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SAMPLING ANDANALYSIS OF INDOORMICROORGANISMS

CHIN S. YANG

P&K Microbiology Services, Inc.Cherry Hill, New Jersey

PATRICIA A. HEINSOHN

Micro Bios Pacifica, California

InnodataFile Attachment9780470112427.jpg


CHIN S. YANG

P&K Microbiology Services, Inc.Cherry Hill, New Jersey


Micro Bios Pacifica, California

Copyright # 2007 by John Wiley & Sons, Inc. All rights reserved.

Published by John Wiley & Sons, Inc., Hoboken, New Jersey.

Published simultaneously in Canada.

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Library of Congress Cataloging-in-Publication Data:

Sampling and analysis of indoor microorganisms/[edited by] Chin S. Yang,Patricia A. Heinsohn.

p. cm.

ISBN-13: 978-0-471-73093-4

ISBN-10: 0-471-73093-9

1. Buildings. 2. Microbial ecology. 3. Molds (Fungi). 4. Environmental sampling.

I. Yang, Chin S. II. Heinsohn, Patricia A.

QR100.S26 2007

5790.17--dc22 2006024643

Printed in the United States of America

10 9 8 7 6 5 4 3 2 1

http://www.copyright.comhttp://www.wiley.com/go/permissionhttp://www.wiley.com/go/permissionhttp://www.wiley.com

CONTENTS

PREFACE xi

CONTRIBUTORS xv

1. INTRODUCTION TO MICROBIOLOGICAL GROWTH ANDCONTAMINATION INDOORS 1

Chin S. Yang and Patricia A. Heinsohn

1.1. Introduction / 1

1.2. Health Effects of Indoor Fungal and Bacterial Growth / 3

1.3. Team and Individual Expertise / 4

1.4. Approach of This Book / 8

1.5. Conclusion / 10

2. CONDUCTING BUILDING MOLD INVESTIGATIONS 15

Patricia A. Heinsohn


2.2. Baseline Investigation / 17

2.2.1. Physical Inspection / 19

2.2.1.1. Visual Inspection / 20

2.2.1.2. Documentation / 22

2.2.1.3. Moisture and Moisture Mapping / 29

2.2.2. Sampling Design / 36

2.2.2.1. Air Sampling Methods / 36

2.2.2.2. Selection of Air Sampling Instruments / 37

2.2.2.3. Air Sampling Flowrate, Pump Calibration, and

Sampling Duration / 38

v

2.2.2.4. Number of Indoor and Outdoor Air Samples, Air Sampling

Locations, and Order of Sampling / 40

2.2.2.5. Sampling Techniques / 41

2.2.2.6. Surface Sampling Techniques and Analysis / 41

2.2.2.7. Laboratory Selection / 43

2.2.2.8. Prior Notification / 45

2.3. Destructive Testing Investigation / 46

2.4. Sampling during Mold Remediation Oversight

and Clearance / 48

2.5. Conclusions / 48

3. MICROBIOLOGICAL SAMPLING STRATEGIES ININDOOR ENVIRONMENTS 51

Philip R. Morey


3.2. Sampling Strategy / 52

3.3. Spatial or Location Variables / 53

3.4. Temporal (Time) Variables / 54

3.5. Indoor/Outdoor Comparisons / 55

3.6. Complaint and Noncomplaint Zones / 56

3.7. Source and Air Samples / 58

3.8. Bulk Samples / 59

3.9. Dust Samples / 61

3.10. Surface Sampling / 63

3.11. Culture Plate Impactions and Liquid Impingers / 65

3.12. Spore Trap Samplers / 66

3.13. Sampling by Filtration / 68


4. MICROSCOPIC ANALYTICAL METHODS FOR FUNGI 75

De-Wei Li, Chin S. Yang, and F. Harrington


4.2. Principles and Usage of Microscopes / 76

4.2.1. Microscopes / 76

4.2.2. Microscope Objectives / 79

4.2.3. Photomicrographic Accessories / 80

4.3. Aseptic Technique and Biosafety / 81

4.4. Sample Preparation for Spore Count / 81

4.5. Materials Needed for Preparing Samples / 82

vi CONTENTS

4.6. Staining and Mounting Techniques / 83

4.7. Procedures for Identification and Quantification of Spore Traps / 85

4.8. Techniques for Spore Count Analysis / 87

4.9. Background Particulates (Nonspore Miscellaneous Materials) / 90

4.10. Limits of Detection / 91

4.11. Data Presentation / 91

4.12. Variation of Replications and Duplications / 91

4.13. Sample Preparation for Direct Examination or from Cultures / 92

4.13.1. Bulk Samples / 92

4.13.2. Bulk Dust Samples / 93

4.13.3. Swab Samples / 93

4.13.4. Tape Lift Samples / 94

4.13.5. Culture Samples / 95

4.14. Evaluation of Fungal Infestation / 96

4.15. Training of Microscopy Analysts / 98

4.16. Quality Assurance/Quality Control Procedures / 99

4.17. WEB Resources / 100

5. CULTURE-BASED ANALYTICAL METHODS FORINVESTIGATION OF INDOOR FUNGI 105

Florence Q. Wu

5.1. Advantages and Limitations of Culture-Based Analytical

Methods / 105

5.2. Factors Influencing the Results of Culture-Based Analysis / 107

5.2.1. Ecological Considerations / 107

5.2.2. Viability of Fungal Spores / 108

5.2.3. Selection of Culture Media / 109

5.2.4. Concentration Variations / 109

5.2.5. Sampler Performance, Sampling Time, and Culture

Preparation / 110

5.2.6. Experience of the Analyst / 111

5.3. Culturable Sampling Considerations / 112

5.3.1. Air Samples / 112

5.3.2. Surface Samples / 112

5.3.3. Bulk Samples / 113

5.3.4. Dust Samples / 114

5.4. Sample Preparation Methods / 114

5.5. Identification of Filamentous Fungi / 116

5.6. Data Reporting / 117

CONTENTS vii

5.7. Data Interpretation / 118

5.8. Laboratory Quality Assurance / 119

6. AIRBORNE BACTERIA IN INDOOR ENVIRONMENTS 123

Linda D. Stetzenbach


6.2. Bacterial Aerosols / 123

6.3. Selected Bacteria-Associated Health Effects / 124

6.4. Analysis of Samples for Airborne Bacteria / 125

6.5. Summary / 127

7. GENETICS-BASED ANALYTICAL METHODS FORBACTERIA AND FUNGI IN THE INDOOR ENVIRONMENT 133

Richard A. Haugland and Stephen J. Vesper


7.2. Genetics-Based Analytical Techniques / 134

7.2.1. In Vitro Nucleic Acid Amplification / 134

7.2.2. Hybridization Probes / 137

7.2.3. Nucleic Acid Sequencing / 138

7.2.4. Microbial Strain Typing / 139

7.3. Applications of Genetics-based Methods for Indoor

Microbiological Analyses / 139

7.3.1. Detection of Pathogenic, Allergenic, and Toxigenic

Bacteria and Fungi Using Nucleic Amplification and Hybridization

Probe Techniques / 139

7.3.2. Microbial Strain Typing and Identification by Nucleic

Acid Sequencing / 141

7.4. Quality Control/Quality Assurance and other Challenges / 142

7.5. Outlook for the Future / 144

8. WOOD IN THE BUILT ENVIRONMENTCONDITIONSFOR MOLD AND DECAY 155

Susan E. Anagnost


8.2. Molds and Wood Decay Fungi / 155

8.3. Moisture Requirements for Growth of Molds and Wood

Decay Fungi / 158

8.4. Water and WoodRelative Humidity versus

Moisture Content / 159

8.4.1. Wood Moisture Content and the Fiber Saturation Point / 161

viii CONTENTS

8.5. Moisture Requirements for Fungal Growth / 164

8.5.1. Moisture Requirements for Wood Decay Fungi / 164

8.5.2. Moisture Requirements for Mold Fungi / 166

8.6. The Effect of Changing Moisture and Temperature Conditions / 167

8.7. Susceptibility of Building Materials to Mold / 168

8.7.1. Visible versus Concealed Mold / 168

8.8. The Effects of Building Design and Construction / 170

8.9. Techniques for Assessment of Wood Decay

and Mold in Buildings / 172

8.9.1. Nondestructive Testing / 174

8.9.2. Destructive Testing / 175

8.10. Summary / 176

9. USE OF STATISTICAL TOOLS FOR DATAPRESENTATION AND ANALYSIS OFINDOOR MICROORGANISMS 179

Stella M. Tsai

9.1 Introduction / 179

9.2 Descriptive Analysis / 180

9.3 Correlation / 183

9.4 t-test and Nonparametric Methods / 185

9.4.1. Using t-test to Test Differences between Two Means

(Two Groups of Samples) / 186

9.4.2. Nonparametric Test for Two Groups of Samples / 186

9.5 Analysis of Variance (ANOVA) and Chi-Square Statistics / 186

9.6 Discussion / 189

10. ECOLOGY OF FUNGI IN THE INDOOR ENVIRONMENT 191

Chin S. Yang and De-Wei Li


10.2. Factors Affecting Fungal Growth Indoors / 191

10.2.1. Biotic Factors / 192

10.2.2. Abiotic Factors / 193

10.2.3. Interactions Between Temperature, Moisture,

and Fungi Indoors / 199

10.3. Ecological Interactions Between Fungi and

Environmental Factors / 200

10.3.1. Indoor Fungal Ecology / 200

10.3.2. Specific Ecological Niche of Some Common Indoor

Fungi / 202

CONTENTS ix

10.4. Release and Dispersal of Fungal Spores / 206

10.4.1. Fungal Spore Types and Their Release / 207

10.4.2. Fungal Spore Dispersal / 208


11. A RETROSPECTIVE AND FORENSIC APPROACHTO ASSESSMENT OF FUNGAL GROWTH IN THEINDOOR ENVIRONMENT 215

Chin S. Yang


11.2. Microbial Forensics / 216

11.3. Rationales and Mycological Backgrounds / 216

11.4. Other Signs and Information / 220

11.5. Case Studies / 223


12. MICROBIAL REMEDIATION IN NONINDUSTRIALINDOOR ENVIRONMENTS 231

Philip R. Morey


12.2. Mold Remediation Principles / 232

12.3. Mold Removal Methods / 233

12.4. Dust Control during Mold Remediation / 233

12.5. Air Sampling for Molds / 234

12.6. Clearance by Measurement of Residual Dust / 235

12.7. HVAC System Mold Remediation / 236

12.8. Remediation of Room Contents in Moldy Buildings / 237

12.9. Quality Assurance during Mold Remediation / 237

12.10. Microbial Remediation of Legionellas and Biofilms / 238

12.11. Remediation of Sewage Contamination / 239

APPENDIX COMMON AIRBORNE AND INDOOR FUNGIAND THEIR SPORES 243

De-Wei Li

INDEX 265

x CONTENTS

PREFACE

Over the last two decades (since the mid-1980s), concerns of human exposures to

microbiologicals originating in the indoor environment due to water intrusion or

moisture control problems have been centered on fungi, which include molds. It

is, however, well understood that fungal growth is not the only group of microorgan-

isms thriving in such conditions. Bacteria are particularly common in the indoor

environment, with growth possibly resulting from water damage. Because of their

ubiquity and small size, bacteria react to water intrusion or excessive moisture

much faster than do fungi. Bacterial growth can produce endotoxins and other

biogenic toxins. Some environmental bacteria are also infectious. In fact, two legio-

nelloses are associated with waterborne bacteria. Legionnaires disease was first

reported in the outbreak in the 1976 American Legion convention in Philadelphia.

Pontiac fever is a nonpneumonic legionellosis, believed to represent reaction to

inhaled Legionella antigens rather than bacterial invasion. Legionella bacteria are

now known to thrive in building water systems such as cooling towers or hot-water

systems.

Although fungal spores are ubiquitous, fungal growth is considered unusual in a

properly maintained building. Because of the ubiquitousness, fungal spores can

germinate and grow into colonies when there is access to moisture. During their life-

cycle, fungi produce a wide variety of chemicals, including secondary metabolites,

some of which are toxic to animals and humans; hence the term mycotoxins.

Fungal spores are known to contain allergens and fungal glucans. Exposure to

fungal allergens and mycotoxins in a water-damaged mold-infested indoor environ-

ment is generally believed to pose an increased risk over that posed by outdoor

exposures.

Because of the concerns and interest in indoor fungal contamination issues,

several guidelines and reference books have been published. Conventional and

xi

newly invented sampling and analytical methodologies have routinely been

employed in the assessments and investigations of microbiologics in the indoor

environment. Some of the methodologies have never been properly evaluated and

validated by scientific methods. Furthermore, results and data derived from the

methodologies are often misused or misinterpreted. In 2004, the Institute of

Medicine (IOM) of the National Academies reported that currently used sampling

and analytical methodologies were inadequate for microbial exposure assessment

in the indoor environment because of widespread uncertainty from potential

errors and bias in the methodologies as well as complexities from large temporal

and spatial variability and diverse microbial species and metabolites that are pro-

duced in such environments. The IOM further recommended that existing exposure

assessment methods for fungal and other microbial agents need rigorous validation

and further refinement to make them more suitable for large-scale epidemiologic

studies. Before such recommendations are fulfilled, it is our desire and opinion

that investigators and laboratory managers be thoroughly familiar with the details

of investigation, including sampling, and analytical methodologies so that they

understand the advantages and limitations of sampling and analytical methods,

utilize methods properly and appropriately, and interpret the results accordingly.

The approach to assessment and investigation is toward evaluating the conditions

of growth and contamination in the indoor environment rather than assessment of

human exposure.

In 2004, a group of scientists at the National Institute for Occupational Safety and

Health (NIOSH) studied employees in 13 college buildings. They reported that

building-related respiratory symptoms can be predicted with semiquantitative

indices of exposure to dampness and mold by classifying water stains, visible

mold, mold odor, and moisture using semiquantitative scales. They then estimated

individual exposure indices weighted by the time spent in specific rooms. The use

of surface coverage area of visible fungal growth to guide and design mold remedia-

tion requirements for human and environmental protections was detailed in guide-

lines and documents published by the New York City Department of Health,

the U.S. Environmental Protection Agency (USEPA), and Health Canada. The

New York City Guidelines available in 1993 and 2000 and the USEPAs Mold

Remediation in Schools and Commercial Buildings published in 2001 used square

footages to define requirements for personal protective equipment and isolation

containment. This implies the relationship between visible mold area and exposure.

In 2004, Health Canada published a document titled Fungal Contamination in

Public Buildings: Health Effects and Investigation Methods, in which it suggested

that there is doseeffect relationship in that more visible mold yielded more symp-

toms. Although the relationship requires further refinement, investigators should

always keep this in mind when conducting an assessment, an investigation, or an

inspection of fungal growth.

Although we understand the complexity of microbiological problems in a

water-damaged environment and the knowledge gap in practices of sampling and

analysis, we think there is sufficient information and knowledge in microbiological

sampling and testing available to properly assess and inspect an environment for

xii PREFACE

microbiological contamination. Clearly, any new advance will greatly add to the

tools and knowledge available for better methodology to sampling and testing for

microbes indoors. In light of the IOM publication, Damp Indoor Spaces and

Health and the University of Connecticuts Guidance for Clinicians on the Recog-

nition and Management of Health Effects Related to Mold Exposure and Moisture

Indoors (http://www.oehc.uchc.edu/clinser/MOLD%20GUIDE.pdf), the impactof microbial growth and contamination in a water-damaged indoor environment

on human health is minimally discussed in this book. Readers are referred to the

documents cited above as well as those published by governmental agencies, such

as New York City Department of Health, USEPA, NIOSH, or Health Canada.

Readers are also encouraged to stay abreast of the scientific literature in this area

as it is published.

Finally, we would like to thank chapter authors for their efforts and contributions

to this book. We are also very grateful for Drs. Francis Harrington, De-Wei Li,

Michael Berg, and Philip R. Morey for reviewing manuscripts. Dr. Keith

Wheatstone of Severn Trent Laboratories is thanked for his support to Chin S. Yang.

CHIN S. YANG

Cherry Hill, New Jersey


Pacifica, California

PREFACE xiii

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SAMPLING AND ANALYSIS OF INDOOR MICROORGANISMS€¦ · SAMPLING AND ANALYSIS OF INDOOR MICROORGANISMS ... stored in a retrieval system, ... Quality Control/Quality Assurance and other