Sampling By Filtration

Bioaersols can be collected on filters and analyzed by methods including direct microscopy, growth of propagules directly on the filter medium, elution of particles from the filter, and dilution plating.18 In addition, specialized methods are available for analysis of dust collected on filters for endotoxins, glucans, and mycotoxins, and by PCR. Personal and area air sampling for hardy fungal and bacterial spores can be carried out using presterilized cassettes with cellulose ester or...

Temporal Time Variables

Sampling results in a building are potentially affected by variables including human activity patterns, HVAC system operation, and climatic or seasonal variations such as alternating rainy and dry periods. For example, sampling for culturable fungi during a prolonged dry season will be less likely to detect water indicator fungi from leak-prone envelope walls than will sampling during a rainy period when the building envelope is leaky and wet.18 Similarly, sampling for human-shed bacteria in a...

Outlook For The Future

Despite technical problems that still persist, the use of genetics-based analytical methods is already well established and is widely expected to play an increasingly important role in clinical diagnostic microbiology and environmental microbiology as well as other areas in the future. Further improvements and progress toward the elimination of many of these technical problems will undoubtedly continue to occur. Future progress in the development and use of molecular methods for indoor...

Training Of Microscopy Analysts

Miller et al. indicated that the skill of analysts in quantifying and identifying fungal structures, especially for small colorless spores, is exceptionally important.48 A basic knowledge of mycology is one of the prerequisites for all analysts. New analysts should be trained in mycology until they have enough experience and background to perform analyses. The training should cover general mycology and familiarity with the most common indoor fungi. The minimal educational requirements of...

Contributors

Anagnost, Ph.D., Faculty of Construction Management and, Wood Products Engineering, State University of New York College of Environmental Science and Forestry, Syracuse, NY 13210 (email seanagno esf.edu) Francis Harrington, Ph.D., 15 Hinman Street, Meriden, CT 06450 (email odyharrington aol.com) Richard A. Haugland, Ph.D., National Exposure Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH 45268 (email haugland. rich epamail.epa.gov) Patricia Heinsohn, Ph.D.,...

Materials Needed For Preparing Samples

First and foremost, all samples should be prepared in a clean area, preferably in a containment hood to avoid any contamination. The slides prepared from the environmental samples are temporary or semipermanent, and may last for only several days to less than 3 months. The rule of thumb is that a freshly prepared sample will always yield a better image and is much easier to examine for minute fungal structures. In addition, the sooner the slides are analyzed, the possibility of deformation or...

Introduction

A building investigation for mold is warranted if a building becomes water-damaged by water intrusion through the building envelope, leaks occur within the building envelope, or the building suffers from chronic excessive humidity. Water can penetrate the building envelope by diverse means, such as natural disasters, construction defects, or lack of building maintenance. Multiple sources may contribute to the total moisture burden. Leaks within the building envelope such as plumbing leakage can...

Indoor Fungal Ecology

The observations and reports of fungi growing indoors are not new. Wood decay in buildings has been studied for generations.21 In addition, fungi as biodeteriorating agents have been known. Aspergillus versicolor was reported as commonplace on the wallpaper surfaces in New Zealand public buildings in 1945.64 Growth of Scopulariopsis brevicaulis in buildings leading to poison gas and illness in occupants was reported in the nineteenth century. Morton and Smith65 reviewed the literature on S....

Limits Of Detection

The detection limit (DL) depends on the percentage of the sample trace counted and is defined as one spore divided by the percent of the trace analyzed. For example, if 25 of the trace is analyzed, the detection limit of the analysis is 25 (or 0.25) 4. The quantitation limit (QL) depends on the total air volume (AV in liters) collected and the detection limit or the percentage of the sample analyzed. It can be calculated with the following equation of sample area analyzed or QL DL x 1000 L AV...

Team And Individual Expertise

A modern indoor environment is designed to accommodate many human needs and comfort, and has many built-in components and systems to fulfill such needs. Because of the complexity of such an environment, a team of professionals with various areas of expertise is often necessary in a comprehensive investigation and assessment, including sampling and analysis for microbiological organisms. Individual professionals and their expertise and experience in such activities are discussed below. Many...

Quality Controlquality Assurance And Other Challenges

It is well recognized that genetics-based microbial detection, identification, and characterization methods can pose unique challenges that must be addressed to ensure acceptable data quality. PCR and other techniques employing nucleic acid amplification are particularly susceptible to both false-positive results, namely, detection of target sequences when none are present in the original sample, and false-negative results, specifically, nondetection of target sequences when they are present in...

References

Burge, Biostatistics and Bioaerosols, in Bioaerosols, H. A. Burge, ed., Lewis Publishers, Boca Raton, FL, 1995, pp. 269-307. 2. Macher, J. M., Data analysis, in Bioaerosols Assessment and Control, J. Macher, ed., American Conf. Governmental Industrial Hygiene (ACGIH), Cincinnati, OH, 1999, pp. 13-1-13-16. 3. AIHA, Planning and conducting a survey, in Field Guide for the Determination of Biological Contaminants in Environmental Samples, L. L. Hung, J. D. Miller,...

Bulk Samples

In addition to the basic materials used to examine the samples of spore traps, a roll of transparent tape (19.0 mm x 32.9 m), and a dropper bottle of 70 ethanol is needed to process bulk, dust, tape, and swab samples. Do not use either packing tape (too wide and too sticky to handle) or frosted tape (not clear and will reduce the resolution and clarity of the images) for the microscopic examination. Remember to use aseptic techniques and clean all instruments between every sample. The following...

Tape Lift Samples

Although field investigators may prepare tape samples using different methods, it is important that only clear adhesive tape (i.e., sticky cellophane tapes) be used to collect samples. Most tape samples are fixed or taped to a glass slide or taped within a plastic bag for shipping to the environmental laboratory. Commercially prepared kits have become common and popular since 2002. Becasue of the composition of the tape adhesive, it is important that the tape sample be examined as soon as it is...

Bulk Dust Samples

The following steps are recommended 1. Shake the dust cassette by hand or place on a vortex instrument to produce a more homogenous mixture. 2. Leave the cassette on a bench to allow the dust to settle. 3. Open the cassette (cut the tape with a weighing spatula pry apart). 4. Subsample the dust by grabbing a portion using filter forceps or with a micro-spatula, then place it on a slide. 5. Add a drop of 70 of alcohol or isopropyl alcohol to wet and fix the sample, then spread the dust with an...

FTest And Nonparametric Methods

Each indoor microbial contamination investigation is conducted to answer several questions. One of the most common questions is Is the indoor microbial concentration detected from the area with complaints different from the no complaint areas In order to answer this question, sufficient numbers of samples should be collected to represent microbial levels in both areas. The standard t-test can be conducted to test the hypothesis if the following three assumptions are met. The first assumption is...

Conclusions

There has been tremendous interest in the effects of microbial contamination in the indoor environment. It is well understood that bacteria, fungi, and possibly other biologics, such as mites and insects, grow in water-damaged indoor environments. An understanding of the moisture dynamics is the crucial step in dealing with microbial growth and contamination indoors. An investigation without an understanding of moisture issues in the building and their impact on microbial growth and...

Evaluation Of Fungal Infestation

Mastering microscopy alone does not guarantee a quality microscopic analysis because the analysis involves the combination of adept microscopic abilities, 4.14. EVALUATION OF FUNGAL INFESTATION 97 well-prepared slides, and a strong background with experience in fungal taxonomy, biology, and ecology. The latter is the most important factor for consistent, reliable analyses. The goal of the analysis is to identify and characterize the fungi or fungal spores from the samples so that the results...

Complaint And Noncomplaint Zones

Occupant complaints in buildings may be environmentally based (dampness, water leaks, visible mold growth, MVOCs, etc.), health-related (e.g., upper respiratory irritation, allergy, infection), or both of these. It should be recognized that results of microbial sampling alone cannot be used to determine whether adverse health outcomes have occurred (microbial PELs or TLVs are unavailable). However, careful inspection of a building for dampness moisture damage can lead to a microbial sampling...

Destructive Testing

A probe or pick test is useful if there is no concern with destruction or appearance of the wood. The probe, similar to an ice pick, is inserted into the wood across the grain, and tilted to lift a small piece of the wood. A splintering, uneven failure indicates sound wood, while a brash, smooth failure indicates weakened wood. The relative amount of pressure applied to fracture the wood can be an indication of decay. Cores can be bored out of wood for culturing and or microscopic analysis....

Release And Dispersal Of Fungal Spores

Fungi reproduce by means of spore production. There are two basic categories of spores, sexual and asexual. Sexual spores include ascospores, basidiospores, and zygospores. Asexual spores include mainly conidia and sporangiospores. Spores serve as propagules and are often released and dispersed through various means. Fungal spores may be grouped into two types dry or wet and slimy. Some spores are dry and powdery, while some are wet and slimy. Whether spores are dry or wet may affect their...

Sampling During Mold Remediation Oversight And Clearance

Once the nature, the location(s), and the extent of fungal growth in a building have been determined, an investigator can prepare technical specifications for mold remediation. It is obvious that without this knowledge, mold remediation contractors cannot know what building materials to remediate, how to remediate them, or what constitutes acceptable clearance verifying effective mold remediation. Thus, technical specifications for mold remediation must be building specific to this level of...

Molds And Wood Decay Fungi

Many wood decay fungi (brown-rot and white-rot fungi) are in the taxonomic group Basidiomycetes, while molds include Hyphomycetes, Coelomycetes, Zygomycetes, Sampling and Analysis of Indoor Microorganisms, Edited by Chin S. Yang and Patricia A. Heinsohn Copyright 2007 John Wiley & Sons, Inc. TABLE 8.1. Fungal Taxonomic Groups that Contain Mold Fungi and Wood Decay Fungi TABLE 8.1. Fungal Taxonomic Groups that Contain Mold Fungi and Wood Decay Fungi or Ascomycetes (Table 8.1). The group...

Case Studies

Background The new owners of a 20-year old townhouse took possession and found a decayed and rotten wood subfloor, between the first and the second floors, within the first month of ownership during a renovation. The decay was such that second floor could be seen from the first floor through two openings. In addition to the wood decay, Aspergillus versicolor, Chaetomium globosum, and Stachybotrys chartarum were identified from moldy drywall samples in two other locations in the townhouse....

Principles And Usage Of Microscopes 421 Microscopes

Most introductory microscopy topics, such as (1) anatomy of a microscope, (2) basic microscopy and image analysis, (3) magnification, resolution and contrast, (4) numerical aperture, (5) illumination techniques, (6) depths of fields of focus, and (7) microscope maintenance, are not discussed here but can be found in a number of reference books on microscopes and on the internet at specific manufacturers' Websites, usually with interactive tutorials, illustrations, and in-depth instructions for...

Microscope Objectives

The compound microscope can be set in a number of ways to greatly increase the contrast images of environmental specimens through objectives and other accessories for specialized applications. Generally, most optical microscopes are equipped with a pair of 10x ocular lenses, or occasionally with 15x eyepieces. Ninety percent of all optical microscopy examinations are conducted using standard achro-mat or plan achromat objectives, which are inexpensive and easily available.9 Standard brightfield...

Factors Affecting Fungal Growth Indoors

Many environmental parameters can influence fungal growth indoors. Some are biotic factors, and others are physical and chemical, or abiotic factors. Biotic factors include the presence of fungal propagules or spores, viability of spores, the nature of the fungal species, and competing fungi and other organisms. Sampling and Analysis of Indoor Microorganisms, Edited by Chin S. Yang and Patricia A. Heinsohn Copyright 2007 John Wiley & Sons, Inc. Abiotic factors include nutrients, temperature,...

Advantages And Limitations Of Culturebased Analytical Methods

Using spore-trapping samplers to collect fungal spores followed by direct microscopy in the laboratory to identify and enumerate the spores is frequently done in indoor air quality (IAQ) surveys. This practice has its practical merits, but it is not without limitations. One problem is the direct examination of the samples by microscopy, which results in uncertainty in the identification of the fungal spores. Only a small number of fungal spore types can be identified to generic level with...

Common Airborne And Indoor Fungi And Their Spores

Proper identification of fungi is challenging and the most important part in microscopic analysis. For spore count, only spore morphological characteristics are available. For microscopic analysis on other samples, additional fungal structures may be available. It is important for analysts to understand how to utilize available fungal structures for accurate identification and to recognize the significance of presence of certain fungi and fungal structures from perspectives of fungal biology...

Sample Preparation Methods

The general principle for fungal recovery is to provide fungal spores with the nutrients and optimal conditions that they require for germination, growth, and sporulation. In an environmental sample, there is often a mixed population of fungal species. The choice of recovery and isolation methods should be based on the ecology of the targeted group (e.g., mesophiles or thermopiles), the need for selective isolation (e.g., for pathogens), and the objective of the sampling (e.g., investigator's...

Destructive Testing Investigation

Destructive testing is done only when there is a high index of suspicion that water intrusion has occurred. Destructive testing enables a forensic water intrusion investigator to ascertain the cause of water intrusion and recommend a repair. Destructive testing enables a mold investigator to determine the nature and extent of fungal growth that is hidden from view during a baseline investigation. Destructive testing in search of hidden fungal growth is justified if (1) there is an active leak...

Physical Inspection

Physical inspection requires access to all areas of the subject building unless there is a clearly defined, justifiable reason not to inspect one or more areas. The underlying reason for this is that a moisture problem in one area of a building can impact another. This includes occupied areas and unoccupied areas such as attics, hot-water-heater closets, storage rooms, garages, and crawlspaces. In part, all of the relevant areas investigated depend on the type and use of the building, such as,...

Surface Sampling

Cellotape, sterile swab, or surface contact plate sampling can be used to document the type and or concentration of microbial agents found on the tested surface.18 As with other types of microbial sampling, analytical results alone cannot be directly related to health outcomes (absence of microbial TLVs or PELs). Cellotape sampling together with direct microscopy analysis provides a convenient method for (1) identifying the types of molds that may be present on a surface and (2) documenting...

Spore Trap Samplers

Spore traps are devices that collect both viable and nonviable spores as well as other morphologically distinct particles (pollens, hyphal fragments, skin scales, plant hairs, textile fibers) by impaction onto a sticky glass or tape surface. Information on sources of commercially available spore traps is found elsewhere.18,32 The particles collected in a spore trap are examined morphologically by direct optical microscopy with or without staining in order to determine kinds of fungal taxa or...

Abiotic Factors

Fungi are achlorophyllous and nonphotosynthetic. Their survival and growth rely heavily on their ability to obtain nutrients, such as sugars, amino acids, vitamins, and macro- and micronutrients, from the substrates. Different species of fungi have different abilities to access and utilize simple or complex forms of carbohydrate, organics, and mineral nutrients.16 Some fungi, such as species of Aspergillus and Penicillium, are called sugar fungi because they are fast-growing and...

Viability of Fungal Spores

Most of the microbial IAQ sampling methods collect fungal spores and hyphae. Spores are minute propagative units (propagules) that function as seeds, usually with some food reserve, but not containing a preformed embryo. The fungal spores that are important in IAQ investigations are generally between 2.0 and 10 mm in length. The mechanism by which spores are produced is the basis for classifying fungi. This is why identification using culture analysis is the primary method of choice. Spore...

Procedures For Identification And Quantification Of Spore Traps

Reference data and relevant calculations are as follows 1. Calibrate the measurements of different magnifications of all objective lenses with a stage micrometer and an ocular micrometer. Attach these measurements to the microscope base for future reference. The magnifications of the microscope should be calibrated at least yearly. The full length of the ocular micrometer under 40 x, 60 x, and 100 x lenses and the diameters of these lenses should be measured also. For instance, the Olympus BH2...

Baseline Investigation

Building investigations do not start with sampling for mold. They start with getting as much relevant background information as possible. Possible sources of relevant information could include the following Building maintenance personnel Building architect or architectural plans Other consultants, including various engineers or environmental consultants The kind of information needed may vary somewhat depending on the nature of water intrusion but commonly includes the following The Leak...

Techniques For Spore Count Analysis

The following steps are recommended 1. Scan the slide. Use a 10x or 20x objective lens to scan the entire trace of the deposit. Determine whether the spore deposit is random or evenly distributed. If clustering and clumping of certain spore conidial types are observed, note it on the datasheet. If the trace is heavy and thick, rank the load of background particulates on the datasheet. Do not focus the spore counting on a certain section or portion of the adhesive band. Scan for spores over the...

Techniques For Assessment Of Wood Decay And Mold In Buildings

A first step to diagnosing a mold or decay problem can be to look for any visible signs of fungal growth such as fungal structures (rhizomorphs, even fruiting bodies in some cases) or discoloration, and signs of associated moisture, such as condensation, water stains, peeling paint, and warping. For molds, fungal colonies can be light or dark either white, gray, black or brownish black or brightly colored, such as green, red, yellow, or pink. Colonies of wood decay fungi are typically white or...

Moisture Requirements for Wood Decay Fungi

It is generally accepted that the moisture content of wood must be above the fiber saturation point for decay to occur. Optimal conditions for decay are when wood moisture content is 40-80 MC.19 In a building when wood moisture content is below the fiber saturation point, the lower limit of RH for wood decay fungi to grow has been shown to be at aw > 0.97, or when RH is > 97 .11 This corresponds to a wood moisture content of about 25-27 (Table 8.3). When free water is present and the wood...

Specific Ecological Niche of Some Common Indoor Fungi

Most, if not all, common fungi found growing indoors are saprophytes, biodeterior-ation agents, or both. Species of Aspergillus and Penicillium are, in particular, well-known saprophytes and documented biodeteriorating agents of a wide variety of materials fruits, foodstuff, produce, textiles, paints, and wood and paper pro-ducts.5,10,74-76 These two genera include more than 180 species for Aspergillus and more than 220 species for Penicillium.77 With each genus, they include both xerophiles...

Microbial Forensics

Microbial forensics is a term that is applied to an analysis tracing a microbe to the source(s) of origin using molecular and genetic techniques.3'4 The most common technique used is by comparing genetic variation and relatedness within and between species.5 Although microbial forensics is relatively new, there are government surveillance systems and gene databanks that store and make DNA microbial fingerprints and gene sequences available for medical and economical investigations. In response...

Quality Assurancequality Control Procedures

Proper QA QC is important to ensure the quality of the microscopic analyses. There are many aspects and areas where QA QC will play a role in a microbiology laboratory. However, the present section will deal only with the issues and steps related to the microscopic analysis. As stated previously, no standard protocols are available for laboratory analyses and no numeric standards or guidelines exist for interpreting laboratory results of airborne fungal structures. It is unlikely that such...

Sampling Design

After obtaining all relevant information, the mold investigator develops a sampling design that will fulfill the goal to determine the nature and extent of fungal growth and contamination. Execution of the sampling design follows the physical inspection. This chapter includes recommendations for various investigation sampling designs. Consult other chapters in this book that specifically address statistics, sample analysis, and fungal ecology to complement discussion here. A recommended...

Fungal Spore Types and Their Release

In conidial fungi or molds, dry conidia are often released by passive mechanisms or by disturbance such as gravitation force, convection currents, deflation force, or mechanical disturbance. Some spores are actively released by osmotic force built up with water. Movement and feeding of arthropods, such as insects and mites, are also a common release-dispersal mechanism.97-99 Drifting mist or tiny water droplets in air current can serve as a secondary process to carry and disperse spores of...

Dust Samples

A sampling strategy involving the analysis of settled dust especially from above-floor surfaces33,37 provides an indication of microbial agents that were likely once airborne. Settled dust is heterogeneous, consisting of particles from people (e.g., skin scales), pets (e.g., Fel d1 allergen), textiles, paper, cooking, and the outdoor air. The composition of settled dust can vary depending on the location in a room or building. Settled dust may be collected by filter cassette minivacuum,34...

Sampler Performance Sampling Time and Culture Preparation

There are variations in performance among the culturable samplers that are commercially available, such as the inertial impactors including the Andersen or similar samplers, Burkard viable sampler, the Surface Air System (SAS) samplers, the Reuter Centrifugal Samplers (RCS), the AGI-30 impinger, or filter cassettes. Investigators should be familiar with the proper usage and performance of the air sampler that they choose to use.6 Sampling time is another crucial factor affecting the results....

Analysis Of Samples For Airborne Bacteria

The concentrations of airborne bacteria are determined by volumetric air sampling,11 but air sampling alone does not verify that an area is free of biological contamination.50 Airborne cells settle onto surfaces and may not be present in the air at the time of sampling, although they are reaerosolized during routine activity.51,52 Therefore, surface sampling in conjunction with air sampling assists in determining areas of contamination, identifying the source of biocontamination, and...

Rationales And Mycological Backgrounds

Fungal spores are microscopic and invisible to the naked eye. Furthermore, developing fungal microcolonies may be small and inconspicuous in the early growth stages. Fungal growth may have occurred for a period of time and be hidden in the HVAC system, in wall cavities, behind baseboard, or underneath vinyl wallpaper. These make the detection of vegetative and reproductive growth in their early stage of development extremely difficulty, although not impossible. Some information can be obtained...

Analysis Of Variance Anova And Chisquare Statistics

The t-test or its nonparametric equivalent is used to test the mean differences of two groups of samples. If more than two groups of samples are in the study design, analysis of variance ANOVA is used to test whether all groups have the same mean. This method has the advantage of testing whether there are any differences between the groups with a single probability associated with the test. The same assumptions for a t-test should be met when conducting an ANOVA that is, all groups of data must...

Air Sampling For Molds

Air sampling is not required in most buildings in order to demonstrate that mold remediation has been effectively carried out.3,4 However, air sampling can be useful as a part of the quality assurance sampling. For purposes of documenting effective remediation, it is generally adequate to verify that visually moldy materials including hidden mold have been removed according to industry-standard quality assurance measures25 and that residual dusts have been removed by HEPA vacuum cleaning so...