Bioaerosols - Aerosols: Measurement, Dosimetry, and Health Effects - page 324

12.6.4.2 Cultivation-Based Methods for Viruses, Bacteria, and Fungi
In cultivation-based (culture- or growth-based) methods, airborne microorganisms are collected
in a liquid, on a ilter, or directly on a semisolid growth medium. After an appropriate incubation
period, the resulting plaques (viruses) or visible colonies (bacteria and fungi) can be counted and
the isolates identiied. One of the primary limitations of cultivation-based methods is that they
provide a measure of only those organisms that are able to grow in the laboratory. Furthermore,
aggregation of bacterial or fungal cells may lead to errors in the enumeration of the total number
of culturable organisms. This is especially true with direct agar impactors as opposed to samplers
that collect particles into liquid, that is, a CFU may result from the growth of one or a cluster of
several cells.
12.6.4.3 Biological Assays
The effects of bioaerosols on other biological systems can be measured by different methods,
among them whole animal exposure, infectivity assays for viruses, bioassays utilizing prokary-
otic or eukaryotic cell lines, and nonspeciic toxicity, cytotoxicity, and genotoxicity assays. One
of the most commonly used biological assays, the LAL assay, measures bacterial endotoxin and
fungal glucans. The LAL assay is an in vitro biological test that uses a lysate of blood cells from
the horseshoe crab ( Limulus polyphemus ). The lysate contains a serine protease that triggers an
enzyme cascade when activated by endotoxin or glucan. Kits for kinetic chromogenic and turbi-
dimetric assays are available commercially. The enzyme activation with endotoxin and glucans
occurs through different pathways (Factor C and D, respectively). Therefore, in commercially
available kits, the competing pathway is depleted to make the assay sensitive only for endotoxin
or glucan.
Variations have been found between LAL preparations from different manufacturers and within
reagent lots from single producers. Variation between laboratories is even larger than within laborato-
ries, but can be decreased with standardized extraction protocols (Section 12.6.4.4). 371 LAL response
varies for LPS molecules from different microorganisms, and the amount of glucan detected per
fungal spore varies widely between species. 372,373 The assumption that responses to different endo-
toxins correspond with toxic potencies has not been proven. A recombinant factor C (rFC) assay that
uses a reagent produced from the cDNA of the Mangrove horseshoe crab ( Carcinoscorpius rotundi-
cauda ) is an alternate, although less utilized assay, for endotoxin. 374 Recent studies have shown that
the rFC and LAL assays give similar estimates of endotoxin concentration. 375,376 Furthermore, the
results were found to correlate with 3-OHFA measurements (a surrogate for total endotoxin) from
gas chromatography-mass spectrometry (GC-MS) analysis (Section 12.6.4.5). 377
A method for detection and quantiication of fungal biomass based on luorogenic detection of
β- N -acetylhexosaminidase enzyme activity has been developed (the MycoMeter test, Mycometer
Inc., Copenhagen, Denmark) 378 and used to measure mold contamination. 379 Another rapid method
for the quantiication of microbial biomass is based on the detection of adenosine triphosphate
(ATP), a basic energy molecule present in all living organisms. This method has been applied in
laboratory-based tests where other living cells do not interfere. 380
A variety of other assays (many of which use continuous cell lines) have been developed to detect
overall toxicity, cytotoxicity, or mutagenicity and to screen for the presence of particular toxins,
for example, alatoxin from A. lavus in air samples from food-processing plants. 381 A boar sperm
cell, motility inhibition assay also has been used to detect bacterial depsipeptide and other toxins
in foods and indoor environments. 382,383 The assay is sensitive to mitochondrial toxins that inhibit
sperm motility (e.g., valinomycin) but is relatively insensitive to toxins that affect protein or nucleic
acid synthesis (e.g., many mycotoxins). A protein synthesis inhibition assay was used to study the
trichothecene toxicity of airborne particles in a home heavily contaminated with Stachybotrys
species. 384 The highest toxin activity was found during renovation, but detectable air concentrations
also were detected before and after these activities.