungi commonly observed in culturable air samples, we generally identify taxa to the species- or species-group level, or to genus-level, wherever is possible within our routine analytical constraints. For example, typically we are able to report most species of Aspergillus to the species-group level (e.g. Aspergillus ustus group, which includes the morphologically very close species A. ustus in the strict sense, A. puniceus, A. panamensis and A. deflectus that cannot be readily differentiated one from the other without specialized procedures). Other taxa that can regularly be reported to the species-group level include Acremonium, Cladosporium, Eurotium, Fusarium, Paecilomyces, Scopulariopsis and Stachybotrys.

By contrast, we routinely identify members of the genus Penicillium to the subgenus-level. This is because identifying species within this group is extremely difficulty in routine practice, and cannot reliably be undertaken by someone who lacks extensive research expertise in this group. Although many environmental mycology laboratories routinely report species-level identifications for this genus, these identifications are dubious at best and cannot be relied upon for any critical purpose. Mostly, they are based on morphological and/ or abbreviated physiological tests given in early monographs, such as Pitt (1979), or less comprehensive treatments including many of the commonly used identification manuals for indoor fungi, including Flannigan et al. (2001), Howard (2003) and Samson et al. (1996) [FULL REFERENCES >]. Filamentous fungi that do not sporulate in culture or otherwise produce morphologically distinguishing features are reported as STERILE MYCELIUM. Non-filamentous fungi are reported as yeasts. Other levels of reporting detail are available on an as-need basis for special projects.

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