How To Identify Microorganisms in the Home and Workplace
By Dr. Rajiv Sahay, CIAQP, FIAS, Environmental Diagnostics Laboratory (EDLab) Director
Mold is ubiquitous in nature. Filamentous fungi often produce mold in indoor environments. Excessive moisture, a carbon source, a moderate temperature (25ºC), and dampness, besides other factors, are supportive elements for the growth of mold indoors. The nature and characteristics of indoor molds are much more variable. Sometimes one can see mold growth in indoor environments with the naked eye. However, it is hard to assess health and hygiene effects just by looking at it. Therefore, it is essential to study the mold in order to understand its impacts.
There are a number of techniques available today to isolate and identify the mold from indoor environments. No single technique fits in every scenario, but rather, it should be case specific. Although mold can be examined and evaluated in various ways, an integrated approach to detect mold in indoor environments is described below:
1. Mold sampling
To study the airborne fungi from indoor environments
I Air samples, air samples are collected. Some popular mechanisms are described below for collecting mold/fungal samples from the ambient air.
a. Drum Trap (DT)
Airborne fungal elements are collected on an adhesive tape mounted on a rotating disc powered by an electric motor in an air sealed drum with an orifice. The rotation of the disc is fixed with that of the exposure time. Hirst spore trap, Tilak air samplers, etc. are some common commercially available samplers in this category.
b. Electrostatic Trap (ET)
Fungal/mold samples are collected by drawing air with a constant flow rate and exposure time over media under the influence of an electrostatically charged environment. Charged particles are collected on their appositively charged electrode. An Electrostatic Sampling Device (ESD), SASS® 3100, Portable Biohazard Sampler, etc. are good commercially available samplers under this technique.
c. Filterer Trap (FT)
Air samples are drawn on a filter mounted within a closed, airtight chamber by pulling the air through it with a constant airflow rate and exposure time. Micro-orifice uniform deposit impactor (MOUDI), filter made out of cellulose ester, polyvinyl chloride, and polycarbonate are widely used for mold/fungi sampling.
d. Impinger Trap (IP)
In this method, the sample is collected by dissipating the air into an air tight flask containing the media with a constant airflow rate and exposure time. Some common IP samplers include, but are not limited to, Greenberg-Smith impinger, AGI-30, etc.
e. Pore Trap (PT)
Air samples for mold/fungal evaluation are collected on media in an air-tight cylinder by collecting air through a perforated metal plate with a constant airflow rate and exposure time. Anderson’s, Burked, Bio-culture, and Button Aerosol Samplers are routinely used based on this technique.
f. Rotorod Trap (RT)
The airborne fungal particulates are collected on a strip of sticky tape or surface mounted on a mechanical arm/surface attached to a spindle powered by an electric motor that can rotate with a specific number of rotations per minute for a determined exposure time. Rotorod sampler by Sampling Technology, Inc. is one of the most widely used samplers of this category.
g. Spore Trap (ST)
Commonly in this method, a gel-coated glass slip is employed inside an air sampling device and air is pulled out with a constant air flow for a predetermined exposure time depending on the project goals. Flow rate is verified in the field utilizing an in-line flow meter. Air is passed over the coated slide causing airborne fungal particles to adhere to the gel. Some commercially available devices of this category are Air-O-Cell, Micro 5, Allergenco-D, M2, Burkard volumetric samplers, etc.
h. Thermal Trap (TP)
The air samples are collected on a glass slip by placing it around a hot body into ambient air.
II Surface samples
Environmental surfaces are collected to evaluate the mold/fungal infestation in and around indoor environments. Some practical methods for collecting a surface sample are given below.
a. Bulk Sample (BSAM)
Bulk samples are made by collecting, scraping, or cutting a representative of the material/dust suspected for mold/fungi by using aseptic techniques. These samples are transferred to the laboratory in a sterile container for further analysis.
b. Surface Imprint Sample (SISM)
Environmental samples are collected with the help of sticky tape. The sticky side of the tape is placed over the test area and an imprint is taken in order to collect a surface sample for a mold/fungal evaluation. Bio-Scan400 is the most accurate (cts/m2) and one of the more commonly used products for collecting surface samples for mold/fungi.
c. Swab Sample (SSAM)
Swab samples are made by swabbing a selected area by using sterile techniques. The collected specimens are transported to the laboratory for further enumeration. A number of companies make cotton or polyester swabs which are available in the market for environmental surface sampling for collecting mold/fungi samples.
d. Vacuum Sample (VSAM)
Dust samples are collected from environmental surfaces suspected for mold/fungi with a dust collecting cassette and/or a vacuum sample device under aseptic conditions. The collected samples are transported to the laboratory in a sterile container for further evaluation. Dust sock®, Dust collector, etc. are available in the market for collecting environmental surface samples for mold/fungi.
e. Wipe Sample (WSAM)
Environmental surface samples are collected by means of wiping the selected area suspected for mold/fungi with a sterile gauze pad by following sterile techniques. A leak proof container should be used for transporting these aseptically collected specimens to the laboratory for mold/fungi evaluation. Sterile gauze can be procured in test kits, drug stores, and various other sources to collect environmental samples for testing mold/fungi.
No one sampling method can be used as an absolute standard for collecting environmental samples for the detection and identification of mold/fungi. The best way to select a sampling method is to explore the performance of the sampling mechanism and it’s suitability for the intended project.
2. Mold examination/identification
Isolation of mold/fungi collected from environmental samples is challengeable. Depending on the project needs, the trapped particles are isolated by using a suitable buffer such as phosphate buffer saline (PBS), distilled water, etc. Sometimes the collected specimens are directly examined. Some common methodologies are described below for the isolation and identification of mold/fungi from samples collected from the environment.
a. Non-culture method
Microscopic techniques are used to examine and identify the mold/fungal elements from the collected sample. This is a rather inexpensive method with a quick turnaround time. However, many times identification is limited to a particular taxon.
b. Culture method
In this method, the isolated mold/fungal inoculums on microbiological media are incubated at a required temperature and time for growing the culture. After obtaining the developed culture, microscopic or biochemical techniques are employed for the identification of mold/fungi. While this may be a time taking process, the identification of fungi is often possible both on the genus as well as the species level. Some fungal organisms are media specific; therefore, the selection of microbiological culture media may influence the outcome.
c. Molecular method
Polymerase Chain Reaction (PCR) or other molecular diagnostics methods are used for the identification of mold/fungi from environmental samples. The advantage to this method is a higher accuracy in the identification with a faster turnaround time. However, experimental set up is expensive and requires specific training.
d. Biochemical method
In this method, the isolated mold/fungal elements are subject to react with certain biochemicals and after a reaction is observed, a pattern is obtained. In other words, a “Metabolic Fingerprint” is obtained in order to identify the targeted mold/fungi.
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