1. SAMPLING FOR CONTAMINANTS
OF BIOLOGICAL ORIGIN
PRESENTED BY

2. MOLD IN THE NEWS!

3. BIOLOGICAL CONTAMINANTS
DEFINING THE ISSUE

4. BIOLOGICAL CONTAMINATION As defined by ACGIH
Aerosols, gases, and vapors of biological origin of a type and concentration likely to cause disease or predispose persons to experience adverse health effects
Inappropriate indoor levels of bioaerosols typically found outdoors
Biological indoor growth of particles that may become airborne and have an adverse effect on exposed individuals

5. BIOLOGICAL CONTAMINANTS AEROSOLS
Airborne particles of biological origin
bacteria, fungi, pollen, viruses
By-products
endotoxins and mycotoxins
Other fragments
insect parts and excreta, skin scales, hair

6. BIOLOGICAL CONTAMINANTS VAPORS
Fungi and bacteria in indoor environments produce microbial volatile organic compounds (MVOCs) as a by-product of their metabolism.
The odors of MVOCs are good indicators of microbial growth even when growth is not visible.
Knowledge is limited. MVOC research will attempt to answer the questions:
What role do MVOCs play in health effects?
Do certain microorganisms produce a typical MVOC fingerprint?

7. BACTERIA FOUND IN THE WORKPLACE
Legionella bacteria can grow in water systems including cooling towers and air conditioners.
Pseudomonas bacteria can grow in water-based metal working fluids.
Staphylococcus aureus, bacteria including the methicillin-resistant superbug (MRSA), can be transmitted by direct skin-to-skin contact with infected individuals.

8. BACTERIA BY-PRODUCTS
Gram-negative bacteria may contain harmful substances in their outer membranes called endotoxins.
Endotoxins are referred to as pyrogens because they will induce fever. They will also cause respiratory distress and even death at high levels.
In the workplace, the most common route of endotoxin exposure is through inhalation of aerosolized bacteria including Pseudomonas found in metalworking fluids.

9. ENDOTOXIN EXPOSURES WORKPLACE OTHER Can be found in sewage treatment
plants, cotton textile
mills, fiberglass
production plants,
poultry/swine facilities,
and in industries using
metal-working fluids
OTHER
Have been found in air
conditioning units, spa
water, and swimming
pools. They can also
be found on water
damaged material
following a water
intrusion event

10. FUNGI THE USUAL SUSPECT IN IAQ
Primary biological contaminant implicated in indoor air complaints
Fungi found most often in the indoor environment include Penicillium, Aspergillus, and Cladosporium.
Described as saprophytic because they can grow on any nonliving organic material if adequate moisture is present.

11. TOXIC MOLD PRODUCES MYCOTOXINS
Mycotoxins are natural by-products of fungal metabolism.
They are produced by some species of Aspergillus (versicolor), Fusarium (moniliforme), and Stachybotrys (chartarum).
Chemical structures and health effects of mycotoxins are quite diverse.

12. MYCOTOXIN EXPOSURES WORKPLACE OTHER Inhalation of aerosolized fungal
spores or other fungal
structures in water-
damaged building
materials
OTHER
Ingestion of moldy
food products by
animals and people
Aflatoxin and
trichothecenes have
been found in mold-
contaminated animal
feed and cereal grains.

13. BIOLOGICAL CONTAMINANTS
WHO’S EXPOSED

14. WORKERS POTENTIALLY EXPOSED TO BIOAEROSOLS
Agricultural Workers
Grain handlers
Tobacco and cotton handlers
Farmers
Livestock producers
Food Handlers and
Processors
Meat packing plants
Poultry processors

15. WORKERS POTENTIALLY EXPOSED TO BIOAEROSOLS
Industrial Workers
Pulp and paper mills
Textile mills
Wastewater and sewage treatment plants
Machinists
Industries with cooling towers

16. WORKERS POTENTIALLY EXPOSED TO BIOAERSOLS
Healthcare workers
Military personnel
Construction and maintenance personnel
Remediation workers
Office workers in humidified indoor air …

17. BIOLOGICAL CONTAMINANTS
HEALTH EFFECTS

18. SKIN INFECTIONS Staph bacteria, including MRSA, can
cause skin infections
that look like a pimple
or boil. Serious
cases can lead to
bloodstream infections
or pneumonia. Mold may
also induce skin infections
like ringworm or rashes. .

19. EYE, NOSE, AND THROAT IRRITATION
ACGIH reports that
most health
complaints in indoor
environments are due
to eye, nose, and
throat irritation,
headache, and fatigue
from unknown
causes.
Sick Building Syndrome

20. INHALATION FEVERS HUMIDIFIER FEVER PONTIAC FEVER
Flu-like symptoms that
arise 4 to 8 hours after
exposure and subside
within 24 hours. Possibly
related to endotoxins
PONTIAC FEVER
A self-limited, flu-like
illness caused by
contamination of water
systems with
Legionella bacteria

21. HYPERSENSITIVITY DISEASES
Result from exposure
to specific antigens in
the environment that
trigger an
immunological
response
Dust-mite and animal
allergens are common causes in residences.

22. HYPERSENSITIVITY DISEASES IN THE WORKPLACE
Hypersensitivity pneumonitis characterized by acute, recurrent pneumonia with fever, cough, chest tightness, and a progression of symptoms
Building-related asthma characterized by chest tightness, wheezing, coughing, and shortness of breath that is worse on work days and improves on weekends

23. INFECTIOUS DISEASES Influenza (H1N1) and SARS - viral illnesses
Legionnaires’ Disease - pneumonia caused by Legionella pneumophila bacteria contaminated water sources
Tuberculosis - lung disease caused by Mycobacterium tuberculosis and spread from person to person

24. HEALTH EFFECTS FROM MYCOTOXINS
Mycotoxins will reduce the effectiveness of the immune system by interfering with or killing macrophages.
This results in increased susceptibility to infectious diseases and a reduction in defense against other contaminants.

25. WIDE-RANGING EFFECTS FROM MYCOTOXINS
VASCULAR SYSTEM
Increased vascular fragility
Hemorrhage
DIGESTIVE SYSTEM
Vomiting
Intestinal hemorrhage
Liver effects
RESPIRATORY SYSTEM
Respiratory distress
Bleeding from lungs
NERVOUS SYSTEM
Tremors
Lack of coordination
Depression
Headache

26. BIOLOGICAL CONTAMINANTS
CONDUCTING AN INVESTIGATION

27. ACGIH RECOMMENDS A STEPWISE APPROACH
Gather information through occupant interviews, surveys, and building inspections.
Formulate a hypothesis on the cause of the complaints/illness by using the information gathered.
Test the hypothesis by collecting samples.
Make recommendations for controls by using sampling data and professional judgment.

28. INFORMATION GATHERING OCCUPANT INTERVIEWS
Before embarking on a program of air measurements, survey the area and the people involved in the complaint.
Use this time for information gathering.
Look around and listen.
Complaint vs non-complaint areas
Date when problem was first noted
Days or times when problem is noted more and less
Seek input to formulate a hypothesis on the root cause of the complaints.

29. INFORMATION GATHERING BUILDING INSPECTION
Examine the physical structure, maintenance, and occupancy patterns.
Look for potential sources of biological contaminant and evidence of water damage.

30. BUILDING INSPECTION MOISTURE INDICATORS
Water marks on ceiling tiles and other surfaces
Visual presence of mold
Musty smell of microbial VOCs
White, powdery, or crystalline substance on the surface of concrete, plaster, and masonry, which are soluble salts dissolved from the building materials

31. BUILDING INSPECTION MOISTURE METERS
Used to survey moisture in any non-conductive, porous material to which the probes can be applied
Ceiling Tiles
Gypsum Board
Carpeting
Wood
Plaster
Concrete

32. MOISTURE METERS FROM SKC
ECONOMY MODEL
Operates by measuring the electrical conductance between two probes inserted into the test material
Useful for construction, renovation projects, or other situations when the test surface can be punctured by the probes
SKC Cat. No

33. MOISTURE METERS FROM SKC
PINLESS MODEL
Measures resistance between two low-frequency signals transmitted from conductive pads without the need for insertion into the test material.
Specialty models available for testing concrete
SKC Cat. Nos /102

34. DATA INTERPRETATION MOISTURE METERS
Moisture levels can be compared from wall to wall to determine where moisture intrusion is occurring.
Once the location of the moisture is found, an investigation can be made as to the cause and a control strategy can be developed.

35. DATA INTERPRETATION MOISTURE METERS
The Western Wood Products Association (WWPA) has prepared a technical guide on preventing and controlling mold in lumber.
See
WWPA recommends that the moisture content of the wood be kept below 20%.

36. DATA INTERPRETATION MOISTURE METERS
Greenguard Environmental Institute (GEI) has received ANSI approval for a standard covering the management of moisture (and mold growth) during building construction.
See

37. BUILDING INSPECTION HVAC SYSTEM CHECKS
OUTDOOR AIR SUPPLY
Inadequate amounts of outdoor air often leads to building-related complaints and health-related symptoms.
Ensure outdoor air supply meets ASHRAE or other appropriate standards.

38. BUILDING INSPECTION HVAC SYSTEM CHECKS
LOCATION OF AIR INTAKES
Air intakes on rooftops can draw in bioaerosols from cooling towers, sanitary vents, building exhausts and animal waste.
Air intakes at street level can draw in moisture, vehicle emissions, and odors.

39. BUILDING INSPECTION HVAC SYSTEM CHECKS
CONDITION OF AIR
FILTERS
HVAC filters are not designed to protect equipment or occupants from heavily contaminated air.
Filters may promote the growth of microorganisms if they become damp.

40. BUILDING INSPECTION HVAC SYSTEM CHECKS
SUPPLY AIR
Ductwork should not be coated with excessive debris.
Dirt mixed with moisture can support microbial growth.
Ensure that cold air leaving a diffuser does not produce condensation and the potential for microbial growth.

41. CHOOSING A SAMPLING METHOD TO TEST THE HYPOTHESES
Data Interpretation Tips

42. SAMPLING FOR BIOLOGICAL CONTAMINANTS
WHY:
To test your hypothesis on the cause of the problem
To positively confirm the absence/presence of contaminant
To identify the type of microbe
Genus/species
To confirm the effectiveness of decontamination
HOW:
Bulk samples
Surface samples
Air samples
Followed by analysis
at a qualified
environmental
microbiology laboratory

43. BULK SAMPLING
Portions of materials in the building can be tested for mold or other biological contaminants.
Typical test materials include sections of wallboard/wallpaper, carpet pieces, return-air filters, duct lining, and settled dust.
These are very useful because air sampling may miss some contaminants due to temporal variations.

44. BULK SAMPLING
Portions of the test material are typically placed in a sealable plastic bag for transport to the lab.
In some cases, sterile jars for dry items or sterile bottles for water or metalworking fluid samples may be required.
Settled dust can be collected using conventional vacuum cleaners and a new vacuum cleaner bag for each sample.

45. SURFACE SAMPLING MICROVACUUM CASSETTES
Carpeting is an effective reservoir for fungal spores and sampling this surface can reveal the history of mold in the building.
Sample fungal spores in carpeting using a vacuum-style cassette.
0.45 µm polycarbonate filter loaded into a 3-piece styrene cassette with 2-inch tubing nozzle
Sample at flows up to 16 L/min to vacuum a defined area.
Work the inlet tube as deep as possible into the carpeting to collect a good sample of the dust.

46. CARPET SAMPLING KIT
SKC Cat. No

47. DATA INTERPRETATION CARPET SAMPLES
An 2003 AIHCE paper by MidWest
Microbiology gave some numerical guidelines
for fungal spores on surfaces like carpet using
microvacuum cassettes:
Normal - < 5000/1000 cm2
Borderline - 25,000/1000 cm2
Elevated - 75,000/1000 cm2

48. SURFACE SAMPLING STERILE WIPES
A swab or filter wetted with sterile water or wash solution is used to wipe a specified area.
The swab is then used to inoculate an agar plate for growth culture.
This technique is often used for MRSA testing.
SKC Cat. No

49. DATA INTERPRETATION SWAB SAMPLES
The November 2001 AIHA Synergist
guidelines for fungal spores in swab
samples:
Normal: < 10,000 cfu/in2 or < 1500 cfu/cm2
Probable Contamination: > 10,000 cfu/in2 or > 1500 cfu/cm2

50. SURFACE SAMPLING LIFT TAPE
Collected by placing clear adhesive, packing tape, or commercially available sampling strips onto a surface and removing it with slow, steady force
Following collection, the tape is attached to glass slides and examined using light microscopy to view mold spores.

51. SURFACE SAMPLING LIFT TAPE ON A SLIDE
Flexible plastic microscopic slides with a sticky adhesive sample area can be used like lift tape.
Press on the test surface, place the slide in the provided mailer, and send to a qualified laboratory.
Stick-to-It Slides
SKC Cat. Nos /9

52. DATA INTERPRETATION LIFT TAPE SAMPLES
The November 2001 AIHA Synergist
guidelines for fungal spores in tape
samples:
Normal: No significant fungal material or biomass
1 to 5% spores
Probable Contamination: 25 to 100% spores

53. AIR SAMPLING WHY AND HOW
Air Sampling for
Bioaerosols
Involves the use of:
Impactors
Filters
Liquid-based (impinger-type) devices
Like with chemical
sampling, air sampling
for biological
contaminants is done
for the purpose of
evaluating actual
human exposures.

54. AIR SAMPLING SPORE TRAP CASSETTES
Easy, inexpensive screening device
Use with a pump at 15 to 30 L/min for up to 10 minutes.
Spores impact onto a microscopic slide with a sticky surface.
Slide is stained and analyzed microscopically.

55. VERSATRAP CASSETTES
SKC Cat. Nos /1

56. AIR SAMPLING PUMPS FOR USE WITH SPORE TRAPS
Constant flows from
10 to 30 L/min
User selectable sampling times
Lithium-ion battery powered up to 4 hours
Indefinite run time from AC adapter
Optional sampling wand
SKC Cat. No

57. DATA INTERPRETATION SPORE TRAPS
Spore trap analysis will provide the total number of spores and the genus of the spores found.
Information can be used to compare the complaint area to non-complaint areas of the building and outdoors.
Genus of the spores should be similar inside and outside.
Numbers should be lower inside.

58. DATA INTERPRETATION SPORE TRAPS
November 2001 AIHA Synergist guidelines for
Air samples:
Residential Buildings:
Normal: < 5000 spores/m3
Probable Contamination: > 10,000 spores/m3
Commercial Buildings:
Normal: < 2500 spores/m3

59. AIR SAMPLING VIABLE CASCADE IMPACTOR
Specified in NIOSH Methods 0800 and 0801
Used with a pump at 28.3 L/min for typical sample times of 2 to 5 minutes
Mold impacts onto growth medium (agar)
Agar plates are shipped
to a microbiology laboratory for growth culture
SKC Biostage
Cat. Nos /11

60. SKC BIOSTAGE SAMPLER PREPARATION

61. SKC BIOSTAGE WITH QUICKTAKE 30 PUMP
Sample Assurance Tip:
Impactor must be cleaned with isopropyl alcohol before each use
Evaluate blank samples of agar plates

62. AIR SAMPLING FILTERS
Collection of microorganisms is achieved by passage of air through a porous medium, typically a membrane filter.
Polycarbonate, mixed cellulose ester, or polyvinyl chloride filters may be used depending upon the application.
Gelatin filters will help to maintain viability by minimizing dehydration of the spores.

63. GELATIN FILTERS WITH SKC BUTTON SAMPLER
An AIHA Journal article reported that 25-mm filters used with the SKC Button Sampler provided collection efficiencies close to 100% for enumeration of airborne spores.
SKC offers sterile gelatin filters in 25 or 37-mm diameters. (Cat. Nos /2)
SKC Cat. No
INHALABLE SAMPLING at 4 L/min

64. VIABLE AIR SAMPLING FILTERS
Filters, support pads and cassettes should be sterile.
Samples are collected with a portable pump at 1 to 4 L/min for 5 to 30 minutes.
After sampling, the material collected on the filters is inoculated onto agar plates.

65. AIR SAMPLING COLLECTION INTO LIQUID
Pumps are used to pull mold spores into glass impingers filled with a liquid collection medium, typically a dilute buffer solution or mineral oil.
Portions of the collection liquid can be placed onto nutrient agar and incubated or analyzed using other methods.
BioSampler
SKC Cat. No
ADVANTAGE: 8-hour sampling

66. OPERATION OF THE BIOSAMPLER
Particle collection is achieved
by drawing the aerosol through
3 nozzles directed at an angle
toward the inner sampler wall.
The collection liquid swirls upward
on the inner surface and removes
the collected particles.

67. ANALYSIS OPTIONS Growth culture Microscopic Bioassay Immunoassay
PCR (using water
as collection media)

68. DATA INTERPRETATION VIABLE AIR SAMPLES
November 2001 AIHA Synergist guidelines:
Residential Buildings:
Normal: < 500 cfu/m3
Probable Contamination: > 1000 cfu/m3
Commercial Buildings:
Normal: < 250 cfu/m3

69. AIR SAMPLING FOR MVOCs
Sorbent tubes/pumps
Stainless steel canisters

70. AIR SAMPLING FOR MVOCs Photoionization detectors (ppb RAE)
Man’s best friend
See

71. DATA INTERPRETATION
GENERAL GUIDELINES

72. WHAT ABOUT OELs?
Few established guidelines/standards for biological contaminants except for wood dust, cotton dust, etc.
ACGIH indicates that a “general exposure limit for concentrations or countable biological agents is not scientifically supportable.”

73. WHY??
Biological contamination typically is a complex mixture of many types of microorganisms.
The health effects for various microorganisms vary greatly between individuals.
There is no single sampling method appropriate for all types of biological contamination.
There is insufficient scientific evidence to support a dose-response relationship from which an exposure standard could be derived.

74. ACGIH RECOMMENDS
“Gather the best data possible and use knowledge, experience, expert opinion, logic, and common sense to interpret information, design control, and remediation strategies.”

75. GENERAL GUIDELINES FOR DATA INTERPRETATION
Fungi found in indoor air should be the same species as that found in outdoor air, but the levels should be lower.
If there is a dominant fungal species in indoor air that is not present in outdoor air, it is probably growing from biological contamination within the building and is reducing air quality.

76. GENERAL GUIDELINES FOR DATA INTERPRETATION
Comparisons of indoor/outdoor air or problem/non-problem areas should be made at the species level, not just at the genus level.
Some species may be considered indicator organisms indicating specific problems such as E. Coli as an indicator of sewage contamination.

77. GENERAL GUIDELINES FOR DATA INTERPRETATION
In order to compare sampling results from indoor/outdoor areas or from different zones, an identical sampling protocol for each zone including the sample media, sampling duration, sampler type, and laboratory analysis is required.

78. CONTROL AND REMEDIATION
OF MICROBIAL CONTAMINATION

79. GENERAL CONTROL RECOMMENDATIONS
Within 24 to 48 hours after floods or leaks, dry materials and discard sewage-contaminated materials.
Have an effective drainage plan to prevent penetration of rain, snow, and groundwater through materials.
The relative humidity of an air-conditioned building should not exceed 60%.

80. GENERAL CONTROL RECOMMENDATIONS
Install appropriate insulation to prevent large temperature differences between air and surfaces.
Maintain and inspect HVAC components such as filters and drain pans to avoid accumulation of water or debris.
Pay close attention to the location of the outside air intake. Air intakes should be as far as possible from cooling towers, standing water, bird droppings, and vehicle emissions.

81. MOST IMPORTANT ISSUES IN REMEDIATION
Identify the conditions that contributed to microbial growth in the building.
Determine if mold is an allergen or a toxin for appropriate remediation steps.

82. TO REMOVE EXISTING CONTAMINATION
Vacuum with high-efficiency filters.
Discard materials with “extensive” microbial growth.
Surface areas greater than 3 m2
Wash with a dilute biocide (such as a 1:10 dilution of bleach) and with detergent followed by thorough drying.

83. TO REMOVE EXISTING CONTAMINATION
Soda Blasting
Similar to sand
blasting, but the soda
material will not
damage building
materials

84. HOW DO YOU KNOW WHEN YOU HAVE FINISHED REMEDIATION?
Water problem is fixed
Moldy materials removed
Mold similar in type/number inside and outside
No new growth or water damage
No health problems on re-entry

85. ESSENTIAL REFERENCES
Field Guide for the Determination of Biological Contaminants in Environmental Samples
By AIHA
Bioaerosols: Assessment and Control
By ACGIH

86. THANK YOU FOR YOUR ATTENTION!
YOU ARE NOW AN
OFFICIAL SKC
MOLDBUSTER!
SKC