Volume 3 | Issue 3
I hope that you are enjoying spring and that you will find the attached articles about endotoxins and
Rhizopus helpful. Separately, I am pleased to introduce the newest member of our team, Dr. John
Shane, who will lead EMLab's Midwest region. Dr. Shane is a well known instructor in the field of spore
trap analysis for environmental particles and mold service in our industry. For the past 10 years Dr. Shane
was Professor and Director of Research of the McCrone Research Institute in Chicago, IL. He will be a
regular contributor in future Environmental Reporters, as well as an instructor at upcoming Mold University
With best wishes,
Harriet Burge and Dave Gallup
are bacterial products that are ubiquitous in the environment and, although they do take part in some
disease processes, exposure at some level is probably essential for the normal development of the human
(and animal) immune systems.
The nature of endotoxin
Bacteria produce two general types of toxins: exotoxins and endotoxins. Exotoxins are soluble proteins
that are excreted from the bacterial cell. Anthrax, tetanus, botulism, and toxic shock syndrome are a few
of the diseases caused by bacterial exotoxins. Endotoxins, on the other hand, are lipopolysaccharides (LPS)
that are part of the bacterial cell structure. The toxin is minimally soluble and effects occur directly
from contact with the bacteria. Endotoxins are produced by Gram negative bacteria such Escherischia
coli, Salmonella, Shigella, Pseudomonas, Neisseria,
Haemophilus, and many others. All Gram negative bacteria have lipopolysaccharides as part of the
cell wall. However, the lipopolysaccharides are not always toxic and those that are vary in potency.
Endotoxins are potent
inflammatory agents, stimulating the release of many chemicals that activate the immune system. In high
concentrations, such as would occur during a Gram negative bacterial infection, fever, inflammation,
coagulation of platelets, hemorrhage, and shock may result. At the much lower concentrations encountered
during inhalation exposures, inflammation is the primary outcome.
In some work environments in which organic material is handled or large amounts of water are used (e.g.,
swine confinement, fiberglass manufacturing) work-related respiratory illnesses may occur. These illnesses
are characterized by fever, chills, muscle aches, shortness of breath, and cough. These symptoms may be
worse on Monday and may gradually abate during the week, only to return following a weekend of no exposure.
In residential and office environments, effects are less clear. Some studies relate dust or air levels of
endotoxin to respiratory inflammation.
In residential environments, endotoxin exposure appears to be related to a decrease in the risk of
developing asthma. This effect is called the hygiene hypothesis, which considers that the reduction in
exposure to endotoxin caused by increasing standards for home hygiene has led in part to the rising
incidence of asthma.
Sampling and measurement technologies
The most commonly used analytical method for endotoxin detection and measurement is the Limulus amoebocyte
assay. This assay measures endotoxin in units of toxic activity as measured by the ability to clot the
blood of Limulus (the horseshoe crab). Results from this assay are highly dependent on the lot of Limulus
lysate being used, and the assay is affected by many factors, including materials that may be present in a
sample. In general, this means that data from different laboratories cannot be directly compared and, even
within a laboratory, data from different days or different batches of chemicals are difficult to compare.
The ACGIH Bioaerosols Committee has proposed a relative standard for airborne endotoxin based on the
Limulus assay that requires outdoor measurements for comparison. The comparisons are made on data produced
by the same assay using the same lot of reagents.
Total lipopolysaccharides can be measured using gas chromatography. This method is stable and
interlaboratory comparisons can be made. However, the method measures both toxic and non-toxic
lipopolysaccharides. Since the non-toxic forms may be immunostimulants, this approach may actually be the
most relevant in the long run. LPS is measured in concentration of the actual compound (e.g.,
nanograms/gram of dust). Endotoxin is measured in units of potency-endotoxin units (EU). In some
literature, endotoxin concentrations are listed as nanograms (ng) or micrograms (ug)/ unit of measure. In
these cases, either GCMS has been used to measure total LPS, or the authors are referring to units of the
reference LPS standard used in the assay. However, because the type of lipopolysaccharide in the unknown
sample may differ from the standard, EU is the preferred unit for Limulus assay data.
Samples for endotoxin analysis can be collected from air or dust. For air samples, endotoxin-free membrane
filters can be used. For dust, a variety of vacuum-based methods have been used with variable results. It
is important to use the method by which any reference data that is to be used for comparison was
Distribution in the environment
Endotoxin is always present in outdoor air. Measured levels have ranged from 0.01-more than 5
EU/m³ of outdoor, with geometric means generally in the 0.02-0.5EU/m³. Endotoxin concentrations
in outdoor air near farming activities can be much higher, reaching levels in excess of 1,000 EU/m³.
In residential and other supposedly "clean" environments, measured levels of airborne endotoxin are rare.
Concentration ranges for the few available studies are in the range 0.5-2,500 EU/m³, with the higher
part of the range possibly indicating unusually high levels. Most residential measures are dust
concentrations, which range from 8,000-250,000 EU/g of dust. Repeated measurements of endotoxin in
residential dust tend to be correlated. The presence of a dog in the house tends to be strongly correlated
with endotoxin concentrations in dust.
Most of the data on endotoxin exposure comes from occupational environments where organic material or
water is used in the manufacturing process. In these types of environments, extremely high endotoxin
concentrations have been measured. A summary of measured levels are listed in Table 1.
.5-3; 350-460; 2500
Outdoor Feedlots (USA)
Machining fluid mist
Role in environmental investigations
In occupational environments where organic material is handled, or water is a part of the manufacturing
process, airborne endotoxin measures are important and air samples should be collected in both the
occupational environment and in outdoor air. There is a large amount of literature on such occupational
exposures, and reference to the medical literature is an important component of interpretation. The
National Library of Medicine is a free and very useful resource: www.pubmed.org.
There is little indication that endotoxin levels in residential and office environments are likely to be
related to ongoing problems. Measuring endotoxin in house dust has been done often enough that there is
reference data that can be used for interpretation of whether levels are low, moderate, or high. How the
data is interpreted with respect to health is less clear.
Fungus of the
By: Dr. Payam
In past issues of the Environmental Reporter we have discussed fungal species that belong to a large class
of fungi known as the Ascomycetes. The types of spores they produce are known as ascospores (spores
produced sexually) and conidia (spores produced asexually). In this issue, members of a totally different
and smaller class of fungi called the Zygomycetes are discussed. The Zygomycetes are distinguished from
other classes of fungi by the production of sexual spores known as zygospores and by the production of
asexual spores called sporangiospores The sporangiospores are usually produced in an enclosed capsule
called a sporangium (sporangia- pl.). Each sporangium may contain up to 100,000 sporangiospores or as few
as 50. Sporangiospores are the most abundant spores produced and are primarily disseminated by wind.
Animals, insects, and rain can also contribute to dissemination. The sexual zygospores are large and
considered to be resistant to environmentally adverse conditions therefore, they are not widely
disseminated. Perhaps the most striking characteristic of this class of fungi is the presence of a
coenocytic mycelium (a mycelium that lacks regular septation, i.e. no cross walls). This is an especially
important characteristic of identification during direct microscopic examinations where mycelium from many
different fungi may be present and there are few spores.
In indoor environments, one of the most common zygomycete genera detected is Rhizopus. The genus
Rhizopus includes approximately 10 species, the most common of which is R. stolonifer. The
identification of this species in culture or on direct exam is straightforward. The combination of rhizoids
(root-like hyphae) and striations on sporangiospores are the most easily identified characteristics. The
sporangiospores of this species occasionally appear on spore traps and, because of their relatively
distinct shape and surface texture, an experienced analyst can identify the spores. Other members of the
genus Rhizopus do not have this spore characteristic. Although experienced analysts can recognize
Rhizopus stolonifer spores, counts of these spores tend to underestimate cultural recoveries.
In culture, Rhizopus species are usually very fast growing. Culture plates may be entirely
covered with this fungus within 24 to 48 hours. The fast growth often causes problems with cultures because
it may inhibit other fungi from growing in the Petri dish. Additionally, during analysis of the sample,
colony counts and identification of other fungi may be difficult because the massive amount of mycelia from
Rhizopus can substantially obscure and hide other colonies of fungi. The radial growth rate of
Rhizopus (but not spore germination) is inhibited by high water activity media such as DG-18,
which can be used in environments where Rhizopus is expected to be abundant.
Rhizopus species are common on wet fruits such as berries (the grey fuzz that spoils strawberries
so quickly is often Rhizopus). The fungus is also capable of growth on a variety of other
substrates including soil, grains, vegetables, and wallboard paper when sufficient water is present.
It is worth noting that some Rhizopus species are used to increase the digestibility of vegetable
materials like soybeans, wheat, and rice. These materials are inoculated with Rhizopus, which
partially digests the proteins and carbohydates. The resulting metabolites can produce a meat-like flavor.
Miso, made in Japan from rice, and tempeh and sufu, made in Indonesia and China from soybeans, are foods
produced by digestion with Rhizopus.
The data and other information contained in this newsletter are provided for informational purposes only
and should not be relied upon for any other purpose. Environmental Microbiology Laboratory, Inc. hereby
disclaims any liability for any and all direct, indirect, punitive, incidental, special or consequential
damages arising out of the use or interpretation of the data or other information contained in, or any
actions taken or omitted in reliance upon, this newsletter.
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