Centers for Disease Control

and Prevention

National Institute for Occupational 

Safety and Health

NIOSH Manual of Analytical Methods (NMAM), 5th Edition

Measurement and Characterization of Fibrous Particles 

in Workplace Atmospheres

by Alan Dozier, Ph.D., and Elizabeth Ashley, NIOSH

1 Version 2 updates FI-2

2 Introduction FI-2

3 Fiber dimension considerations FI-9

4 Phase contrast light microscope counting (PCM) FI-10

5 Polarized light microscopy (PLM) of bulk materials FI-17

6 Electron microscopy FI-20

7 Optical detection (light scattering) FI-23

8 Fiber separation methods FI-24

9 Other techniques FI-25

10 Conclusions FI-27

11 References FI-28
Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

1 Version 2 updates

NMAM Chapter FI is based upon a previous version written by Paul A. Baron [NIOSH 

2003] that was adapted from a review article [Baron 2001]. The following are in this 2022 

revision:

■ Chapter title was updated

■ References were updated

■ Introduction was updated

■ Adoption of elongate mineral particle terminology and made congruent with NIOSH

Current Intelligence Bulletin 62, Asbestos Fibers and Other Elongate Mineral Particles:

State of the Science and Roadmap for Research [NIOSH 2011]

■ Information on the effects of over-grinding was added to the Sample Preparation

Section within the Polarized Light Microscopy (PLM) of Bulk Materials Section

■ Information on using commercially available equipment for fiber classification was

added to Fiber Separation Section

■ Additional information on removing long fibers in air flow streams was added to the

Fiber Separation Section

■ Other techniques were moved from a subsection within the Polarized Light

Microscopy (PLM) of Bulk Materials Section to a separate section with expanded

information on identification

2 Introduction

Disease from exposure to fibrous particles in workplace atmospheres has provided much 

of the impetus for regulations governing such exposures. Exposure to fibrous minerals, 

such as asbestos, nonasbestos minerals, and some zeolites, where the material is insoluble 

in vivo, can cascade in a number of biological events that lead to disease [Aust et al. 2011; 

EPA 2020; Khaliullin et al. 2020; Lippman 2014; Mazurek et al. 2017; Nielson et al. 2014; 

NIOSH 2011]. Workers also face possible exposures to other commercially introduced 

synthetic fibers, such as mineral wools, ceramic, carbon nanotubes (CNT), and carbon 

nanofibers (CNF) [Lippman 2014; NIOSH 2013]. The concern with biopersistent 

synthetic fibrous materials is that they can produce similar biological responses as 

asbestos, however, epidemiological studies indicate that they may be less hazardous than 

asbestos [Lippman 2014]. This is an active area of research.

Natural organic fibers have been shown to not produce the long-term health effects of 

some of the inorganic fibers unless they are industrially processed and contain harmful 

additives [Jarvholm 2000]. Natural cellulose can be processed to produce several fibrous 

types, such as bacterial cellulose (BC), microcrystalline cellulose (MC), microfibrillated 

cellulose (MFC), and cellulose nanocrystals (CNCs). These materials are gaining wide 

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Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

use in industry [Camarero-Espinosa et al. 2016]. Strategies for risk assessment have been 

laid out for cellulose nanomaterials [Shatkin and Kim 2015], and an update was published 

stating that conclusions cannot yet be made about any long-term health consequences 

[Ede et al. 2019].

The sections of this chapter cover fiber dimension considerations, NIOSH methods for 

phase contrast microscopy (PCM), polarized light microscopy (PLM), electron microscopy 

(EM), optical detection, fiber separation, and other developing techniques, followed by 

conclusions. The chapter is not meant to be a comprehensive review of these subjects but 

rather an introduction with extensive references for further reading and education.

a. What is a fiber?

The NIOSH Bulletin 62 on Asbestos Fibers and Other Elongate Mineral Particles: 

State of the Science and Roadmap for Research [NIOSH 2011] has provided a 

much-needed framework for terminology related to fibrous particles. The reader is 

encouraged to review this “roadmap” for much greater detail into these topics.

Common English dictionaries define a “fiber” as “a long threadlike substance or 

material.” Unfortunately, the term “fiber” has been used inconsistently and in widely 

varying contexts in scientific literature, which has led to ambiguous exposureresponse relationships and exposure measurements. The roadmap addresses these 

issues with inconsistent terminology [NIOSH 2011]. Note that the terminology used 

in this chapter is consistent with the roadmap.

An elongate particle (EP) is any particle (of any material) having an aspect ratio 

greater than 3:1. This definition is desirable because fibers are subdivided into many 

categories, which may not be easily determined or well defined. For example, a single 

mineral particle or fiber may be in a transition state and can vary in characteristics 

from one portion of itself to another. These subcategories become very important in 

defining asbestos. The definition for asbestos as an elongate mineral particle (EMP) 

follows [NIOSH 1990, 2011]:

An acicular single crystal or similarly elongate polycrystalline aggregate particles. 

Such particles have macroscopic properties, such as flexibility, high aspect ratio, 

silky luster, and axial lineation. These particles have attained their shape primarily 

because of manifold dislocation planes that are randomly oriented in two axes but 

parallel in the third. Note: Upon microscopic examination, only particles that 

have a 3:1 or greater aspect ratio are defined as fibers. Other macroscopic 

properties used to define fibers cannot be ascertained for individual particles 

examined microscopically.
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Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

NIOSH, using these definitions, and specifically EMP, issued an updated 

recommended exposure limit (REL) and a clarification [NIOSH 2011]:

■ a countable elongate mineral particle (EMP) is any fiber or fragment of a

mineral longer than 5 μm with a minimum aspect ratio of 3:1 when viewed

microscopically with use of NIOSH Analytical Method 7400 (‘A’ rules) or its

equivalent; and

■ a covered mineral is any mineral having the crystal structure and elemental

composition of one of the asbestos varieties (chrysotile, riebeckite asbestos

[crocidolite], cummingtonite-grunerite asbestos [amosite], anthophyllite asbestos,

tremolite asbestos, and actinolite asbestos) or one of their nonasbestiform

analogs (the serpentine minerals antigorite and lizardite, and the amphibole

minerals contained in the cummingtonite-grunerite mineral series, the tremoliteferroactinolite mineral series, and the glaucophane-riebeckite mineral series).

This clarification of the NIOSH REL for airborne asbestos fibers and related EMPs results 

in no change in counts made, as defined by NIOSH Method 7400 (“A” rules) [NIOSH 

2019]. However, it clarifies definitionally that EMPs included in the count are not 

necessarily asbestos fibers.

Asbestos and asbestiform will be discussed further in the Asbestos Section.

b. Mineral and nonmineral fibers

The term “mineral fiber” has been frequently used by nonmineralogists to 

describe EMPs found in the thoracic cavity (sometimes referred to as thoracic 

EMPs), originating either from an asbestiform habit (e.g., as asbestos fibers) or a 

nonasbestiform habit (e.g., as needle-like [acicular] or prismatic crystals), as well 

as EMPs that result from the crushing or fracturing of nonfibrous minerals (e.g., 

cleavage fragments). “Asbestiform” is a term applied to minerals with a macroscopic 

habit similar to that of asbestos. The lack of precision in these terms and the difficulty 

in translating macroscopic properties to microscopically identifiable characteristics 

contribute to miscommunication and uncertainty in identifying toxicity associated 

with various forms of minerals. Deposits may have more than one mineral habit and 

transitional minerals may be present, which make it difficult to describe the mineralogy 

clearly. EPs also come in a wide variety of nonmineral types from organic (plant and 

animal), synthetic such as polymer, glass, carbonaceous fibers, boron nitride fibers, etc.

c. Nonasbestos fibers with known health effects

There are “fibers of concern,” not covered by asbestos policies, where there have been 

documented health effects. Erionite is perhaps the most worrisome example [NTP 

2004]. An epidemic of malignant mesothelioma affecting several villages in Central 
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Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

Turkey has been studied for several decades [Baris and Grandjean 2006; Baris et al. 

1981]. Homes and other buildings in those villages were traditionally constructed of 

blocks of local volcanic stone containing erionite, a fibrous zeolite mineral. A recently 

published prospective mortality study has documented that mesothelioma accounts 

for over 40% of deaths among those residing in the affected villages. Although 

no clear epidemic of erionite-caused disease has been documented elsewhere, 

the mineral occurs in the intermountain west of the United States, and a recent 

publication reports a case of erionite-associated malignant mesothelioma in North 

America [Kliment et al. 2009].

On the basis of studies in rats, palygorskite (attapulgite) fibers longer than 5 

micrometer (μm) were determined to possibly be carcinogenic to humans 

(Group 2B) [IARC 1997]. In experimental animals, the evidence was limited 

for the carcinogenicity of long sepiolite fiber (>5 μm) and inadequate to assess 

carcinogenicity of nonerionite fibrous zeolites (including clinoptilolite, mordenite, 

and phillipsite) and wollastonite (Group 3) [IARC 1997]. These Group 3 

determinations highlight the need for additional research on nonasbestiform EMPs.

Occupational health interest in EMPs other than asbestos fibers has been focused 

primarily on fibrous minerals exploited commercially (e.g., wollastonite, sepiolite, 

and attapulgite) and mineral commodities that contain (e.g., Libby vermiculite) or 

may contain (e.g., upstate New York talc) asbestiform minerals [NIOSH 2014; Van 

Gosen 2007a]. Exposure to airborne thoracic EMPs generated from the crushing 

and fracturing of nonasbestiform amphibole minerals has also garnered substantial 

interest. The asbestos minerals, as well as other types of asbestiform minerals, are 

typically associated with other minerals in geologic formations at various locations 

in the United States [Van Gosen 2007b]. The biological significance of occupational 

exposure to airborne particles remains unknown for some of these minerals and can 

be difficult to ascertain given the mixed and sporadic nature of exposure in many 

work environments and the general lack of well-characterized exposure information.

There are man-made EPs of concern as well. Particularly important examples are CNTs

and CNFs. NIOSH issued a REL for these of 1 microgram per cubic meter (1 μg/m3

) 

[NIOSH 2013]. These EPs have been shown in animal studies to produce inflammation

in the lungs similar to crocidolite asbestos, migrate to the intrapleural space to produce

mesothelial lesions [Xu et al. 2012], instigate systemic responses including an increase

in inflammatory mediators in the blood, oxidant stress in aortic tissue, and increase

plaque formation in an atherosclerotic mouse model [Erdely et al. 2009; Li J. et al.

2007, Li Z. et al. 2007]. CNTs and CNFs can be encountered in facilities ranging from

research laboratories and production plants to operations where CNT and CNF are

processed, used, disposed, or recycled. The findings of these studies and others indicate

the need to limit worker exposure to CNTs and CNFs [NIOSH 2013].
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Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

d. Asbestos

“Asbestos” is a term used for certain minerals that have crystallized in a particular 

macroscopic habit (asbestiform) with certain commercially useful properties. The 

fibers of all varieties of asbestos are long, thin, and usually flexible when separated. 

One variety of asbestos, chrysotile, is a mineral in the serpentine group of sheet 

silicates. Chrysotile fibers consist of aggregates of long, thin, flexible fibrils that 

resemble scrolls or cylinders. The dimensions of individual chrysotile fibers depend 

on the extent to which the material has been manipulated. Five varieties of asbestos 

are minerals in the amphibole group of double-chain silicates—riebeckite asbestos 

(crocidolite), cummingtonite-grunerite asbestos (amosite), anthophyllite asbestos, 

tremolite asbestos, and actinolite asbestos [Virta 2002].

These amphibole varieties occur in an “asbestiform” habit, though not all 

“asbestiform” minerals are asbestos. “Asbestos” and “asbestiform” are two commonly 

used terms that lack mineralogical precision. When certain minerals were marketed 

or regulated as asbestos, the mineral names had definitions that might have been 

imprecise at the time and might have changed over time. In particular, the mineral 

name “amosite” was a commercial term for a mineral that was not well defined at first. 

The definitions of amosite in the Dictionary of Mining, Mineral, and Related Terms 

[U.S. Bureau of Mines 1996] and in the Glossary of Geology [American Geological 

Institute 2005] allow for the possibility that amosite might be anthophyllite asbestos, 

although it is now known to be a mineral in the cummingtonite-grunerite series. This 

is one source of confusion in the literature.

A further source of confusion comes from the use of the geological terms for a 

mineral habit. Minerals of the same chemistry, differing only in the expression of their 

crystallinity (e.g., massive, fibrous, asbestiform, or prismatic), are not differentiated 

in geology as independent species. Thus, tremolite in an asbestiform crystal habit is 

not given a separate name (either chemical or common) from tremolite in a massive 

habit. It has been suggested that crystals grown in an “asbestiform” habit can be 

distinguished by certain characteristics. Examples of this are parallel or radiating 

growth of very thin and elongate crystals that are to some degree flexible, the presence 

of bundles of fibrils, and, for amphiboles, a particular combination of twinning, 

stacking faults, and defects [Chisholm 1973].

The geological conditions necessary for the formation of asbestiform crystals are not 

as common as those that produce other crystal habits. These other habits may occur 

without any accompanying asbestiform crystals. However, amphibole asbestos may 

also include additional amphiboles that, if separated, are not asbestiform [Brown 

and Gunter 2003]. The mineralogical community uses many terms, including fibril, 

fiber, fibrous, acicular, needlelike, prismatic, and columnar, to denote crystals that 

are elongate. In contrast, in sedimentology, similar terms have been more narrowly 

defined with specific axial ratios. Thus, it is not clear, even from a single reference 
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Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

source, exactly what range of morphologies are described by these terms and the 

degree of overlap, if any. For example, the Dictionary of Mining, Mineral, and 

Related Terms defines fibril as “a single fiber, which cannot be separated into smaller 

components without losing its fibrous properties or appearance,” but also defines a 

fiber as “the smallest single strand of asbestos or other fibrous material” [U.S. Bureau 

of Mines 1996].

Asbestos has been the EMP type most associated with adverse health effects and 

disease for some time [Henderson and Leigh 2011], with many reviews [ATSDR 1990, 

2001; Craighead and Gibbs 2008; Dement 1990; Institute of Medicine 2006; Rajhans 

and Sullivan 1981; Santee and Lott 2003; Selikoff and Lee 1978; Weill et al. 2018; WHO 

1986, 2012, 2014]. The three primary diseases associated with asbestos EMP exposure 

are asbestosis, the result of inflammation and collagen formation in lung tissue; lung 

cancer; and mesothelioma, a rare cancer of the linings of the internal organs, mostly 

seen in the lung lining, although it may also occur in the tissues lining the abdominal 

cavity and its organs. EMPs have demonstrated similar health effects to asbestos 

[Castranova et al. 1994; Lippman 2014; NIOSH 2011]. Determination of the toxicity 

of EMPs of nonasbestiform varieties, such as cleavage fragments, is an ongoing area of 

research [IARC 2012, 2017; Khaliullin 2020; Manning et al. 2002].

e. Toxicity aspects of fibers

A current theory describing the toxicity of EPs indicates that dimension, dose, surface 

chemistry, and biodurability in lung fluid are the four primary factors determining EP 

toxicity [Ishida et al. 2019; Lippmann 1990, 2014, 2020]. The first thing to consider is 

how EPs are deposited in the lungs. This depends on all the indicated parameters in 

Figure 1 in a complex fashion including aerodynamic properties. Both EP diameter 

and length are important in the deposition of EPs in the lungs and how long they 

are likely to remain in the lungs. EP diameter affects aerodynamic behavior, which 

favors small diameter EPs, to travel and deposit into the airways of the lungs and 

the gas exchange regions. However, larger diameter particles are affected more by 

gravitational settling, impaction, and interception, resulting in greater deposition 

further up in the respiratory tract. The saddle points, or carinae, in the branching 

respiratory tree are often a focal point for deposition of larger diameter EPs.

EP removal from the lungs is primarily affected by the cilia lining the non-gasexchange regions (conducting airways) of the lungs. The cilia push the mucus 

produced in the lungs, along with any EPs trapped in the mucus, out of the lungs 

and into the gastrointestinal tract in a matter of hours or days. Finally, roaming 

macrophages in the gas exchange regions ingest EPs deposited there for removal 

through the lymph system and may recruit other macrophages into the region. Human 

macrophages are approximately 17 μm in diameter and can only ingest particles 

smaller than they are. This causes frustrated phagocytosis, resulting in the release 

of inflammatory cytokines and other chemicals into the lungs [Barlow et al. 2017; 
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Blake et al. 1997; Padmore 2017; Stayner et al. 2008; Turkevich et al. 2014], including

the production of cells with double nuclei [Ishida 2019]. This and other cellular

interactions with long lasting biodurable EPs appear to trigger collagen buildup in the

lungs, known as fibrosis, and, over a longer period, can produce cancer as well.

The surface properties of EPs are also thought to have an effect on toxicity [Groppo 

et al. 2005; Occella and Maddalan 1963]. The reason being that it can be directly 

associated with the number of available reactive sites on the EP’s surface. Exposed 

ions on the surface can easily react with cell membranes, causing membranolysis 

[Fantauzzi et al. 2010]. EPs with low biodurability that dissolve in lung fluid in a 

matter of weeks or months, such as certain glass EPs, appear to be somewhat less 

toxic than more insoluble EPs [NIOSH 2011].

f. Exposure evaluation

Asbestos exposure assessment techniques have been in existence since the 1930s 

[Dement et al. 1983], and some additional techniques began to be used in the 1960s 

[Rajhans and Sullivan 1981]. Earlier than this, it was not widely recognized that the 

fibrous nature of asbestos was intimately related to its toxicity, so many techniques 

Figure 1. Schematic of mechanisms that affect EP deposition 

and retention in the lungs.
Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

involved collection of airborne particles and counting all large particles at low 

magnification by optical microscopy. Thermal precipitators, impactors (konimeters), 

impingers, and electrostatic precipitators were all used to sample asbestos. The 

primary technique in the United States (U.S.) and the United Kingdom (UK) during 

this early period was the liquid impinger. The liquid impinger impacted an air stream 

from ambient air at 2.7 liters per minute (L/min) into a liquid reservoir that collected 

dust particles larger than about 1-μm aerodynamic diameter [Rajhans and Sullivan 

1981]. After sampling, an aliquot of the liquid was placed on a slide in a special cell, 

particles larger than 5-μm size were counted, and the results were reported in millions 

of particles per cubic foot. Dissatisfaction with this approach stemmed from the 

lack of correlation between the measured particle concentration and disease in the 

workplace [Lippmann 1988].

3 Fiber dimension considerations

EPs are often characterized or counted using microscopy methods according to their 

aspect ratio, i.e., the ratio of the large dimension to one of the small dimensions. The 

distribution of EP dimensions in a sample can usually be characterized by assuming a 

cylindrical geometry (i.e., the two small dimensions are identical) and measuring the 

length and diameter of an individual EP. The distribution of airborne EP sizes generated 

by grinding bulk material or by mechanically releasing particles into the air often 

results in a two-dimensional (bivariate) lognormal distribution. Such a distribution 

is characterized by five parameters: the geometric mean length, the geometric mean 

diameter, the length and diameter geometric standard deviations, and a correlation 

term that relates length to diameter [Schneider et al. 1983]. In addition, several other 

parameters that are a function of length and diameter, such as aerodynamic diameter, can 

also be characterized by a lognormal distribution [Cheng 1986].

Often the discussion of EPs assumes they are straight cylindrical objects that can be well

defined by idealized geometry parameters. However, many real-world EPs are not so simple

to describe. EPs can be contaminated by the attachment of other dust particles, creating a

complex structure with aerodynamic behavior not matching that of cylindrical EPs. The

complexity of EP shapes affects all of the measurement and separation techniques described

below, frequently making it difficult to compare one method to another; for example, ISO

and ASTM mass determination methods can use square shapes for amphiboles.

For EMPs, their detailed features can aid in their identification. For example, asbestos

EMPs are often curved, have splayed ends, or differ in other ways from a cylindrical

shape (the majority of asbestos is chrysotile and a cylinder, which is an approximation

that can be used for mass estimation for other types) [Champness et al. 1976; Langer et

al. 1974; McCrone 1980; Spurny et al. 1979]. An asbestos mineral is composed of fibrils

[NIOSH 2011] (about 0.03-μm diameter) that are packed together. This fibrillar structure

is characteristic of asbestiform minerals. When the mineral is broken apart mechanically, 

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the material separates primarily on the major axis between fibrils, and the resulting fibers 

are usually narrower bundles of fibrils. The ends of these narrower bundles can be further 

broken apart, with smaller individual fibrils spread apart yet still be part of the bundle.

There are many types of EP materials being produced for commercial purposes. These 

include fibrous glass, mineral wool, refractory ceramic fibers, wood and other plant 

fibers, and synthetic organic fibers. Most of the fibers of these materials generally have 

larger diameters than asbestos fibers. On the other hand, CNTs are very thin (<0.005-μm 

diameter), and because of their high tensile strength, high conductivity, and other special 

properties, show great promise as a commercial material [Liu et al. 1998; DeVolder et al. 

2013]. The complexity and variety of structures make CNT fiber counting a challenge. 

Measurement techniques must be tailored to the size distribution and physicochemical 

properties of these commercial materials.

4 Phase contrast light microscope counting (PCM)

As asbestos-induced disease became widely studied in the 1960s, cellulose-based 

membrane filter sampling was applied to asbestos sampling in combination with high 

magnification phase contrast light microscopy (PCM) for counting EMPs. This technique 

involves air sampling, resulting in a relatively uniform distribution of particles, EPs, and 

EMPs over the surface of a cellulose ester filter. The filter or a segment of the filter is then 

placed on a microscope slide, made transparent, and the particles observed on the filter 

with a high magnification (~450X) phase contrast light microscope. Over the years, many 

researchers have tried to improve and standardize the PCM method. One researcher, 

W.H. Walton, discussed many aspects of this technique in a review [Walton 1982]. The 

high variability of the analysis results and the method’s dependence on operator technique 

made method improvement and research difficult. The PCM method does not typically 

measure all asbestos EMPs; only those approximately >0.25-μm diameter are visible and 

only those >5-μm length are counted by protocol. Therefore, the PCM method is only an 

index of exposure and uses the assumption that what is detected is correlated with the 

EMPs actually causing disease [Lippman 1988].

The PCM method does not allow differentiation of asbestos EMPs. This is an important

limitation when the method is used in settings where EP concentrations with a substantial

nonasbestos fraction may occur. This should be remembered when considering some of

the parameters discussed next. The aim of evaluating changes to the PCM technique may

depend on whether consistency with other laboratories within a country or throughout the

world is more important than making measurements that are more closely related to health

effects. Several PCM EP counting methods have been published by national [HSE 1990,

2006; NHMRC 1976; OSHA 1998] and international organizations [ASTM 2006; ISO 2014;

WHO 1997]. Most countries have methods similar to those referenced here. A number of

factors which influence analysis results have been investigated, including the following:

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a. Microscope-related parameters

1.) Microscope magnification

The exact level of microscope magnification depends on microscope design, with

most current methods using 450X (±10%) total magnification. Pang and coworkers

investigated using 1250X magnification to improve EP detectability, but this has not

been adopted in any established methods [Pang et al. 1989]. Pang also investigated

the effect of using lower magnification (400X) and found that counts were lower for

chrysotile asbestos by 25%, but amosite EMP counts were unaffected [Pang 2000].

2.) Phase contrast optics

This contrast enhancement technique allows detection of asbestos EMPs down to

about 0.25-μm diameter for chrysotile and about 0.15 μm for amphiboles. Other

techniques such as dark field microscopy may offer improved detectability but also

increase the background from nonfibrous particles.

3.) Test slide to check optics

A test slide was developed to allow a check of proper alignment and magnification

in the microscope [LeGuen et al. 1984]. This ensures a reasonable level of

uniformity in microscope setup and operation, including the operator’s visual

perception. Improper setup can reduce detectability of EMPs. There have also

been cases where the optics were “too good,” and results were obtained that were

higher than the reference count.

4.) Counting area in microscope field

Some early measurements with the phase contrast microscope were made using a

rectangular graticule for defining the counting area, while others were made using

the entire microscope viewing area. It was found that larger viewing areas resulted

in lower counts, so the Walton-Beckett graticule [Walton and Beckett 1977] was

developed, which nominally gave a 100-μm diameter counting area (the area is

calibrated precisely for each microscope) and has been incorporated in all current

methods.

b. Sample preparation techniques

1.) Filter type

Virtually all measurements are made using 0.8-μm pore size mixed cellulose ester

(MCE) filters. Some measurements are made using 1.2-μm pore size filters when

sampling low concentrations to allow a higher flow rate through the filter. Smaller

pore size filters are used to ensure that EPs are deposited as near the surface of the

filters as possible. This results in EPs ending up in the same plane so that they can

be readily viewed with a minimum change of focus during EP counting. Pore sizes
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Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

smaller than 0.8 μm are only used with line-operated pumps because of limited 

suction power available with personal sampling pumps.

2.) Selection of the liquid for making filter transparent

A liquid is placed on the filter that closely matches the filter refractive index 

yet has an index that is as far as possible from that of the EMPs being detected. 

Rooker et al. [1982] showed that the refractive index difference between a 

cleared filter and EMPs translated directly into greater detectability of small 

diameter EMPs. A viscous solution of dimethyl phthalate and diethyl oxalate 

mixed with cellulose filter material was commonly used in the 1970s and early 

1980s. However, it did not result in a permanent sample, with crystallization 

of the mount and movement of EPs often occurring several days after sample 

preparation. Permanent slides were needed for quality assurance purposes, and 

the sample preparation technique was also slow and required some skill. A rapid 

acetone-based filter clearing technique was developed that could be used safely in 

field situations [Baron and Pickford 1986]. After clearing, filters were coated with 

triacetin to surround the EPs. This resulted in a longer lasting sample (typically 

months to years) and is the technique currently specified in most methods. 

Another technique uses a resin called Euparal to surround the EPs, resulting in a 

permanent slide preparation [Ogden et al. 1986]. This method has been adopted 

in the NIOSH [2019] PCM method.

3.) Filter loading

The number of EPs on a filter is usually specified to be within a certain loading 

range to ensure consistent counting. Cherrie et al. [1986] demonstrated using 

a serial dilution technique where counting efficiency was a function of the 

concentration of EPs on the filter. At very low filter loadings, i.e., <100 EPs per 

square millimeter (mm2

), there was a tendency to count high relative to an 

intermediate range of concentrations (100–1300 EPs/mm2

), where the counts 

were a linear function of loading. This “overcounting” was apparently due to 

greater visibility of EPs in a clean visual field. This effect was noted for both 

human counters and an image analysis system. At high filter loadings (>1300 EPs/

mm2

), undercounting occurred due to the overlap of EPs with other EPs and with 

nonfibrous particles. Most published methods indicate that optimum counting 

occurs within the 100–1300 fibers/mm2

 range, while some restrict the range further 

to less than 650 fibers/mm2

.

4.) Fiber counting rules

The EP counting rules for most current optical methods indicate that a countable 

EP should be longer than 5 μm, narrower than 3 μm, and have an aspect ratio 

greater than 3:1. These rules were selected because shorter EPs were difficult to 

detect by optical microscopy, and the 3:1 aspect ratio was used to discriminate 

between fibrous and nonfibrous particles in occupational settings. There has been 
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Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

a great deal of controversy over these rules. The use of a longer EP cutoff, e.g., 

15–20 μm, has been suggested, based on two separate arguments. First, that most 

asbestos EMPs are relatively long and thin (with high aspect ratio), and the longer 

EP cutoff would discriminate better toward EPs that were truly asbestos EMPs 

according to mineralogical definitions [Wylie 1979]. Second, that EPs that enter 

the lungs are removed readily by macrophages if they are shorter than about 15 

μm [Blake et al. 1997]. Longer EPs cannot readily be engulfed by macrophages, 

thus staying in the lungs for a long period, causing continuing fibrosis. However, 

it is possible that high doses of short fibers could be sufficient enough that 

the macrophages cannot efficiently remove them through natural clearance 

mechanisms, and this could lead to an enhancement of fibrosis.

The aspect ratio criterion has also been questioned because many nonasbestiform 

particles have length and width distributions that include EPs with aspect ratios 

greater than 3:1. Because asbestos and other minerals often contain particles, 

cleavage fragments, and EMPs not in the asbestiform habit, it has been argued 

that these should not be counted. However, the Occupational Safety and Health 

Administration (OSHA) has supported the 3:1 minimum aspect ratio through 

legal precedent. The National Institute for Occupational Safety and Health 

(NIOSH) has noted that because of the great difficulty in differentiating whether 

individual high aspect ratio particles are cleavage fragments or asbestiform fibers, 

all such particles should be counted. These high aspect ratio particles may cause 

disease whether or not they are asbestiform. As stated previously, the method 

provides an “index” of exposure that is useful in determining occupational 

exposure [Lippman 1988]. See NIOSH REL in the Introduction Terminology 

Section earlier in this chapter [NIOSH 2011].

Other aspects of EP counting have been investigated, including how to count 

nonstandard EP shapes, overlapping EPs, overlapping compact particles on EPs, 

and bundles of EPs. Each of these factors can have a noticeable effect on the 

final count. Cowie and Crawford [1982] investigated the effect of some of these 

factors and estimated most of them made a difference in the final count on the 

order of 20%. Many of the methods currently in use have slight variations in their 

interpretation of which EPs to count and thus can contribute to variation in results 

between countries and organizations.

NIOSH Method 7400 contains two sets of counting rules: the A and the B rules. 

The A rules are used for asbestos and are consistent with counting rules in 

previous NIOSH methods [NIOSH 2019]. The A rules conform to the OSHA 

3:1 aspect ratio rule and do not have an upper diameter limit for which EPs are 

counted. The B rules were introduced as an alternative to the A rules when Cowie 

and Crawford [1982] found that they provide better reproducibility between 

laboratories. The B rules are offered by some commercial laboratories for use 

with EPs other than asbestos for informational purposes because the B rules have 
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Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

3 μm as an upper diameter limit. This upper diameter limit substantially reduces 

the counting of typically large-diameter EPs, e.g., glass and cellulose, which are 

unlikely to deposit in the lungs [Breysse et al. 1999].

c. Quality assurance approaches

1.) Sample recounts

Most methods require individual counters to recount about 10% of the field

samples to ensure consistent counting procedures and alert the analyst in the case

of problem samples. It is also recommended that counters have samples that are

routinely recounted to ensure consistent counting within a laboratory over time

[NIOSH 2019].

One of the difficulties in analyzing errors made by analysts during PCM counting

is that individual fields are difficult to relocate after the analyst has finished

counting a slide. Differences in counts between analysts have often been ascribed

to local variations in loading on the filter. Pang’s development of a slide coverslip

that defines counting areas on the sample solves this problem [Harper et al. 2009;

Pang 2000]. Areas on the coverslip are vacuum coated with a thin layer of gold

and platinum, using an electron microscope grid as a mask. This leaves defined

areas on the coverslip that can be located by grid index marks. Thus, specific

fields in a sample can be readily located. Using this grid mapping approach, the

location, orientation and shape of each EP can be noted, and differences in counts

can be reconciled on an EP-by-EP basis. The coverslips have been used to study

EP counting accuracy by comparing routine counting of specified fields to counts

agreed upon by a group of competent counters. It was found that the principal

differences for chrysotile EMP sample counts were due to missing EMPs close to

the visibility limit, while the principal differences for amosite EMP samples were

caused by incorrectly sizing of EMP length near the 5-μm limit. The chrysotile

samples were therefore typically undercounted (negative bias), while the amosite

samples had increased variability with individual counters being biased either high

or low. Both these errors can be reduced by training counters with pre-counted

reference slides prepared using Pang’s coverslips [Pang 2000]. In addition, these

reference slides can be used on a routine basis to ensure consistency in counting.

Pang’s coverslips or modified versions show great promise for training analysts and

perhaps for improving quality assurance schemes [Harper et al. 2009].

2.) Interlaboratory sample exchanges

Crawford et al. [1982] found that using sample exchange programs was more

important in ensuring agreement between laboratories than similarity in details

of the counting rules. Thus, the exchange of field samples between laboratories

is commonly performed to improve reproducibility of counting. A description

of several quality assurance techniques for asbestos EMP counting is described
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Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

by Abell et al. [1989]. To fulfill NIOSH Method 7400 requirements for an 

interlaboratory sample exchange, Tombes and Calpin [2002] described a simple 

approach using appropriate statistical tests.

3.) Quality check samples

In order to get agreement between laboratories within a country or internationally, 

several programs send out identical samples to participating laboratories to assess 

their relative performance [Arroyo and Rojo 1998; Crawford et al. 1992; Harper 

et al. 2009; Kauffer 1989; Schlecht and Shulman 1986]. These programs provide 

valuable data that allows laboratories to ensure that their results are similar to that 

of other laboratories using reference samples.

d. Qualitative fiber analysis (optical)

The use of identification techniques is not allowed in reporting EP counts using 

NIOSH Method 7400 to ensure that laboratories are using the same methods for 

counting and eliminates a source of analyst subjectivity. Considerable confusion has 

been caused by individual laboratories using qualitative identification techniques to 

change the counting procedure and, hence, the final results. Despite this, work has 

been done to find simple rules based on EP dimensions to be able to differentiate 

asbestiform from nonasbestiform EPs with some success using reference standards 

[Harper et al. 2008]. Other techniques are available for providing at least tentative 

identification of EP type—use of these techniques is commonly called differential 

counting, which uses polarized light to distinguish some nonasbestos EPs. Polarized 

light techniques are based on the optical properties of the materials, including 

refractive index and crystallinity. These techniques can provide reasonably certain 

positive identification for EMPs.

EP shape criteria can also be used to differentiate EP types of interest. Glass fibers 

tend to be straighter, with smoother sides than chrysotile EMPs, for instance. 

These techniques are often used in the analysis of bulk materials [NIOSH 1994a]. If 

differential counting is used in reporting a PCM result, any alternative method or 

modifications to NIOSH Method 7400 that were used in developing the count should 

be reported to avoid any confusion.

e. Sampling volume for asbestos abatement applications

Sampling for asbestos after abatement requires the selection of a sampling volume that 

produces high confidence that the air concentration meets standards. The following 

approach is an example of how to calculate the sampling volume needed to produce 

a high level of confidence that a target exposure standard (e.g., NIOSH REL, OSHA 

PEL, EPA clearance standard) is met.

The U.S. Environmental Protection Agency (EPA) authorizes the use of PCM for 

some clearance monitoring applications, specifying that a level of 0.01 fibers per 
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Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

milliliter (mL) be met. The term “fibers” is used in this example because the methods 

are written using the term “fibers.” NIOSH Method 7400 indicates that the limit 

of detection (LOD) for PCM analysis is 7 fibers/mm2 based on interlaboratory 

variability. Under the heading “Evaluation of Method, Interlaboratory Comparability,” 

NIOSH Method 7400 provides the formulas for calculating the confidence limits on a 

single analysis result. The interlaboratory variability (see Figure 1 in [NIOSH 2019]) 

at the LOD (7 fibers/mm2) is such that the upper 95% confidence limit on a measured 

value is 4 times greater than the measured value, which is the basis for using the 

factor of 4 in the equation below.

The equation in the sampling section in NIOSH Method 7400 [NIOSH 2019] can be 

used to estimate the sampling volume:

Rearranging:

With the appropriate values inserted, the equation becomes

Where:

f = fibers

L = liter

LOD = Limit of Detection in fibers per millimeter squared

Limit = the target air concentration in fibers per cubic centimeter (cc)

Solving this equation for sampling volume gives 1,078 L. This is the minimum volume 

that will give a result, allowing a single sample to indicate compliance with the 0.01 

fibers/mL limit with 95% confidence. It requires that the sample gives a result less 

than or equal to the LOD or 5.5 fibers per 100 fields. A higher fiber count may still 

indicate that the concentration meets the target level, but not with the same level 

of confidence. This is likely to be a low estimate of concentration and additionally 

ensure compliance with the standard because the fiber concentration is low, and low 

fiber loadings are usually overestimated as described in the Filter Loading Section. 

time

LOD eff.area of filter 

flow rate L

4 2

2

* *

min *

f

mm

L

mm

imit f

cc

cc *

L 1000

sample volume

LOD eff.area of filter 

Limit

4 2

2 * * f

mm

f

cc

mm

*1000 cc

L

sample volume

7 385 

0.01

4

1000

2

2

* *

*

f

mm

f

cc

cc

L

mm
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Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

However, the background concentration of nonfibrous dust on the filter must also be 

low to ensure that fibers are not obscured.

5 Polarized light microscopy (PLM) of bulk materials

PLM is often used to determine the asbestos content in representative samples of bulk 

materials. Not all EPs in a bulk material may be asbestos. PLM allows for the identification 

of asbestos, which is why PCM is not used. The asbestos EMPs in bulk material can 

be released and become airborne when the bulk material is disturbed. For this reason, 

it is desirable to measure the asbestos content of bulk samples. The EPA [EPA 1987a] 

has defined asbestos-containing material (ACM) as material containing more than 

1% asbestos using the PLM method, which effectively estimates concentration by area 

observed. Some confusion exists regarding the units of the asbestos percentage. EPA 

originally indicated that the limit for ACM was 1% by mass [EPA 1987a], but because 

of the difficulties in determining corrections for differences in material density and in 

determining particle volumes, the limit was changed to 1% by area as determined by the 

PLM method [EPA 1990c]. OSHA does not specify units for percent asbestos in bulk 

materials in its regulations [OSHA 1994]. It does state that insulation materials containing 

greater that 1% asbestos are ACM. However, any amount of asbestos in a material may 

result in exposures to asbestos in excess of its OSHA permissible exposure limit (PEL) and 

are regulated even if the amount is less than 1% [Baron 1993].

Several PLM techniques are used for identifying EMP types as well as semi-quantifying 

the percent fibrous material (usually asbestos) in a sample [EPA 1990a; ASTM 2016; Beard 

and Rook 2001; ISO 2014; McCrone et al. 1978; Middleton 1979; NIOSH 1994a; OSHA 

1995; Perkins and Harvey 1993; Vallero and Beard 2009]. These techniques depend on 

particle shape, the refractive index, and other optical properties of individual particles. 

Many of these PLM techniques require visual observation of color in the EP and become 

less reliable for EPs thinner than about 1 μm [Baron 1993; Vaughan et al. 1981].

a. Sampling

Several procedures have been suggested for obtaining representative bulk samples of 

ACM that prevent unnecessary exposure to asbestos aerosol [EPA 1985a,b; Jankovic 

1985]. Representative sampling of commercial ACM is often problematic as these 

materials may vary substantially in asbestos concentrations between nearby locations 

and even at different depths at the same location. Sampling from multiple locations 

and compositing samples helps improve the likelihood of obtaining a representative 

sample. Different types of materials will most likely also require different mechanical 

means of sampling, such as how a hard versus soft material is sampled.

The material should be wetted or sealed during sample removal. A small coring 

device, such as a cork borer, can be used to obtain a sample from the full depth of the 
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Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

material. At least three samples per 1000 square feet (ft2) of ACM should be taken 

[EPA 1987a]. The sample should be placed in a well-sealed, rugged container. Finally, 

the sampled area should be repaired or sealed to minimize further EMP release.

Surface sampling has been proposed by several groups, but there is no relationship 

between airborne EMPs and those found on surfaces [Chatfield 2000]. Therefore, 

surface sampling for EMPs is not recommended.

b. Sample preparation for analysis by PLM

Sample preparation for a PLM analysis involves grinding the material to the optimum 

particle size range (1–15 μm diameter) and dispersing the particles in a liquid of 

known refractive index on a glass slide [Perkins and Harvey 1993]. It is important 

to not over-grind asbestos-containing materials, as over-grinding can lead to the 

deformation of the morphological features of EMPs and may induce a variable 

degree of lattice distortion [Langer et al. 1978; Van Orden et al. 2012]. This can 

result in a lack of crystallinity at the particle surface [Groppo et al. 2005; Occella and 

Maddalan 1963]. For example, a round-robin evaluation of the CARB 435 preparation 

procedures demonstrated that “...the finer a rock is ground, the less asbestos is 

reported by a laboratory (either because the asbestos is too small to be observed or 

because the grinding destroys the asbestos characteristics” [Van Orden et al. 2012].

Particle size uniformity in the prepared sample is extremely important in obtaining 

a representative sample. A few large chunks of material, which may not be 

representative, may contain more or less asbestos than hundreds of much smaller 

particles. Friable material, which is crumbly or can be crushed by hand, may readily 

release EMPs and is considered more hazardous. Friable materials are generally easier 

to prepare for analysis than some other ACMs, such as vinyl asbestos floor tiles, 

which may require dissolution or ashing of the matrix material so that the EMPs are 

separated and visible in the microscope. Before and after preparation, the sample is 

observed with a stereomicroscope at 10X–100X magnification to evaluate sample 

uniformity and observe whether fibrous material is present.

c. Fiber identification using PLM

EPs are immersed in a fluid selected to have a known refractive index. When particles 

are placed in a liquid whose dispersion is different from that of the particle, the 

particle may exhibit a color caused by the refraction of light. This technique requires 

the use of special “dispersion staining” optics [McCrone 1980]. Also, when an EP 

has a larger refractive index than the surrounding fluid medium, the bright halo 

(Becke line) around that EP appears to move into it as the microscope focus is raised; 

when the EP has a smaller refractive index, the Becke line moves out of it. Placing 

the fibrous material into several fluids with different refractive indices allows the EP 

refractive index to be bracketed [Frandsen 2016].
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Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

Once the sample has been uniformly dispersed on a slide in the appropriate refractive 

index liquid, specific EP types, e.g., asbestos, can be identified and the percent asbestos 

estimated. Two approaches are typically used: visual comparison with prepared 

reference slides or pictures and point counting. When attempting to estimate whether 

a material is an ACM (i.e., >1% asbestos), the visual comparison technique is adequate 

when more than about 10% of the particles observed are asbestos. Point counting 

is used for lower concentration samples to provide higher accuracy [EPA 1990b]. It 

involves observing 400 or more randomly selected “points” (identified with a reticle 

crosshair) in the sample. The number of points containing asbestos is divided by the 

total number of points observed to give the percent asbestos. A combination of these 

approaches balances the analysis time and accuracy of the results [Webber et al. 1990].

EP morphology, i.e., the shape of the EP, can be used to assist in its identification. 

For instance, chrysotile EMPs tend to be curved or curly, while amphibole EMPs 

are straight, especially when they are shorter than 50 μm. Asbestos EMPs often have 

frayed or split ends, while glass or mineral wool EPs are typically straight or slightly 

curved with fractured or bulbous ends. Many plant EPs are flattened and twisted, with 

diameters between 5–20 μm. Note that it is not recommended to identify EPs solely 

based on morphology.

EP refractive index and other crystalline properties can be used to identify EP type 

with reasonable certainty. Several techniques for determining these properties can be 

used in a polarized light microscope. When viewed in the microscope with crossed 

polarizing filters, isotropic (isometric or amorphous) EPs appear consistently bright 

when rotated, while anisotropic (uni- or biaxial crystal structure) EPs appear bright, 

but disappear when rotated to their extinction angle, which is a function of crystal 

structure. Thus, amorphous materials such as glass or mineral wool EPs can easily be 

discriminated from asbestos EMPs.

Elongation of an anisotropic EP can also be determined while using crossed 

polarizing filters. An accessory plate with a known slow light vibration direction, such 

as a quartz plate or a compensator, can be inserted, and changes in the interference 

colors in the EP are then noted. When the longest dimension of the EP is situated in 

the same direction as the accessory’s direction of vibration, the wavelength of light 

will change, either increase or decrease. EPs with lower wavelengths of light along the 

longest dimension of the EP have positive signs of elongation (“length-slow”). EPs 

with higher wavelengths of light along the longest dimension of the EP have negative 

signs of elongation (“length-fast”).

Additional optical properties can be used to identify EPs, including the color/

pleochroism or birefringence of the EP. Pleochroism and birefringence are both 

characteristics of anisotropic materials. Materials can demonstrate pleochroic effects 

if the specimen’s brightness and/or color changes when the microscope stage is 

rotated in plane-polarized light. Birefringence is a measure of the difference between 
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Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

the high and low refractive indices of an anisotropic material. The refractive indices 

are measured or observed quantitatively or qualitatively through observation and 

interpretation of the dispersion staining colors or observation of the interference color 

(retardation) relative to the particle’s thickness [Delly 2019].

Other materials present on the slide can sometimes hamper EPs’ accurate 

identification. Materials that interfere with identification either by their similarity 

or by covering up the EPs can be removed by physical treatment of the sample. For 

instance, organic materials, such as cellulose EPs or diesel soot, can be removed by 

low temperature oxygen-plasma ashing [Baron and Platek 1990]. Leather EPs and 

chrysotile have a similar appearance and refractive index. The leather can also be 

removed by ashing at 400°C [Churchyard and Copeland 1988].

PLM analysis is primarily used for qualitative identification of EP type. Accurate 

identification of asbestos and other crystalline EPs and EMPs requires proper training 

in the crystallographic properties of particles, as well as training and familiarization 

with PLM. As with EP counting, a laboratory quality assurance program is 

necessary to ensure consistently accurate results. The National Voluntary Laboratory 

Accreditation Program (NVLAP), operated by the National Institute for Standards 

and Technology (NIST), inspects laboratories for proper practice and provides 

reference samples that are unknown to the laboratory four times a year to check their 

performance in EP identification. Under a predecessor to this program, approximately 

350 laboratories correctly classified 98.5% of the samples as asbestos and correctly 

identified the specific asbestos types in approximately 97% of the samples. A blind 

test of 51 laboratories resulted in 97.5% correct classifications and 79.1% correct 

identifications [EPA 1986]. The American Industrial Hygiene Association (AIHA) 

Proficiency Analytical Testing Program (PAT) provides similar PLM bulk asbestos 

audit samples to laboratories. Some common interferences for bulk analysis by PLM 

include sepiolite, vermiculite, and cleavage fragments of nonasbestos amphiboles.

PLM has been cast in a quantitative measurement role by the EPA requirement of 

determining whether a school building material meets the 1% asbestos level defining 

ACM. Many variables, including particle size, density, and shape, are not adequately 

controlled or measured in the analysis and contribute to errors in the percent mass 

estimate. However, because the regulatory requirement has only one significant figure, 

with careful analysis and certification requirements, it is adequate.

6 Electron microscopy

Scanning electron microscopy (SEM) has not been the focus of as much method 

development as either light microscopy or transmission electron microscopy (TEM) 

in asbestos EMP identification. PCM found favor because of the low equipment cost 

and lower training level required for analysis when there is certainty that asbestos is 
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Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

present. When there is uncertainty, TEM analysis should be used. TEM is also preferred 

for environmental and research studies because it offers the highest resolution and the 

most robust identification capabilities. TEM allows visibility of asbestos EMPs as small 

as individual fibrils, electron diffraction for crystal structure identification, and energy 

dispersive X-ray analysis for elemental measurement. SEM typically has intermediate 

resolution, with many instruments of this type not able to see all asbestos fibers. However, 

many modern SEMs have the capability of detecting asbestos fibrils. Energy dispersive 

X-ray analysis is also available for many SEMs, providing some qualitative information

of EP type. However, since electron diffraction typically cannot be performed by SEM,

particle mineralogy cannot be identified.

a. Scanning electron microscopy (SEM)

Particles are observed in the SEM when a beam of electrons is focused onto the 

sample surface and scanned over an area. Some of the electrons are scattered from the 

surface and detected above the surface synchronously with the beam scan rate, and 

an image of the scanned surface is created. Thus, the SEM detects the surface particles 

on a substrate. The best image can be obtained on conducting objects deposited on a 

smooth, conducting substrate. Particles are often deposited on aluminum or carbon 

planchets (disks) that fit directly into the SEM or onto polycarbonate membrane 

(track-etched) filters. The samples are usually coated with gold or carbon to increase 

conductivity.

Some SEM methods have been developed for EP counting [ASTM 1996; ISO 2002, 

2019c; WHO 1985]. These methods are primarily used for inorganic, man-made EPs 

that have larger diameter EPs than can occur with asbestos. They can also be used for 

asbestos identification using X-ray analysis. This identification is not as definitive as 

the TEM methods because of the lack of electron diffraction analysis.

b. Transmission electron microscopy (TEM)

The transmission electron microscope (TEM) allows detection of particle 

morphology, elemental composition using energy dispersive X-ray analysis (EDX), 

and electron diffraction analysis down to the smallest asbestos EMPs. Although TEM 

analysis is potentially very powerful and accurate, the process of sample collection, 

preparation, and details involved in sample analysis can degrade the quantitative 

accuracy of the technique. Several more specialized techniques, such as electron 

energy loss spectroscopy and secondary ion mass spectrometry, have been used for 

analyzing particles and can also be applied to identification of EPs [Fletcher et al. 

2001; Fletcher et al. 2011].

Airborne EP samples for TEM analysis are typically collected onto a filter, usually a 

polycarbonate membrane or MCE membrane filter. For the latter filter type, the filter 

is chemically collapsed to form a smooth upper surface on which collected EPs are 
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Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

trapped. Sometimes the surface is etched using a low temperature asher to expose 

the EPs collected on or near the surface of the original filter. The filter is coated with 

a carbon film that entraps EPs exposed on the filter surface and the filter material 

is then dissolved away. The carbon film is transferred to a TEM grid (usually 3-mm 

diameter) and the sample can be placed in the TEM for analysis.

For NIOSH Method 7402 [NIOSH 1994b], the surface is not ashed because some 

EPs, e.g., cellulose, may be removed and give an inaccurate total EP count [Baron and 

Platek 1990]. Ashing can thus affect the measurement of the asbestos EMP fraction 

when used in conjunction with NIOSH 7400 [NIOSH 2019].

The above approach to preparing MCE filters for TEM analysis is called the directtransfer approach, since EPs are transferred to the carbon film with minimum 

disturbance to the way they were collected. An alternative technique is to dissolve 

the entire filter in liquid, ultrasonicate the suspension to disperse the particles, and 

deposit an aliquot of the particle suspension onto a polycarbonate filter for final 

transfer to the carbon film. This is called the indirect-transfer technique. With the 

indirect technique, the optimum particle loading of the TEM sample can be obtained 

and soluble particles can be removed from the sample. However, the suspension 

process can change the apparent size distribution of the particles and EPs by breaking 

apart agglomerates or even breaking apart asbestos EMPs into smaller EMPs or fibrils 

[Sahle and Laszlo 1996]. The breakup problem can be especially severe for chrysotile, 

causing a large increase in EMP count.

The process of sample collection and preparation is complex and subject to factors 

that can bias the final result. Because only small portions of the filter are observed 

during TEM analysis, sampled EPs that deposit nonuniformly onto the filter due to 

inertial, gravitational, and electrostatic effects will be assessed inaccurately [Chen and 

Baron 1996]. EPs that penetrate the filter surface and are not transferred to the carbon 

film will be lost. If the filter is incompletely dissolved away from the carbon film, the 

sample will be difficult to analyze.

Many of the sources of bias and variability noted in sampling and counting by PCM 

also apply to TEM analysis. EP counting in a TEM can also introduce biases and 

variability in the final result. There is a tendency to use the high magnification of 

the TEM to look for the smallest EPs, while ignoring some of the larger ones. Even 

so, EPs shorter than 0.5 μm tend to be missed because they are difficult to see in the 

background clutter of the sample [Steel and Small 1985]. Taylor et al. [1984] found 

that TEM counting gave poorer precision than counting the same sample by PCM 

and recommended that the fraction of asbestos EMPs counted by TEM be applied 

to the PCM count as indicated in NIOSH Method 7402. This combined PCM/TEM 

approach gave better precision than counting by TEM alone.

In addition to morphology, qualitative analysis of EPs by TEM primarily involves 

two techniques, energy dispersive X-ray analysis and electron diffraction. X-ray 
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Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

analysis produces responses for each of the elements (typically atomic number >6 

but is instrument dependent) present in a particle; the responses occur as peaks in an 

energy spectrum. Specific asbestos EMPs can be identified using peak intensity ratios 

observed in standard samples and as specified in the method.

The crystal structure of individual EPs is evaluated using electron diffraction. 

Typically, selected area electron diffraction (SAED) is used to select a single EP to 

produce a diffraction pattern consisting of a number of spots. The spot locations 

depend not only on the EP crystal structure, but also on the geometry of the electron 

beam optics and other instrumental parameters. The diffraction spot locations 

relative to one another give a very specific identification of crystal structure. For easily 

recognized minerals, such as chrysotile, the visual identification of the diffraction 

pattern is often sufficient. However, to identify fibers not fitting the electron 

diffraction analysis pattern for standard asbestos EMPs, careful measurement, or 

indexing, of the diffraction spots is important.

The combination of X-ray analysis and electron diffraction gives a highly definitive 

identification of specific minerals. However, as with any analytical methods, there 

are exceptions that require greater expertise to recognize potential interferences. 

Some minerals that are difficult to differentiate from regulated asbestos minerals 

include nonregulated amphiboles such as those occurring in a nonasbestiform habit. 

Sometimes, these can be distinguished by the shape in the TEM image [Bailey et 

al. 2004; Belluso et al. 2017]. Amphiboles in fibrous talcs can be intermixed with a 

crystallographic relationship [Virta 1985]. Careful TEM analysis was shown by Virta 

[1985] to identify them. It should be noted that the NIOSH REL quoted earlier in 

the introduction [NIOSH 2011] specifically includes the counting of nonasbestiform 

varieties of regulated asbestiforms, so distinguishing regulated asbestiforms from 

nonasbestiforms is not performed in NIOSH methods.

Quality assurance is especially important with TEM analysis of EPs. The NVLAP 

program provides quality assurance accreditation for laboratories performing TEM 

analysis using the Environmental Protection Agency’s Asbestos Hazard Emergency 

Response Act (AHERA) [EPA 1987a] method. Note that data provided under the 

AHERA method cannot be directly compared with counts by NIOSH TEM Method 

7402 because of significant differences in counting rules, the types of structures 

counted as asbestos, and the size range of counted EMPs. There are several other 

established methods for analyzing asbestos EMPs by TEM [ASTM 1998; EPA 1987a,b, 

1994; ISO 2019a,b; NIOSH 1994b].

7 Optical detection (light scattering)

Two types of light scattering detectors are commonly used for measuring airborne dust 

concentrations: the optical particle counter (OPC), which detects and counts individual 
Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

particles, and the photometer (sometimes called a nephelometer), which detects the 

scattering from all particles in a defined detection volume. A standard OPC can be used to 

detect asbestos concentrations in a workplace where the aerosol is primarily fibrous and 

good correlation with EP counts can be obtained [Rickards 1978]. A nephelometer may 

also be used but may have an even greater interference from nonfibrous dusts.

A general fibrous aerosol monitor uses an electrostatic alignment technique by applying 

a field that aligns and rotates individual EPs in a laser beam. The light scattering from the 

EPs uniquely identifies the presence of individual EPs. This allows the specific detection 

of EPs [Lilienfeld et al. 1979] and can be used to measure EP length [Marijnissen et al. 

1996]. Several field tests have indicated fibrous aerosol monitor designs agree reasonably 

well with field measurements of EPs by phase contrast microscopy, though mostly at 

concentrations above ambient levels. They have been used at abatement sites to provide 

rapid feedback and ensure acceptable containment of airborne EPs during asbestos 

removal. New technologies are being developed for asbestos identification in real time 

[Renard et al. 2020]. One commercially available asbestos aerosol monitor exploits the 

paramagnetic properties of asbestos along with light scattering to detect all regulated 

asbestos species in real time and reject nonasbestos EPs with a 99% confidence level 

[Stopford et al. 2013]. Comparison with standard asbestos methods at abatement sites has 

demonstrated very good correlation between results.

8 Fiber separation methods

Fiber diameter is known to be a dominant aerodynamic factor. Esmen et al. [1979] 

showed that the average EP concentration in workplaces decreased exponentially with 

an increase of EP diameter, indicating that the larger diameter EPs settled out more 

quickly than smaller diameter EPs. This is due to the aerodynamic diameter of EPs being 

dependent primarily on EP physical diameter and EP density, with a minor dependence 

on EP length [Baron 1996]. Baron showed that sampling EPs with a thoracic sampler was 

approximately equivalent to counting only EMPs with a physical diameter smaller than 3 

μm [Baron 1996]. Jones et al. [2001] reported that there appeared to be no impediment to 

using a thoracic sampler for EP sampling. They found that several samplers matched the 

thoracic convention, the sample collected by these samplers could be analyzed by standard 

methods, and that field studies indicated equivalence to the current method [Jones et al. 

2001]. Maynard [1999] also found that there appeared to be no variation in penetration 

through these samplers as a function of EP length.

A spiral centrifuge can be used to separate EPs and reference spherical particles to

estimate EP aerodynamic diameter [Stöber 1972]. It was found that the aerodynamic

diameter was directly proportional to physical diameter, proportional to the square root

of the EP density, and proportional to EP length to the 1/6th power. For EMPs having a

density of about 3 grams per cubic centimeter (g/cm3), the aerodynamic diameter was

approximately three to five times the physical diameter of the EP. Behavior of glass EPs in

a cascade impactor was investigated by Burke and Esmen [1978]. A small correction to 

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the aerodynamic diameter was developed to take into account the interception of longer

EPs with the impaction surface [Feigley et al. 1992]. An inertial spectrometer was used to

measure EP aerodynamic diameter and good diameter separation was achieved [Baron

et al. 1994a; Morigi et al. 1999]. Baron et al. [1994b] and Deye et al. [1999] developed a

technique for separating EPs by length using dielectrophoresis. This technique was also

shown to be useful for measuring EP length and diameter distributions [Baron et al. 2000].

It has been shown that aerodynamic size separation of EP aerosols can be accomplished

with commercially available equipment using an aerodynamic aerosol classifier and a

multi-cyclone sampling array [Lee et al. 2020]. Cyclones, impactors, and porous foam

classifiers have been evaluated for efficiency of removing airborne EPs not likely to deposit

in the lungs from a sampling stream [Maynard 1996]. Long EPs can also be effectively

removed from an air flow stream by using a screen configuration method [Ku et al. 2014].

Although current U.S. practice does not use an upper diameter limit for asbestos EMPs, a 

limit of <3 μm is commonly used for man-made EPs outside the United States. It would 

be advantageous for thoracic sampling to become routine for measurement of asbestos 

and other EPs. This approach has several advantages. It aligns the analytical method 

for EPs with conventional practice for dust sampling and counting based on thoracic 

sampling. It removes some of the larger particles in the sample, resulting in a cleaner 

sample for the analyst. It removes the need for imposing an EP diameter limit during 

counting and would align the analytical methods for asbestos and other EPs to those used 

for all dusts in the U.S. and in the global arena. Thoracic sampling has the disadvantage 

of requiring the flow rate for a specific sampler to be fixed. This reduces the flexibility to 

target the loading of the filter by adjusting the flow rate. However, several classifiers can be 

designed to operate at selected flow rates to allow some flexibility in sampling.

9 Other techniques

a. Counting

EP counting by an analyst can produce relatively high biases and variability even 

among the best analysts and even among a single analyst depending on factors 

such as time of day or how long they have been on the microscope. Therefore, 

several researchers have attempted to develop automated counting systems. With 

the increases in computing power over the last 45 years, significant efforts have 

been made to develop such a system. An early attempt was made by researchers at 

Manchester University, in collaboration with the Health and Safety Executive in the 

UK [Kenny 1984]. They carried out a comprehensive study to develop an EP counting 

system. The Manchester Asbestos Program (MAP) was able to give reasonably 

good agreement with manual counting for certain types of samples. It was used 

as a reference analyst for the U.S. and UK reference sample programs for several 

years. Eventually, MAP was dropped as the reference because it was not sufficiently 

consistent for all types of samples.
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Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

Image analysis of asbestos EMPs have the following principal challenges: the 

complexity of many EMP shapes, including bundles, agglomerates, and split EMPs; 

the EMPs often go in and out of the plane of focus; the background includes many 

particles and other nonfibrous shapes; the phase contrast optics produces haloes 

around particles in the sample that can be detected as EMPs; and finally, and perhaps 

most importantly, the contrast between the EMPs and background is poor, and 

many EMPs are near the detection threshold. An evaluation of the MAP program 

indicated that a significant fraction of the EMPs were misidentified as multiple EMPs, 

not detected at all, and groups of compact particles or edges of large particles were 

detected as EMPs [Baron and Shulman 1987].

Inoue and coworkers [1998] developed image analysis software using a 

microprocessor-based personal computer. Initial tests indicated that it works 

approximately as well as human counters. Inoue also evaluated how well human 

counters and the image analyzer did in detecting the same EPs in a sample. They found 

that only about 50% of the EPs were consistently counted by all counters, so the image 

analysis system did approximately as well as the manual counting [Inoue et al. 1999]. 

More recently, Alexandrov et al. [2015] used fluorescence microscopy for improved 

EP detection before automated counting. This did result in better EP/background 

separation, but the automated counting routine used still needed operator intervention 

to obtain accurate results. Biswas [2020] used a machine-learning segmentation 

method to obtain a 95% counting accuracy on SEM asbestos EMP images.

In addition to image analysis, optical microscopy can be enhanced using a personal 

computer to more easily observe the image and to mark and measure fiber 

dimensions with automatic recording of the EPs counted [Lundgren et al. 1995]. This 

does not appear to improve the counting accuracy because the analyst still decides 

which EPs are to be counted.

b. Identification

The current best practice for asbestos EMP identification is stated in [ISO 2019b], “...

(TEM is) the only technique capable of unequivocal identification of the majority 

of individual fibres of asbestos.” However, this is qualified by the statement, from 

the same document, “The method cannot discriminate between individual fibres of 

asbestos and elongate fragments (cleavage fragments and acicular particles) from 

nonasbestos analogues of the same amphibole mineral.” EP identification using a TEM 

is accomplished by evaluating EP morphology, chemical composition, and crystal 

structure using imaging, energy dispersive analysis (EDS), and electron diffraction, 

respectively. The combination of this analytical analysis, identification evaluation, and 

manual fiber/structure counting can be expensive and time consuming.

Recent work explores the use of several other techniques such as spectroscopies 

(Raman, infrared, IR) and SEM electron backscatter diffraction (EBSD) for the 
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Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

identification of asbestos. These techniques have the advantage of minimal sample 

preparation, efficient identification, and the ability to scan larger areas. The 

disadvantage for optical methods still remains the optical resolution. Both visible 

and ultraviolet Raman spectroscopy have been explored [Bard et al. 2004; Petry et al. 

2006; Rinaudo et al. 2003; Rinaudo et al. 2004]. Rinaudo et al. [2004] demonstrated 

that visible Raman could identify all asbestos species when the fibers are in bundles 

of at least 1.5 μm in diameter. Petry et al. [2006] used ultraviolet (UV) Raman and 

obtained unambiguous identification of all regulated asbestos types. UV has the 

advantage in that it does not produce fluorescence, which allows the assessment 

of hydroxyl-stretching vibrations. However, as in Rinaudo, Petry’s data came from 

clusters or bundles of EMPs that produce high signal to noise ratios.

Fourier Transform Infrared Spectroscopy (FTIR) is another vibrational spectroscopy 

that has been demonstrated in both the OH-stretching and in the lattice modes 

regions and can be used for efficient identification of amphibole asbestos types 

[Ventura et al. 2018]. As in the case of Raman spectra, the analysis was done on 

large clusters and bundles of EMPs. In the case of EBSD [Bandli and Gunter 2014], 

transmitted EBSD patterns were used in an SEM. This is in effect a diffraction 

pattern analysis method, subject to similar complications as for the identification 

of amphiboles from SAED patterns obtained by TEM. Nonetheless, the authors 

demonstrated that this technique can distinguish between asbestos phases and other 

interfering mineral phases. It also has an advantage of being able to scan larger areas 

than a TEM with the ability to do direct analysis without having to prepare samples 

utilizing direct transfer or indirect transfer methods. All of these techniques have 

produced encouraging results but have not been tested on field samples.

In the case of spectral methods, spectra of minerals in the sample can interfere with 

the desired spectra, however, in some cases, data processing techniques may be 

able to resolve this. These techniques have not been evaluated with smaller clusters 

or bundles of EMPs, whereby the signal to noise ratios may be too low, hindering 

utility. However, because these techniques use either optical microscopes or SEM, 

they can, in principle, be combined with automated stages and machine-learning 

counting methods to boost efficiency of analysis even further. In addition, the utility 

of spectroscopic identification has been improved using machine-learning methods 

that allow unidentified spectra to be matched to known spectra in a large database 

based on probabilities [Zheng et al. 2018]. Bandli and Gunter [2014] suggest this may 

be possible for EBSD also because some EBSD databases already exist.

10 Conclusions

The capability to measure EP size distributions is available through microscopy and, to 

a much lesser extent, through direct-reading instrumentation. PCM, PLM, SEM, and 

TEM methods often do not produce directly comparable results because of differences in 
Measurement and Characterization of Fibrous Particles in Workplace Atmospheres

counting rules, resolution capability, and the ability to distinguish asbestos from interfering

particles or other EPs. The traditional methods of microscopy are relatively inaccurate

when compared to chemical analysis methods for most other analytes because of the

many sources of error in the sampling and analysis procedure. To improve laboratory-tolaboratory agreement, training, quality control (including the exchange of samples among

laboratories), and proficiency testing are important. Implementation of training through

the use of Pang’s coverslips allows for the investigation of counting errors, and therefore,

the potential improvement of PCM counting accuracy. Thoracic sampling could eliminate

interfering particles and thereby improve measurement methods in the future.

Disclaimer

Mention of any company or product does not constitute endorsement by the National 

Institute for Occupational Safety and Health (NIOSH), Centers for Disease Control and 

Prevention. In addition, citations to websites external to NIOSH do not constitute NIOSH 

endorsement of the sponsoring organizations or their programs or products. Furthermore, 

NIOSH is not responsible for the content of these websites. All web addresses referenced 

in this document were accessible as of the publication date.

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