Indoor Environmental Specialists
Welcome to Environmental Diagnostics Corporation (EDC). Managing indoor environments is easier with the proper documentation from experienced professionals. Our highly commended proactive, reactive, and mold assessments, cutting edge sampling methods, and interpretative tools make EDC a leader in the industry.

What are bioaerosols?
Bioaerosols are airborne particles and gases that are living or have originated from living organisms. The term microbials is often used as a synonym but does not necessarily denote that the contaminant is airborne.

Examples are:
  • Endotoxins (bacterial by-product)
  • Mycotoxins (fungal by-product)
  • Microbial VOCs (fungal and bacterial by-products)
  • Antigens
  • Dust mites
  • Viruses
  • Bacteria
  • Fungi

There are no specific regulations governing surface microbiological contamination or airborne microbiological contaminants in indoor air (bioaerosols). This is in part due to the many variables involved with sampling for microorganisms, dramatic fluctuations in background levels of microorganisms, lack of agreement between researchers about what constitutes a "problem situation". However, academia and industrial hygiene associations have presented guidelines. Microbial concentrations and the exposure to their by-products (spores, antigens, mycotoxins, endotoxins, and volatiles) account for a substantial amount of illness, allergic reaction, and physical discomfort. Litigation, medical diagnosis, and other forces often demand a standard of care that includes microbial testing in indoor air quality (IAQ) investigations. Often more than one type of sampling is suggested by published guidance.
Guidance on bioaerosols is readily available from ACGIH1 and AIHA2 and other organizations. This document is for general information purposes only. Although it may be helpful, it is not intended for use by healthcare or hospital facilities, litigation development, or medical/epidemiological investigations.

What are potential health consequences of exposure to bioaerosols?
The health consequences of pathogenic or allergenic types and relative concentrations of microbiologicals (bioaerosols) in the indoor environment can range from no reaction to severe and insidious health afflictions. Microbial sampling and proper interpretation of results, given that they generally represent only a snapshot in time, must be evaluated within the context of a well conducted IAQ investigation. Special environments such as critical care facilities and institutions housing or caring for immune suppressed individuals must be held to more stringent guidelines. The synergistic effect of exposure to multiple fungal genera, mycotoxins, bacteria, and endotoxins and its relationship to occupant complaints and illness is poorly understood.
Although infection can even occur in an otherwise healthy individual occasionally, contaminated indoor environments are of particular concern with infants, elderly, and immune compromised individuals who are much more susceptible to infections. Health effects can be divided into four general categories: infection, toxicosis, allergy and irritation.

Infection: There are now over 100 fungal species that are known to cause infection in humans. There are three classifications of infection caused by fungi: systemic, opportunistic and dermatophytic. Infection can also be caused by virus, bacteria, and other biological contamination.

Toxicosis: Many fungi produce toxic metabolites called mycotoxins. The health effects from exposures to the levels of mycotoxins that may be encountered in contaminated indoor environments are not yet completely known. Generally mycotoxins are nonvolatile and inhalation exposure usually occurs only after disturbance of a contaminated source. Symptoms of exposure to mycotoxins may include cold and flu like symptoms, headache, nose bleeds, dermatitis and immune suppression. See the mycotoxin section for more information. Toxicosis can also be caused by exposure to endotoxins, which originate from the cell walls of gram-negative bacteria. Reported acute symptoms of inhaled endotoxin exposure include chest tightness, cough, shortness of breath, fever, and wheezing. See the endotoxin section for more information.

Allergy: Allergy is the most common symptom associated with exposure to bioaerosols, including pollen grains, fungal spores, house dust mite feces, and other allergens (cockroaches, cat, dog, mouse, etc.). Any fungus can be allergenic, producing antigenic proteins and polysaccharides that can cause allergic reactions in sensitive individuals. Symptoms include: allergic rhinitis, sinusitis, chronic bronchitis, eczema, atopic dermatitis, allergic contact dermatitis, allergic bronchiopulmonary aspergillosis, hypersensitivity pneumonitis, humidifier fever, and asthma.

Irritation: Fungi and bacteria produce odoriferous microbial volatile organic compounds (MVOC) during degradation of substrates. These compounds, among other contaminants, can irritate mucous membranes, cause headaches and other symptoms.
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What are endotoxins, mycotoxins, and MVOCs?
Sampling and interpretation of endotoxins, mycotoxins, and MVOC's (many of which are quite odoriferous and toxic) is a new science. Adverse health effects potentially caused by these contaminants should not be underestimated. Determining the concentrations of these contaminants, however, can be expensive, time-consuming, and should only be conducted by experienced investigators and labs with the proper techniques, quality control, analysis, and interpretation. In most cases, samples must be interpreted relative to each other and a control therefore, a minimum of three samples must be taken of a particular contaminant category. Expect these specialized samples to take a total of at least three to four hours and cost at least $200 each.

Endotoxin is a term for the highly toxic outer membrane of gram-negative bacteria. Health effects vary, depending on the individual, dosage, and route of exposure. The fact that the bacteria do not have to be alive for endotoxins to pose a serious health threat is the main impetus for concern. Reported acute symptoms of inhaled endotoxin exposure include chest tightness, cough, shortness of breath, fever, and wheezing. Chronic effects have be suggested. Gram-negative bacteria are found everywhere in nature. But elevated airborne levels are prevalent in sewage treatment plants, cotton textile mills, poultry houses, and swine operations. As these bacteria are prevalent in many water systems, particularly ones containing sewage, buildings with humidifiers or water intrusions (especially ones involving sewage) often have elevated levels of gram-negative bacteria.

Airborne endotoxin may pose a much larger risk than formerly thought and as such it is likely to emerge as a major indoor air quality issue in the future. A routine analytical technique for detecting endotoxins has been developed. Results from different labs are not comparable and background control samples must be taken.

In general, little is known about indoor endotoxin sources. The best way to minimize non-industrial endotoxins sources eliminate contamination with GNB and limit aerosolization of material contaminated with GNB. Closely monitor and frequently disinfect standing bodies of water including humidifiers. Address water incursion quickly and properly, especially when sewage is involved.

Fungi are ubiquitous to the environment and many use organic water-damaged building materials as nutrients. During the digestion process fungi secrete enzymes into the nutrient source to break down complex compounds into simpler compounds, which are taken up by the fungi and digested. The digested nutrients are classified into two categories, primary and secondary metabolites. The primary metabolites consist of cellulose and other compounds that are used for energy to grow and reproduce. The secondary metabolites, called mycotoxins, are produced to give fungi a competitive edge against other microorganisms, including other fungi. There are over 200 recognized mycotoxins, however, the study of mycotoxins and their health effects on humans is in its infancy.

Mycotoxins can cause a variety of short term as well as long term health effects, ranging from immediate toxic response to potential long term carcinogenic, teratogenic, cardiovascular, and neurologic effects. Symptoms due to exposure to mycotoxins include dermatitis, cold and flu symptoms, sore throat, headache, fatigue, diarrhea, and impaired or altered immune function, which may lead to opportunistic infection. Research has implicated many toxin producing fungi, such as Stachybotrys, Penicillium, Aspergillus and Fusarium species, to indoor air quality problems and building related illnesses. Mycotoxins are non-volatile and although routes of exposure vary, the most significant concern addressed here is the accumulation of mycotoxin on particulates, especially fungal spores that can become aerosolized and inhaled. Scientifically valid and cost-effective sampling for mycotoxins is not available for indoor air quality studies, at this time.

Microbial Volatiles (MVOC)
Some investigators are beginning to use VOC analysis to search for microbial VOCs (MVOCs). However, only a few laboratories currently can apply such analysis effectively in building investigations. The uncertainties associated with attributing VOCs to microbial sources preclude using this approach for routine investigations. It is difficult to determine what portion of indoor VOCs arise from common fungi, bacteria, and other microorganisms, but MVOCs likely are responsible for only a small fraction of total VOCs, measuring in the low ug/m3 range. However, a severely contaminated building may have a significant MVOC contribution derived from microbial growth. MVOCs nevertheless can contribute to the health effects reported by the occupants of contaminated buildings. Many bacteria, especially anaerobic ones, produce odorous MVOCs that microbiologists use to identify bacterial isolates. However, information on the MVOCs bacteria produce in building environments is even more limited than for fungi. Some primary fungal metabolites are pungent and others evoke unpleasant associations such as "dirty socks" or "locker rooms" or are described as "moldy," "musty," or "mildew-like." Still other fungi release odors that are considered pleasant and are described with such terms as "fruity," "earthy," or "snow-pea-pod-like." At this time, MVOC sampling for non-research IAQ assessments is not a valid option.
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Can EDC sample for antigens; dust mites; viruses; and Legionella?
Sampling and interpretation of antigen (cat, dog, mouse, cockroach proteins) and dust mite concentrations is available and sampling is affordable. Environmental sampling for viruses is not recommended. Air sampling for Legionella is not recommended, only water sampling. Legionella is addressed elsewhere.

Does bacteria play a role in indoor air quality?
Bacteria in the indoor environment are common but certain types and concentrations can be problematic. Bacteria is primarily sampled from suspected source reservoirs (i.e. humidifiers, condensate pans) or air sampling. Isolation of bacteria by laboratory culture remains the primary method of environmental sample analysis (i.e. direct microscopy is not used, in contrast to fungal spore analysis). Endotoxin (from bacterial cell walls) sampling and analysis is a promising science. Bacteria in the environment seldom form discrete visible evidence other than slime, biofilm, or foam, typically found in standing water. Fruity, sour, or putrid odors are often indicators of bacterial growth. The content of some discussion here may not be applicable for Legionella , Tuberculosis, or other types of bacteria where specific medical guidance and sampling already exist.

Gram positive bacteria (GPB):
This human-shed type of bacteria is common indoors. Concentrations depend on number of people, activity, clothes worn, and ventilation rates. High levels may be acceptable depending on the factors above. Extremely high levels of GPB in low-activity areas may indicate over-crowding or poor ventilation. Such problems can be identified, however, by other contaminants such as carbon dioxide or visual observations. Some types of gram positive bacteria are pathogenic but investigation of these typically only occurs in healthcare and hospital settings.

Gram negative bacteria (GNB):
A predominance of this type of bacteria indoors suggests the presence of fecal contamination (from a sewage backflow or overflowed toilet) or standing water (from in humidifier reservoir) or significant flooding that was not cleaned or remediated properly. Serious health effects are possible depending on numerous factors.

Viable bacterial spore sampling and subsequent incubation, identification, and interpretation has numerous limitations including:
  • Refer to the limitations that affect culture-based air sampling.
  • Isolation and identification of bacteria in culture is time-consuming and require repeated transfers and manipulation of cultures.
  • False negatives and underestimation of actual bacterial contamination problems has been documented.
  • Little is known about the potential effects for healthy adults and children from a majority of the bacteria recovered during air and source sampling.

If information about the extent of potential gram negative bacterial contamination is required then endotoxin sampling could be conducted.
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How does fungal biodiversity and concentrations affect environments?
Fungi or mold just like bacteria is everywhere. It is critical to life on earth. However, elevated fungal concentrations and pathogenic types are undesirable in indoor environments. Depending on the environment and human susceptibility even low concentrations of pathogenic types or high concentrations of non-pathogenic types can be harmful. Further information about fungi can be found in other sources that are detailed near the end of this document.

No accepted guidelines exist that formally address microbial genus or species and associated concentrations. But what is known is the possible health consequences and legal/financial implications undesirable bioaerosol exposure. Each situation or suspect environment is typically addressed on a case by case basis where all factors are evaluated in concert. Fungi spores can effectively be sampled by one or more methods discussed below.

What about microbiological sampling methods?
The adverse health consequences of elevated concentrations or pathogenic biodiversity of microbiologicals (bioaerosols) in the indoor environment can be significant and prevented. Microbial sampling and proper interpretation of results must be evaluated within the context of a complete building indoor air quality (IAQ) investigation. All sampling and data interpretation must be made on a case by case basis in a non-alarmist, confidential, and practical manner.

One step to understanding microbial sampling and subsequent results is to recognize the advantages and disadvantages of various methods of microbiological sampling. A comprehensive microbial sampling and analysis strategy should use both culture and non-culture methods, given appropriate financial resources. If resources are limited culture methods could receive less attention than other methods. The top priority should be a thorough visual and olfactory examination by a qualified investigator using written and photo documentation. Real time instruments such as a moisture meter, particle mass monitor, temp/humidity meter, and other "IAQ parameter sensors" should also be used and evaluated in concert with microbial sampling.

The methods discussed here are primary applicable for mold and bacteria. Other methods are available for endotoxin and antigen sampling.

Surface, source, and bulk sampling
Surface sampling consists of placing a medium (i.e. culture swab, agar plate, or clear double-sided tape) in contact with a visually contaminated surface. At a laboratory, the suspected contaminant is then either applied to a growth media (culture method) for subsequent analysis or analyzed by direct microscopy. Surface sampling must be interpreted carefully since it is subject to several limitations. Resulting analysis and identification should appropriately discuss the potential for false negatives, inherent interpretation difficulty, and culture limitations. Results should be qualitative (identify fungal type present) but not quantitative. Generally, surface sampling should not be used for random or proactive purposes but reserved for diagnostic, healthcare, forensic, and water-intrusion investigations. Collection using a direct tape lift and analysis by direct microscopy is faster, less expensive, and more accurate but can only determine genus not species.

Bulk and source sampling can involve collection of material such as contaminated drywall, insulation, or return air filters. Or a quantity of dust be obtained through vacuuming or other collection and sent for analysis. Non-viable and viable analysis methods can be conducted. Interpretation can be difficult but also part of examining specific hypothesis so a proper remediation strategy can be developed.

  • Culture-based air sampling method
    Various types of culture-based sampling exist (such as those that use liquid impingers), however, direct agar impaction is most commonly used. Microbial food or "agar" in small circular plates (as used by Anderson sampler type instruments) or in strips (as used by the RCS sampler) has been used extensively to grow "viable" airborne microorganisms for subsequent identification. This method has been used extensively to obtain information about viable fungal and bacterial contamination.

    Disadvantages of this method include:
  • Microbial food or agar is "selective" meaning the growth of certain microbial type can be inhibited by the type agar itself. Knowledgeable selection of agar is required.
  • Microbial competition, specifically bacteria and fungal elements often compete with each other and among themselves for nutrients (depending on species and genus). For example, Penicillium mold has anti-bacterial properties. Aspergillus has been shown to outgrow and inhibit Stachybotrys.
  • Incubating culturable or "viable" spores requires several days before to identification is possible.
  • Various microbials incubate or "grow" differently at particular temperatures.
  • Non-viable or "dead" spores can not be counted using this method, even though they are a major contributor to occupant health aliments,
  • Overgrowth can kill off or mask colonies resulting in inaccurate concentrations,
  • Condensation from agar can cause cross contamination.
  • Improperly stored or transported agar can affect results.
  • Viable spores can be killed or damaged by the inherent nature of sampling resulting in false negatives and underestimation of contamination.
  • Direct microscopy is often required anyway for proper genus identification of fungi (not available for bacteria).
Advantages of this method include:
  • Species can be identified for medical diagnostic and treatment purposes (keep in mind, that it can be difficult to correctly identify species - i.e. Aspergillus genus has over 160 species).
  • A good amount of data interpretation information is available in literature.

Slit impaction air sampling using direct microscopic analysis
Slit impaction or "spore trap" instruments, such as the Allergenco, Burkhard, or Zefon Air-O-Cell, use a clear sticky substrate (such as stopcock grease) to collect bioaerosols, typically fungi, for subsequent analysis by "nonviable" direct microscopy, typically at 400 magnification. EDC prefers this method as its primary method of airborne fungal sampling.

Disadvantages of this method include:
  • Species can not be determined if such information were needed for medical or legal reasons. Fungal genera is easier to identify.
  • Bacteria can not be analyzed.
  • The viability of organisms can not be determined (however, dead organisms can cause disease too).
  • Currently, there is less concentration guidelines in available literature since counts include both viable and nonviable spores.
Advantages of this method include:
  • No special transporting or handling of samples is needed.
  • Since spore trap samples do not need to be incubated they can immediately be analyzed by direct microscopy. This is ideal for clearance sampling after remediation.
  • There is no concern of selectivity of agar or microbial competition.
  • This method has the ability to allow enumeration and identification of viable and non-viable fungal spores, pollen, fibers, and other bioaerosols.
  • A significant quantitative advantage over culture-based methods has documented for some fungal genus.
  • Instruments (Zefon, Burkhard and Allergenco) samplers are typically lighter, more portable, and quieter.

Sampling for mold in wall cavities
Growth of toxic fungi in wall cavities can occur as a result of a number of circumstances ranging from construction and design defects to accidental water intrusion. Assessment of microbial contamination in wall cavities presents a number of problems. Typical destructive testing (see boroscope exception) performed to gain access for visible inspection and surface sampling should be avoided. In addition to the aesthetic issues associated with destructive testing, there are potential hazards to the technician and occupants if a large penetration hole exposes mycotoxigenic fungi to the ambient indoor air. In response to these issues, the WallChek has been developed. The WallChek is a nondestructive sampling device for assessing microbial contamination in wall cavities. When used correctly, the WallChek device can help identify sources of fungal growth within wall cavities that may not be visible from within the room. As with all indoor microbial investigations, interpretation is subjective and should be based on comparing total numbers and ranking of fungal genera of suspect and control sample locations. (Courtesy of Aerotech Labs)

Visual inspection of wall cavities, given the limitations of obstructing insulation and studs, can be achieved using penetrations as small as 1/2" holes. A borescope can then be used to visually observe if extensive growth is present.

What quality online sources for microbiological guidance does EDC suggest?
For further information EDC finds the following online sources helpful. However, explore at your own risk.

Can EDC provide detailed published guidance on microbials?
Yes, as a service to our commercial clients and members EDC has provided the following sources of microbial guidance. A password is required to enter the members only section. If you are an EDC client or member and do not know your password please contact EDC via phone or email (see top of the screen).
Information that addresses microbials and bioaerosols typically address other aspects including: sampling , data analysis, health relationships, biocides, remediation, and details on specific agents. The sources below, although full of detail, can be used by the layman reader as an overview or reference guide.

Title: Guidelines on Assessment and Remediation of Fungi in Indoor Environments
Author: New York City Department of Health, Bureau of Env. & Occ. Disease Epidemiology
Date, Size: April 2000, (102KB), 6989 words
Download (Members Only)

Title: Bioaerosols: Assessment and Control
Author: ACGIH (American Conference of Governmental Industrial Hygienists)
Date, Size: 1999, Several hundred pages, hardcover
Cost: Contact ACGIH or contact EDC for a copy (EDC charges $135 for the book including shipping)
Summary: A classic. Overall, the best bioaerosols book available. It is highly recommended for anyone that needs accurate guidance.
Chapters: There are 26 chapters such as: Investigation Strategy \ Health Effects \ Building Walkthrough \ Sampling Plan \ Sample Analysis \ Data Interpretation \ Medical Roles \ Respiratory Infection \ Prevention and Control \ Air Sampling \ Source Sampling \ Data Analysis \ Data Evaluation \ remediation \ Biocides \ Source Organisms \ Bacteria \ Fungi \ Amebae \ Viruses \ House Dust Mites \ Endotoxins \ Mycotoxins \ Antigens \ MVOCs

What is Stachybotrys, a toxic mold?
Stachybotrys is a mold that has caused increased nationwide concern especially due to recent (March 2000) media attention to its potentially toxic effects. Although many molds produce mycotoxins, those produced by Stachybotrys, especially T-2 toxin and satratoxin H, are extremely toxic, are suspected carcinogens and are immunosuppressive. Currently, it is documented that the most effective way to confirm airborne presense of Stachybotrys spores is to use nonviable spore trap methods. Bulk and surface sampling from visible contamination is also suggested.
The following sources provide timely guidance regarding Stachy.

Title: Update: Pulmonary Hemorrhage/Hemosiderosis Among Infants - Cleveland, Ohio, 1993-1996
Author: CDC MMWR Weekly
Date, Size: March 2000, (40KB), 2461 Words
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Summary: A review within CDC and by outside experts of an investigation of acute pulmonary hemorrhage/hemosiderosis in infants has identified shortcomings in the implementation and reporting of the investigation described in MMWR (1,2) and detailed in other scientific publications authored, in part, by CDC personnel (3-5). The reviews led CDC to conclude that a possible association between acute pulmonary hemorrhage/hemosiderosis in infants and exposure to molds, specifically Stachybotrys chartarum, commonly referred to by its synonym Stachybotrys atra, was not proven. This report describes the specific findings of these internal and external reviews.

Title: Stachybotrys chartarum: Current Knowledge of Its Role in Disease
Author: Daniel L. Sudakin, MD, MPH
Date, Size: February 2000, (41KB), 2371 Words
Download (Members Only)
Summary: Stachybotrys chartarum is one of several species of filamentous fungi capable of producing mycotoxins under certain environmental conditions. In some observational studies, the growth of this toxigenic mold in the indoor environment has been implicated as a cause of building-related illness. Following reports of a cluster of cases of pulmonary hemosiderosis and hemorrhage associated with exposure to Stachybotrys, public health measures have been recommended which have far-reaching implications. Although the hazards associated with exposure to some mycotoxins have been well studied, the health risks from environmental exposure to Stachybotrys remain poorly defined. The purpose of this review is to critically evaluate the current body of epidemiological knowledge regarding Stachybotrys and to increase physician awareness regarding this emerging environmental health issue.
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Has microbial contamination caused lawsuits?
Absolutely, EDC suggests reviewing the following comprehensive document.
Title: Microbiological Contamination Litigation
Author: Alexander Robertson
Date, Size: 1999, (93KB), 6502 words
Download (Members Only)
Sections: Causes of action \ Common fungi found in water damaged buildings \ Fungi and mycotoxins \ Allergic reactions to fungi \ Destructive and non-destructive testing for mold \ Interpretation of results of microbiological testing \ Recent research on mold \ Expert testimony in mold cases \ Damages recoverable in mold cases \ Statute of limitations for mold cases \ Recent published verdicts & settlements of mold cases in California \ Recent publicized mold cases in other states.

What is the role of moisture?
The documents below provide an extended discussion of moisture at its impact on buildings and wood.
Title: Moisture Dynamics in Building Construction
Date, Size: 2000, (62KB), 4374 words
Download (Members Only)
Sections: How and Where to Look for Moisture \ Factors Contributing to Moisture Problems \ Looking for Signs \ Moisture Transport and Movement

Title: Water and Wood
Date, Size: 2000, (119KB), 8955 words
Download (Members Only)
Summary: Everything you will ever need to know about moisture, wood, and wood floors.
Please contact EDC at 703-352-0488 if we can provide further assistance.

1American Conference of Governmental Industrial Hygienists (ACGIH). Bioaerosols: Assessment and Control, 1999.
2American Industrial Hygiene Associations (AIHA). Field guide for the Determination of Biological Contaminants in Environmental Samples, 1996.



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