**Veterinary Diagnostic using Transmission Electron Microscopy**

### M.H.B. Catroxo and A.M.C.R.P.F. Martins

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61125

#### **Abstract**

Transmission electron microscopy has been an excellent tool, essential for the diagnosis of bacterial and viral animal diseases. Four basic techniques have been widely used: negative staining (rapid preparation), immunoelectron microscopy, immunolabeling with colloidal gold particles and resin embedding. The negative staining technique (rapid preparation) is the most applied, due to its speed, simplicity and specificity and can be used in various clinical specimens – such as feces, semen, urine, serum, organ fragments, crusts, body fluids, cell culture suspension, oral, ocular and fecal swabs, among others –, in which the agents can be directly viewed in large numbers in the samples. The immunoelectron microscopy technique using a specific primary antibody promotes the clumping of particles, also allowing the serotyping of the agents. In the immunolabeling with colloidal gold technique antigen-antibody reaction is enhanced by marking the antigen colloidal gold particles associated with protein A. The method of resin embedding, followed by ultrathin sections of cells or infected tissues can monitor the different stages of maturation viruses or bacteria and their behavior inside of host cells by determining not only the infection, but also the course of the disease in farms. The techniques can be applied to all animal species, either large or small, including aquatic and wild animals. Its implementation allows rapid diagnosis, providing subsidies for the immediate institution of prophylactic measures, and control and prevention of bacterial and viral animal diseases.

**Keywords:** Veterinary diagnostic, Negative staining, Immunoelectron microscopy, Immunolabeling with colloidal gold particles, Resin embedding, Transmission elec‐ tron microscopy

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1. Introduction**

In recent decades there has been a major advance in the diagnosis field, allowing the devel‐ opment mainly of sophisticated techniques at the molecular level, facilitating increasingly improved detection of diseases of viral and bacterial origin.

Despite this progress, electron microscopy remains an excellent and sensitive tool in the application of such techniques, and they are essential for the rapid diagnosis of these agents [1,2,3].

Four basic techniques have been widely used: negatively stained (rapid preparation), immu‐ noelectron microscopy, immunolabeling with colloidal gold particles and resin embedding [4].

### **2. Negative staining (Rapid preparation)**

The negative staining technique is the main method of electron microscopy, as well as the most used, due to its ease in preparation and speed in obtaining results, which are achieved in 5-10 min [1,5,6]. It was developed by Brenner & Horne in 1959 [7], when the viral particles could be viewed at the ultrastructural level [8].

The technique utilizes an electron opaque substance to surround the virus or another biological structure, showing contrast between the electron lucent biological material and the back‐ ground against which it is viewed. The image formation is the result of electron being absorbed or deflected by the stain [9,10].

To develop the samples, copper or nickel grids measuring from 200-400 meshs are used and require plastic film pretreatment before being covered with carbon. Several types of plastic films are used for this purpose, with Formvar, Parlodion and Pioloform being the most common ones [11,12]. In our laboratory we use the Parlodium 0.5% in amyl acetate, followed by deposition of thin carbon under a metallizer.

Various types of contrasting (heavy metals salts) are used to provide better contrast to agents. The most used are the phosphotunstic acid (PTA), uranyl acetate, sodium silicotungstate, methylamine tungstate and ammonium molybdate 11,12].

We chose the 2% ammonium molybdate and pH5.0 as standard contrasting in our laboratory, in fact, in order to provide a soft contrast in all types of samples, clearly showing ultrastructural detail, such as the envelope covered with spikes and tape nucleocapsid shaped "Hering-bone", found in paramyxovirus [4,13,14].

This heavy metal has been used to contrast negatively many virus species [15].

The technique can be applied in various types of samples, such as feces; urine; serum; organ fragments; crusts; vesicular and peritoneal fluid; semen; ocular; nasal and fecal swabs; epithelium; cell culture supernatant; and others [16,17,1,18,19,20,21,22,6].

Small amounts of samples (1 drop of 40 μl) are necessary for the technical processing [6].

**1. Introduction**

[1,2,3].

In recent decades there has been a major advance in the diagnosis field, allowing the devel‐ opment mainly of sophisticated techniques at the molecular level, facilitating increasingly

Despite this progress, electron microscopy remains an excellent and sensitive tool in the application of such techniques, and they are essential for the rapid diagnosis of these agents

Four basic techniques have been widely used: negatively stained (rapid preparation), immu‐ noelectron microscopy, immunolabeling with colloidal gold particles and resin embedding [4].

The negative staining technique is the main method of electron microscopy, as well as the most used, due to its ease in preparation and speed in obtaining results, which are achieved in 5-10 min [1,5,6]. It was developed by Brenner & Horne in 1959 [7], when the viral particles could

The technique utilizes an electron opaque substance to surround the virus or another biological structure, showing contrast between the electron lucent biological material and the back‐ ground against which it is viewed. The image formation is the result of electron being absorbed

To develop the samples, copper or nickel grids measuring from 200-400 meshs are used and require plastic film pretreatment before being covered with carbon. Several types of plastic films are used for this purpose, with Formvar, Parlodion and Pioloform being the most common ones [11,12]. In our laboratory we use the Parlodium 0.5% in amyl acetate, followed

Various types of contrasting (heavy metals salts) are used to provide better contrast to agents. The most used are the phosphotunstic acid (PTA), uranyl acetate, sodium silicotungstate,

We chose the 2% ammonium molybdate and pH5.0 as standard contrasting in our laboratory, in fact, in order to provide a soft contrast in all types of samples, clearly showing ultrastructural detail, such as the envelope covered with spikes and tape nucleocapsid shaped "Hering-bone",

The technique can be applied in various types of samples, such as feces; urine; serum; organ fragments; crusts; vesicular and peritoneal fluid; semen; ocular; nasal and fecal swabs;

This heavy metal has been used to contrast negatively many virus species [15].

epithelium; cell culture supernatant; and others [16,17,1,18,19,20,21,22,6].

improved detection of diseases of viral and bacterial origin.

328 The Transmission Electron Microscope – Theory and Applications

**2. Negative staining (Rapid preparation)**

be viewed at the ultrastructural level [8].

by deposition of thin carbon under a metallizer.

methylamine tungstate and ammonium molybdate 11,12].

or deflected by the stain [9,10].

found in paramyxovirus [4,13,14].

Samples must be collected and maintained immediately under refrigeration (4º C) before being transported and sent to the electron microscopy laboratory to ensure preservation of the morphology of viral particles [19].

In this technique the samples are suspended and drops collected from the surface of the suspension are transferred to metallic grids, which are then negatively contrasted [7,23,24,25, 26,27,28,29,20,15,30,19,6].

The rapid diagnosis of viral agents is accomplished through the comparison between the dimensions and specific morphology of the visualized particles and the other taxonomically combined viral families. The viruses can be morphologically classified according to their symmetry, which can be helical, cubic, icosahedral or complex [31]. The more commonly used criteria for the taxonomic classification are the following: dimension, shape and structure; shape of the viral capsid; presence or absence of the envelope; and surface projections. Complete and incomplete particles, as well as empty capsids, can also be distinguished [4,2].

The most commonly found viruses in cases of diarrhea outbreaks in several animal species are the coronavirus (fig. 1), rotavirus (fig. 2), adenovirus (fig. 3), astrovirus, and parvovirus, which can be seen in large numbers in feces, fecal swabs or intestine fragments [1,4,13,6].

Viruses that cause respiratory diseases such as influenza virus (fig. 4), coronavirus, and paramyxovirus (fig. 5), can also be easily detected in lung fragments, lung washes and nasal discharge [1,13].

Other viruses responsible for important reproductive diseases such as herpesvirus (infectious bovine rhinotracheitis), flavivirus (bovine viral diarrhea virus) (fig. 6), parvovirus (porcine parvovirus), herpesvirus (Aujeszky's disease), and circovirus (circovirosis swine) can be widely found in organ fragments [13].

Negative staining also plays an important role in the rapid investigation of viral diseases that cause skin lesions caused by herpes virus (fig. 7), poxvirus (figs. 8, 9) and papillomavirus (fig. 10) in fragments of lesions, crusts, epithelium and vesicular fluid [1,19,6,12,20,32,33,34].

The large brick-shaped orthopoxvirus can readily be distinguished from the parapoxvirus and the smaller icosahedral herpesvirus [35], being one of the most recommended techniques by the OIE for the accomplishment of the laboratorial diagnosis of the virus in skin lesion [36].

This technique also allows the detection of different viral particles in the same sample [13,6], especially in cases of diarrhea caused by the simultaneous presence of rotavirus and corona‐ virus (fig.11).

The negative staining can also be applied to identify the presence of a few types of bacteria of easy morphological identification, such as Leptospira (fig. 12), mycoplasma (fig. 13) and other bacteria types found in specific cell cultures.

of the virus in skin lesion [36].

rotavirus and coronavirus (fig.11).

of the virus in skin lesion [36].

rotavirus and coronavirus (fig.11).

The large brick-shaped orthopoxvirus can readily be distinguished from the

sample [13,6], especially in cases of diarrhea caused by the simultaneous presence of

bacteria of easy morphological identification, such as Leptospira (fig. 12), mycoplasma (fig.

The large brick-shaped orthopoxvirus can readily be distinguished from the

This technique also allows the detection of different viral particles in the same

The negative staining can also be applied to identify the presence of a few types of

parapoxvirus and the smaller icosahedral herpesvirus [35], being one of the most

recommended techniques by the OIE for the accomplishment of the laboratorial diagnosis

This technique also allows the detection of different viral particles in the same

The negative staining can also be applied to identify the presence of a few types of

parapoxvirus and the smaller icosahedral herpesvirus [35], being one of the most recommended techniques by the OIE for the accomplishment of the laboratorial diagnosis

sample [13,6], especially in cases of diarrhea caused by the simultaneous presence of

13) and other bacteria types found in specific cell cultures.

bacteria of easy morphological identification, such as Leptospira (fig. 12), mycoplasma (fig.

Fig. 1 – Negatively stained coronavirus particles showing characteristic radial projections forming **Figure 1.** Negatively stained coronavirus particles showing characteristic radial projections forming a corona, in feces of *Mazama gouazoubira* (arrow). Bar: 200 nm.

Fig. 2 - Negatively stained rotavirus, showing complete" (big arrow) and "empty" **Figure 2.** Negatively stained rotavirus, showing complete" (big arrow) and "empty" (minor arrow) particles in feces of swine. Bar: 110 nm. Bar: 110 nm.

Fig. 3 - Negatively stained adenovirus particles, exhibiting the hexagonal outline formed by the distinct capsomers (arrow) **Figure 3.** Negatively stained adenovirus particles, exhibiting the hexagonal outline formed by the distinct capsomers (arrow) in feces of pigeon. Bar: 70 nm.

Fig. 4 – Influenza virus in feces of ferret,

showing well defined spikes (arrow). Bar: 80 nm.

 Fig. 5 – Negatively stained paramyxovirus particles, pleomorphic,roughly spherical, containing an envelope covered by spikes (arrow) in feces of parrot. Bar: 140 nm.

in feces of pigeon. Bar: 70 nm.

Fig. 4 – Influenza virus in feces of ferret,

in feces of pigeon. Bar: 70 nm.

in feces of pigeon. Bar: 70 nm.

Fig. 4 – Influenza virus in feces of ferret, **Figure 4.** Influenza virus in feces of ferret, showing well defined spikes (arrow). Bar: 80 nm. showing well defined spikes (arrow). Bar: 80 nm.

Bar: 110 nm.

Bar: 110 nm.

The large brick-shaped orthopoxvirus can readily be distinguished from the

sample [13,6], especially in cases of diarrhea caused by the simultaneous presence of

bacteria of easy morphological identification, such as Leptospira (fig. 12), mycoplasma (fig.

The large brick-shaped orthopoxvirus can readily be distinguished from the

This technique also allows the detection of different viral particles in the same

The negative staining can also be applied to identify the presence of a few types of

parapoxvirus and the smaller icosahedral herpesvirus [35], being one of the most

recommended techniques by the OIE for the accomplishment of the laboratorial diagnosis

This technique also allows the detection of different viral particles in the same

The negative staining can also be applied to identify the presence of a few types of

parapoxvirus and the smaller icosahedral herpesvirus [35], being one of the most recommended techniques by the OIE for the accomplishment of the laboratorial diagnosis

sample [13,6], especially in cases of diarrhea caused by the simultaneous presence of

bacteria of easy morphological identification, such as Leptospira (fig. 12), mycoplasma (fig.

of the virus in skin lesion [36].

rotavirus and coronavirus (fig.11).

of the virus in skin lesion [36].

rotavirus and coronavirus (fig.11).

13) and other bacteria types found in specific cell cultures.

13) and other bacteria types found in specific cell cultures.

Fig. 1 – Negatively stained coronavirus particles showing characteristic radial projections forming a corona, in feces of *Mazama gouazoubira* (arrow).

Fig. 1 – Negatively stained coronavirus particles showing characteristic radial projections forming

a corona, in feces of *Mazama gouazoubira* (arrow).

**Figure 1.** Negatively stained coronavirus particles showing characteristic radial projections forming a corona, in feces

Fig. 2 - Negatively stained rotavirus,

in feces of pigeon. Bar: 70 nm.

**Figure 3.** Negatively stained adenovirus particles, exhibiting the hexagonal outline formed by the distinct capsomers

Fig. 4 – Influenza virus in feces of ferret,

showing well defined spikes (arrow). Bar: 80 nm.

 Fig. 5 – Negatively stained paramyxovirus particles, pleomorphic,roughly spherical, containing an envelope covered by spikes (arrow) in feces of parrot. Bar: 140 nm.

showing complete" (big arrow) and "empty" (minor arrow) particles in feces of swine.

Fig. 2 - Negatively stained rotavirus,

**Figure 2.** Negatively stained rotavirus, showing complete" (big arrow) and "empty" (minor arrow) particles in feces of

showing complete" (big arrow) and "empty" (minor arrow) particles in feces of swine.

Fig. 3 - Negatively stained adenovirus particles, exhibiting the hexagonal outline formed by the distinct capsomers (arrow)

Bar: 200 nm.

Bar: 110 nm.

Bar: 200 nm.

of *Mazama gouazoubira* (arrow). Bar: 200 nm.

330 The Transmission Electron Microscope – Theory and Applications

swine. Bar: 110 nm.

(arrow) in feces of pigeon. Bar: 70 nm.

showing well defined spikes (arrow). Bar: 80 nm.

 Fig. 5 – Negatively stained paramyxovirus particles, **Figure 5.** Negatively stained paramyxovirus particles, pleomorphic,roughly spherical, containing an envelope covered by spikes (arrow) in feces of parrot. Bar: 140 nm.

**Figure 6.** Flavivirus isometric particles in bovine intestine. Bar: 70 nm.

Bar: 70 nm.

Fig. 6 – Flavivirus isometric particles in bovine intestine.

showing enveloped (big arrow) and non-enveloped particles (minor arrow). Bar: 100 nm. **Figure 7.** Herpesvirus in skin lesion of parrot, showing enveloped (big arrow) and non-enveloped particles (minor ar‐ row). Bar: 100 nm. Fig. 7 – Herpesvirus in skin lesion of parrot,

particles (minor arrow). Bar: 100 nm.

showing enveloped (big arrow) and non-enveloped

Fig. 7 – Herpesvirus in skin lesion of parrot,

**Figure 8.** Negatively stained poxvirus showing regular spaced thread-like ridges comprising the exposed surface (big arrow) and enveloped particle (minor arrow). Bar: 320 nm.

Fig. 8 – Negatively stained poxvirus showing

regular spaced thread-like ridges comprising the exposed surface (big arrow) and enveloped

particle (minor arrow). Bar: 320 nm.

Fig. 6 – Flavivirus isometric particles in bovine intestine.

Fig. 6 – Flavivirus isometric particles in bovine intestine.

Fig. 7 – Herpesvirus in skin lesion of parrot,

**Figure 7.** Herpesvirus in skin lesion of parrot, showing enveloped (big arrow) and non-enveloped particles (minor ar‐

Fig. 7 – Herpesvirus in skin lesion of parrot,

Fig. 8 – Negatively stained poxvirus showing

Fig. 8 – Negatively stained poxvirus showing

**Figure 8.** Negatively stained poxvirus showing regular spaced thread-like ridges comprising the exposed surface (big

arrow) and enveloped particle (minor arrow). Bar: 320 nm.

particles (minor arrow). Bar: 100 nm.

particles (minor arrow). Bar: 100 nm.

row). Bar: 100 nm.

showing enveloped (big arrow) and non-enveloped

showing enveloped (big arrow) and non-enveloped

Bar: 70 nm.

332 The Transmission Electron Microscope – Theory and Applications

Bar: 70 nm.

**Figure 9.** Parapoxvirus particles with a distinctive criss-cross filament pattern derived from the superimposition of up‐ per and lower virion surfaces (big arrow) and projections surface (minor arrow). Bar: 110 nm.

**Figure 10.** Negatively stained papillomavirus particles exhibit distinct, isolated capsomers (arrow). Bar: 70 nm.

Fig. 10 - Negatively stained papillomavirus particles

Fig. 11 - Simultaneous presence of particles of rotavirus (big arrow) **Figure 11.** Simultaneous presence of particles of rotavirus (big arrow) and coronavirus (minor arrow) in feces of cattle. Bar: 150 nm.

and coronavirus (minor arrow) in feces of cattle. Bar: 150 nm.

Fig. 12 – Negatively stained *Leptospira interrogans*. Bar: 1000 nm.

Fig. 13 – Negatively stained *Mycoplasma gallisepticum*. Bar: 140 nm.

The immunoelectron microscopy technique is an antigen-antibody reaction which

promotes increased sensitivity in 100-fold when using a specific antibody connected to a

**1.2 - Immunoelectron Microscopy Technique** 

**Figure 12.** Negatively stained *Leptospira interrogans*. Bar: 1000 nm.

**Figure 13.** Negatively stained *Mycoplasma gallisepticum*. Bar: 140 nm.

#### **2.1. Immunoelectron microscopy technique**

The immunoelectron microscopy technique is an antigen-antibody reaction which promotes increased sensitivity in 100-fold when using a specific antibody connected to a known antigen [35,37,11].

It was first developed for quantifying plant virus by Derrick (19730 [38] being subsequently used in various types of clinical specimens [39,40,41,42].

Used when the number of agent particles in a sample is very low, it allows identification of the agent for specific antigen-antibody reaction - such identification can be achieved by its morphology.

The technique does not require purification of antibodies and detects contaminating antigens and/or antibodies [37]. It is also used to serotype morphologically similar (but antigenically distinct) particles [43,9].

Three variations of the method have been described, immune clumping or direct immunoe‐ lectron microscopy [4,44,45] solid phase immune electron microscopy (SPIEM) [38], and decoration [46].

In the immune clumping method, an antigen suspension is blended with an equal volume of specific antibody against the said antigen; the suspension is incubated for 1 h at 37º C and negatively contrasted to allow the execution of the antigen-antibody reaction. The particles of the agent create aggregation through the reaction with its homologous antibody [4,9].

This procedure has been used to facilitate the detection and distinction of small viral particles from the background debris present in clinical specimens [47].

The sensitivity of immunoelectron microscopy can be increased by applying the most used SPIEM method in our laboratory.

In this technique, a grid is previously prepared with colodium film and sensitized with the antibody, cleaned with PBS, incubated with the antigen and negatively contrasted. As a result of the antigen-antibody reaction, the particles of the agent are agglutinated, creating groups on the grid surface (fig. 14) [4,9].

Several types of viruses can be added by this technique, such as flaviviruses, rotavirus, poxvirus, paramyxovirus, and parvovirus, among others.

Immunoelectron microscopy was used to trap particles of equine herpesvirus in organ fragments in cases of abortion [48].

The technique can also be applied to trap some types of bacteria and mycoplasma.

**Figure 14.** Immunoelectron microscopy of parvovirus particles aggregated by antigen-antibody interaction in suspen‐ sion of swine liver. Bar: 120 nm.

#### **2.2. Decoration technique**

**Figure 12.** Negatively stained *Leptospira interrogans*. Bar: 1000 nm.

334 The Transmission Electron Microscope – Theory and Applications

**Figure 13.** Negatively stained *Mycoplasma gallisepticum*. Bar: 140 nm.

used in various types of clinical specimens [39,40,41,42].

The immunoelectron microscopy technique is an antigen-antibody reaction which promotes increased sensitivity in 100-fold when using a specific antibody connected to a known antigen

It was first developed for quantifying plant virus by Derrick (19730 [38] being subsequently

Used when the number of agent particles in a sample is very low, it allows identification of the agent for specific antigen-antibody reaction - such identification can be achieved by its

The technique does not require purification of antibodies and detects contaminating antigens and/or antibodies [37]. It is also used to serotype morphologically similar (but antigenically

Three variations of the method have been described, immune clumping or direct immunoe‐ lectron microscopy [4,44,45] solid phase immune electron microscopy (SPIEM) [38], and

**2.1. Immunoelectron microscopy technique**

[35,37,11].

morphology.

decoration [46].

distinct) particles [43,9].

In the decoration or antigen-coated grid technique, the antibodies are used to "decorate" or coat viruses [37,46].

Carbon-coated electron microscope grids are incubated with a specific antibody that is used to trap antigen particles found in a suspension. After the trapping, the treated grids are washed and negatively stained. The antigen particles covered in a dense and relatively continuous antibody coat, which bypasses the said particles, result in a "decoration". Such a feature indicates a positive reaction [49,50].

This technique can be used to detect very small viruses or to serotype virus [15].

Some types of bacteria such as Leptospira can be perfectly "decorated" by this technique (fig. 15).

**Figure 15.** *Leptospira interrogans* "decorated" by decoration technique. Bar: 1000 nm.

#### **2.3. Immunolabeling with colloidal gold particles by negative staining technique**

Immunocytochemical techniques are powerful tools for the localization of special antigens utilizing a specific marker 51].

Tracers or markers are exogenously administered substances that, when visualized in electron microscopy preparations, provide valuable information about cell compartments, junctional elements, and cells surfaces [24].

The antigen marking plays a fundamental role in the identification of difficult viruses to be visualized or those with a low titer [23].

Two types of markers have been used to detect virus and virus-antibody interaction in liquid preparations, ferritin and colloidal gold. Ferritin is a protein enclosing an iron core and colloidal gold is formed by the reduction of chloroauric acid with sodium citrate [9].

Ferritin conjugated to antibody combined with negative staining methods has been utilized to show the attachment of IgG on influenza virus and on hepatitis B core antigen. The method has also been used to detect rotavirus, adenovirus, and enterovirus [52,53,54,55,56].

The colloidal gold has been the most widely used marker in liquid preparations.

This gold-labeled antibody decoration technique antigen differs by the amount of gold label attached to the antigens [9].

The advantage of colloidal gold compared to other markers can be attributed to the particulate nature of the probe and the availability of different sizes and densities of the electron beam [1,57].

In this technique the antigen-antibody reaction is enhanced by determining the protein-A associated antigen through colloidal gold particles which, when linked to the antigen, allow the latter to be easily visualized through an electron microscope [11]. In our laboratory, we use a method originally developed by Knutton (1995) [51] to identify and characterize proteinaceous filamentous bacterial surface structures, such as fimbriae or pili, which function as adhesins and can also be applied to mark viral particles.

In our laboratory, we use a method originally developed by Knutton (1995) [51] to identify and characterize proteinaceous filamentous bacterial surface structures, such as fimbriae or pili, which function as adhesins and can also be applied to mark viral particles. In this method, the grids are treated with drops of viral or bacterial suspension, incubated with the primary antibody diluted to 1/80, post-incubated in protein A drops in

In this method, the grids are treated with drops of viral or bacterial suspension, incubated with the primary antibody diluted to 1/80, post-incubated in protein A drops in association with gold particles (secondary antibody) and negatively stained. The antigen-antibody interaction is strongly enhanced by the dense colloidal gold particles on viruses, indicating a positive reaction (fig. 16). association with gold particles (secondary antibody) and negatively stained. The antigenantibody interaction is strongly enhanced by the dense colloidal gold particles on viruses, indicating a positive reaction (fig. 16). The method also allows detection and identification of antigen structures induced by

The method also allows detection and identification of antigen structures induced by the virus and its localization in infected cells, serotype viral strains [58], and determines antigenic variants in isolated strains [59]. the virus and its localization in infected cells, serotype viral strains [58], and determines antigenic variants in isolated strains [59].

Fig. 16 – Bovine papillomavirus marked by the particles of colloidal gold (arrow). **Figure 16.** Bovine papillomavirus marked by the particles of colloidal gold (arrow). Bar 100 nm.

#### Bar 100 nm. **2.4. Immunolabeling in ultrathin sections technique**

antibody coat, which bypasses the said particles, result in a "decoration". Such a feature

Some types of bacteria such as Leptospira can be perfectly "decorated" by this technique

This technique can be used to detect very small viruses or to serotype virus [15].

**Figure 15.** *Leptospira interrogans* "decorated" by decoration technique. Bar: 1000 nm.

**2.3. Immunolabeling with colloidal gold particles by negative staining technique**

Immunocytochemical techniques are powerful tools for the localization of special antigens

Tracers or markers are exogenously administered substances that, when visualized in electron microscopy preparations, provide valuable information about cell compartments, junctional

The antigen marking plays a fundamental role in the identification of difficult viruses to be

Two types of markers have been used to detect virus and virus-antibody interaction in liquid preparations, ferritin and colloidal gold. Ferritin is a protein enclosing an iron core and

Ferritin conjugated to antibody combined with negative staining methods has been utilized to show the attachment of IgG on influenza virus and on hepatitis B core antigen. The method

This gold-labeled antibody decoration technique antigen differs by the amount of gold label

The advantage of colloidal gold compared to other markers can be attributed to the particulate nature of the probe and the availability of different sizes and densities of the

colloidal gold is formed by the reduction of chloroauric acid with sodium citrate [9].

has also been used to detect rotavirus, adenovirus, and enterovirus [52,53,54,55,56].

The colloidal gold has been the most widely used marker in liquid preparations.

indicates a positive reaction [49,50].

336 The Transmission Electron Microscope – Theory and Applications

utilizing a specific marker 51].

elements, and cells surfaces [24].

attached to the antigens [9].

electron beam [1,57].

visualized or those with a low titer [23].

(fig. 15).

The immunoelectron microscopy is one of the best methods for detecting and localizing proteins in cells and tissues and to detect virus or viral antigen on the surface of or within ultrathin sections of the cells [9,60,61].

**1.5 - Immunolabeling in Ultrathin Sections Technique**  The type of marker used depends on the type, location, and stability of the antigen under study [9].

The immunoelectron microscopy is one of the best methods for detecting and localizing proteins in cells and tissues and to detect virus or viral antigen on the surface of or within ultrathin sections of the cells [9,60,61]. Various types of markers have been used in ultrathin sections. The best known are the red ruthenium, lanthanum nitrate, horseradish peroxidase, ferritin, colloidal thorium dioxide (thorotrast) and colloidal gold [24].

The type of marker used depends on the type, location, and stability of the antigen

The ruthenium red has been applied to study ultrastructural aspects of retroviruses, such as details of the structure of peplomers, fusion or entry, assembly, release, and budding in infected cells [62,63,64].

Ruthenium red staining also promotes the ultrastructural visualization of glycoprotein layer surrounding the spore of *Bacillus anthracis* and *Bacillus subtilis* [65].

Applications of immunolabeling with ferritin allowed investigation of the complex antigenic interactions induced in cells by infection with herpesvirus [66] and by influenza and vaccinia viruses [67], as well as aspects of maturation and budding of African swine fever virus [68].

This marker facilitated electron microscopic study on the penetration of Newcastle disease virus into cells leading to the formation of polykaryocytes [69]. However, the immunolabeling antibody against the surface hemagglutinin spike on the viral surface of Newcastle disease virus showed that the size of the ferritin particles resulted in confusion with natural ferritin in the cell cytoplasm [57].

Electron microscopic observations of bacteria and Mycoplasma ultrastructure has been made by immunolabeling with ferritin [70,71].

Horseradish peroxidase-labeled antibody was used for localization of viral precursor antigens of reovirus [72], herpes simplex and vaccinia viruses [73], and rotavirus [74] and to study the organization of the endosome compartment of Semliki Forest virus [75].

There has been a recent report on the development of "Apex" (monomeric 28-kDa peroxidase), a genetically encodable EM tag that is active in all cellular compartments that withstands strong EM fixation to give excellent ultrastructural preservation [76].

The application of lanthanum in culture of *Scherichia coli*, allowed the study of the components of the cell envelope, the periplasm, and the energized inner membrane [77].

Ultrastructural aspects of the attachment and penetration of herpesvirus in BHK21 cells [78] and loci of viral ribonucleic acid synthesis of arbovirus [79] were described by immunolabeling with colloidal thorium dioxide.

Combined methods of ferritin tracing, lanthanum staining, and acid phosphatase localization was employed to demonstrate active process of retrovirus phagocytosis [80].

The association of tracers, ruthenium red with lanthanum nitrate was utilized as a marker for scrapie particles [81].

The gold particle labeling technique was first described by Faulk & Taylor in 1971 [82], when they were able to tag gold particles to anti-salmonella rabbit gamma globulins in one step in order to identify the location of the antigens of salmonella.

The use of primary antibodies conjugated with gold particles allows high resolution detection and localization of a multiplicity of antigens, both on and within the cells, revealing the distribution of molecular components at various structural levels [60,83].

This technique allows immunolocalization of viral proteins and their association with cell membranes of infected cells [84].

Two methods of immunolabeling were developed, the pre-embedding method and the postembedding method. The pre-embedding method primarily detects determinants exposed at the surface of infected cells such as virus receptors or envelope glycoproteins of budding viruses that are freely accessible to antibodies and reagents. The post-embedding labeling of thin sections allows access to determinants present in the different compartments of the cell and to internal viral structures since they become exposed at the surface of the section [85].

The major advantage offered by the post-embedding method is that each antigen molecule at the surface of the section should stand the same chance of being immunodetected, regardless of its cellular or sub-cellular localization of bacterial cells proteins [86].

In post-embedding methods of immunogold staining, the cells or tissues are fixed chemically or cryo-immobilized, dehyidrated, and embedded in epoxy or acrylic resins. The sections are then immunochemically stained with primary antibodies raised against antigens exposed on the surface of the sections. The primary antibodies are visualized by staining immunochemi‐ cally with secondary antibodies raised against the species and isotype of the primary anti‐ bodies, conjugated to colloidal gold particles. The ultrathin sections are stained with uranyl acetate and lead citrate [9,60,87].

This technique was used to label glycoproteins GP1 and GP4 of the bovine herpesvirus type 1 epitopes exposed at the surface of the cytoplasmic membrane or the envelope of the budding viral particles [85].

Immunolabeling using VP8-specific antiserum and colloidal gold labeled protein A as the electron-dense marker was applied to identify tegument protein VP8 of bovine herpesvi‐ rus-1 [88].

Salanueva et al. [89] showed that the two types of particles of the porcine transmissible gastroenteritis virus, large annular virus and small dense viruses are closely related, since both large and small particles reacted equally with polyclonal and monoclonal antibodies specific for TGEV proteins.

Another application of this technique showed that the collapse of the endoplasmic reticulum cisternae observed during African swine fever virus infection is dependent on viral envelope protein, J13Lp [90].

#### **2.5. Resin embedding technique**

The ruthenium red has been applied to study ultrastructural aspects of retroviruses, such as details of the structure of peplomers, fusion or entry, assembly, release, and budding in

Ruthenium red staining also promotes the ultrastructural visualization of glycoprotein layer

Applications of immunolabeling with ferritin allowed investigation of the complex antigenic interactions induced in cells by infection with herpesvirus [66] and by influenza and vaccinia viruses [67], as well as aspects of maturation and budding of African swine fever virus [68]. This marker facilitated electron microscopic study on the penetration of Newcastle disease virus into cells leading to the formation of polykaryocytes [69]. However, the immunolabeling antibody against the surface hemagglutinin spike on the viral surface of Newcastle disease virus showed that the size of the ferritin particles resulted in confusion with natural ferritin in

Electron microscopic observations of bacteria and Mycoplasma ultrastructure has been made

Horseradish peroxidase-labeled antibody was used for localization of viral precursor antigens of reovirus [72], herpes simplex and vaccinia viruses [73], and rotavirus [74] and to study the

There has been a recent report on the development of "Apex" (monomeric 28-kDa peroxidase), a genetically encodable EM tag that is active in all cellular compartments that withstands

The application of lanthanum in culture of *Scherichia coli*, allowed the study of the components

Ultrastructural aspects of the attachment and penetration of herpesvirus in BHK21 cells [78] and loci of viral ribonucleic acid synthesis of arbovirus [79] were described by immunolabeling

Combined methods of ferritin tracing, lanthanum staining, and acid phosphatase localization

The association of tracers, ruthenium red with lanthanum nitrate was utilized as a marker for

The gold particle labeling technique was first described by Faulk & Taylor in 1971 [82], when they were able to tag gold particles to anti-salmonella rabbit gamma globulins in one step in

The use of primary antibodies conjugated with gold particles allows high resolution detection and localization of a multiplicity of antigens, both on and within the cells, revealing the

This technique allows immunolocalization of viral proteins and their association with cell

organization of the endosome compartment of Semliki Forest virus [75].

strong EM fixation to give excellent ultrastructural preservation [76].

of the cell envelope, the periplasm, and the energized inner membrane [77].

was employed to demonstrate active process of retrovirus phagocytosis [80].

distribution of molecular components at various structural levels [60,83].

order to identify the location of the antigens of salmonella.

surrounding the spore of *Bacillus anthracis* and *Bacillus subtilis* [65].

infected cells [62,63,64].

338 The Transmission Electron Microscope – Theory and Applications

the cell cytoplasm [57].

by immunolabeling with ferritin [70,71].

with colloidal thorium dioxide.

membranes of infected cells [84].

scrapie particles [81].

The introduction of the epoxy resins for electron microscopy in the 1950s was a major step in the development of thin section electron microscopy for ultrastructural analysis [91].

The resin embedding technique consists of glutaraldehyde or paraformaldehyde fixation (2,5%), osmic acid post-fixation (1%), uranyl acetate en bloc staining, dehiydration with acetone, embedding in epoxy resin, thin sectioning and staining [92,93,94].

Ultrathin sections is an important tool to reveal fine details of the ultrastructure of all types of cells and tissues [95]. In an infectious process, it allows observing pathogenesis of infection and the identification of the agent [43].

The thin sectioning technique has the advantage of allowing the observation of virus cell interaction, which reveals the site of virus replication and maturation in the host cells, a pertinent information in the identification of unknown viruses [96]. Different methods for thin section electron microscopy have been developed for

Many ultrastructural aspects of this interaction can be observed in infections by several genera of poxviruses and papillomavirus. Amorphous, fibrillar, homogeneous, crystalline and A-B types inclusion bodies (fig. 17), nuclei with aspect dentate, membrane-bound vacuoles and mature and immature particles budding from cellular membranes, can be distinctly visualized in ultrathin sections after the resin inclusion of lesion or crust fragments in cases of ecthyma contagious, myxomatosis, swinepox, and avianpox, among others [32,33,34,97,98]. Different methods for thin section electron microscopy have been developed for detection of mycobacteria [100]. detection of mycobacteria [100].

Fig. 17 - Ultrathin section of the scabs fragments infected by Avian pox. **Figure 17.** Ultrathin section of the scabs fragments infected by Avian pox. Type A or Bollinger intracytoplasmic inclu‐ sion bodies, containing in its Interior mature particles (arrow). Bar: 800 nm. Interior mature particles (arrow). Bar: 800 nm.

Type A or Bollinger intracytoplasmic inclusion bodies, containing in its

Fig. 18 - herpesvirus particles in various stages of development **Figure 18.** herpesvirus particles in various stages of development (arrow) in cells inoculated with brain suspension of monkeys infected with herpesvirus type 1. Bar: 660 nm.

Fig. 18 - herpesvirus particles in various stages of development (arrow) in cells inoculated with brain suspension of monkeys infected with herpesvirus type 1. Bar: 660 nm. Several aspects of morphogenesis herpesvirus were seen in Vero cell monolayers inoculated with brain suspension of monkeys infected with herpesvirus type 1 (fig. 18).

with herpesvirus type 1. Bar: 660 nm.

**2.0 - DISCUSSION** 

**2.0 - DISCUSSION** 

(arrow) in cells inoculated with brain suspension of monkeys infected

 Electron microscopy is undoubtedly an indispensable tool in the diagnosis of animal infectious diseases and to investigate the structural analysis of cells and tissues at various

 Electron microscopy is undoubtedly an indispensable tool in the diagnosis of animal infectious diseases and to investigate the structural analysis of cells and tissues at various These ultrastructural details not only determine the infection, but also the course of the disease in the creations.

The embedding technique was used to describe a new *Rickettsia* species isolated from the tick *Amblyomma incisum* from the Southeast of Brazil, the *R. monteiroi*, showing characteristic Gramnegative morphology, with a cell wall and a cytoplasmic membrane separated from the cell wall by the periplasmic space [99].

Different methods for thin section electron microscopy have been developed for detection of mycobacteria [100].

### **3. Discussion**

The thin sectioning technique has the advantage of allowing the observation of virus cell interaction, which reveals the site of virus replication and maturation in the host cells, a

Many ultrastructural aspects of this interaction can be observed in infections by several genera of poxviruses and papillomavirus. Amorphous, fibrillar, homogeneous, crystalline and A-B types inclusion bodies (fig. 17), nuclei with aspect dentate, membrane-bound vacuoles and mature and immature particles budding from cellular membranes, can be distinctly visualized in ultrathin sections after the resin inclusion of lesion or crust fragments in cases of ecthyma

> Fig. 17 - Ultrathin section of the scabs fragments infected by Avian pox. Type A or Bollinger intracytoplasmic inclusion bodies, containing in its

> Fig. 17 - Ultrathin section of the scabs fragments infected by Avian pox. Type A or Bollinger intracytoplasmic inclusion bodies, containing in its

Fig. 18 - herpesvirus particles in various stages of development (arrow) in cells inoculated with brain suspension of monkeys infected

Fig. 18 - herpesvirus particles in various stages of development (arrow) in cells inoculated with brain suspension of monkeys infected

**Figure 18.** herpesvirus particles in various stages of development (arrow) in cells inoculated with brain suspension of

Several aspects of morphogenesis herpesvirus were seen in Vero cell monolayers inoculated

 Electron microscopy is undoubtedly an indispensable tool in the diagnosis of animal infectious diseases and to investigate the structural analysis of cells and tissues at various

 Electron microscopy is undoubtedly an indispensable tool in the diagnosis of animal infectious diseases and to investigate the structural analysis of cells and tissues at various

with herpesvirus type 1. Bar: 660 nm.

with herpesvirus type 1. Bar: 660 nm.

with brain suspension of monkeys infected with herpesvirus type 1 (fig. 18).

**2.0 - DISCUSSION** 

**2.0 - DISCUSSION** 

monkeys infected with herpesvirus type 1. Bar: 660 nm.

Interior mature particles (arrow). Bar: 800 nm.

Interior mature particles (arrow). Bar: 800 nm.

**Figure 17.** Ultrathin section of the scabs fragments infected by Avian pox. Type A or Bollinger intracytoplasmic inclu‐

Different methods for thin section electron microscopy have been developed for

Different methods for thin section electron microscopy have been developed for

contagious, myxomatosis, swinepox, and avianpox, among others [32,33,34,97,98].

pertinent information in the identification of unknown viruses [96].

340 The Transmission Electron Microscope – Theory and Applications

detection of mycobacteria [100].

detection of mycobacteria [100].

sion bodies, containing in its Interior mature particles (arrow). Bar: 800 nm.

Electron microscopy is undoubtedly an indispensable tool in the diagnosis of animal infectious diseases and to investigate the structural analysis of cells and tissues at various levels of resolution [1, 101].

The negative staining is a traditional technique that allows a quick, efficient, simple and conclusive diagnosis. The "open view" of the direct electron microscopy can visualize all agents on the specimen grid, also unknown microbes as well as unsuspected ones [6].

No simple method for an unequivocal and rapid diagnosis of infectious disease is available [19].

The success of the diagnosis depends on the quality of the sample taken, preparation and experience of the ultramicroscopist [102].

A positive detection requires of 105 particles per ml in the diagnostic suspension [4,6].

In the feces, however, the viral particles are present in high concentrations and are easily visualized [35].

One of the main applications of negative staining is the investigation of outbreaks of viral gastroenteritis [11] that cause high mortality in the creations.

This technique has also been used to detect the presence of enteric viruses such as poult enteritis complex (PEC), a disease economically important in poultry, characterized by enteritis, diarrhea, poor weight, and high mortality [103].

The technique also allows the identification of multiple agents in a same sample [19].

Herpesvirus, poxvirus, and papillomavirus particles are also found in large numbers in crusting, blistering, vesicular fluid or epithelium [4,6].

Some viruses may have low viral titer; however, this problem can be circumvented by applying the techniques of immunoelectron microscopy and immunolabeling with colloidal gold.

The electron microscopy has been utilized as a front-line method in emergency infectious diseases and/or in suspect cases of bioterrorism [19, 104].

The sudden appearance of vesicular lesions or respiratory illness in farm animals may be evidence of an emerging disease, a possible zoonosis or an agriterrorist act [19].

Exotic infections in several animal species have also been identified by electron microscopy [20]. Coronavirus particles were detected in ferrets with clinical status of diarrhea [105].

Several outbreaks of viral diseases were detected in wild animals using electron microscopy techniques. An enteric coronavirus was detected in capybaras [106] and the presence of poxvirus and paramyxovirus was confirmed in wild birds [32, 98].

Electron microscopy plays a fundamental role in assisting veterinary clinics and hospitals, Ecological Parks, Zoos, and breeding farms.

The immediate results of examinations of electron microscopy allows the rapid introduction of therapeutic, preventive and control measures in breeding sites and plan strategies for fighting infection, avoiding unnecessary loss of animals and economic damages.

The gain in time is an important factor in the control of infections [1,107].

In the event of new outbreaks caused by infectious agents, electron microscopy allows to assess the possibility of developing specific vaccine for the protection of the creations.

The introduction of electron microscopy techniques in diagnostic routine during outbreaks in farm animals allows to helping determine the risk areas at the site to be studied, collaborating in this way with the National Agribusiness, giving a base for health programs.

Another important use of electron microscopy is the identification of an unknown virus that has been isolated in tissue culture [20] or those viral agents particularly difficult to cultivate [3] and when alternative standard diagnostic methods fail to produce reasonable results [26].

The obtained electron micrographs are widely used to illustrate practically any text scientific papers, monographs, atlases and books in cell biology, anatomy, and pathology [95].

Future applications in this area include negative staining and cryo-negative staining, cryopreparation methods of vitreous sections (CEMOVIS) and digital images for transmission electron microscopy that can be processed by software programs may contribute to the improvement of veterinary diagnostic by electron microscopy [57,101,108,109].

The electron microscope is an expensive (costs about 600,000 euros), sophisticated, and extremely efficient equipment for rapid veterinary diagnosis, the annual cost spent on maintenance, should not be considered a disadvantage.

The main requirement of an Electron Microscopy Laboratory, however, is the need for highly trained operators with knowledge and skills to handle the equipment, prepare the sample, and realize the diagnosis accurately [6,12,26,43,110].

The preparation of a negative staining sample amounts to less than 0.5 euros, which is much less than the costs for alternative molecular tests [6].

The cost to the user is around \$ 16.50 per sample processed by this technique in our laboratory.

Considering the importance of electron microscopy in the diagnosis and research preparation is key to the new generation of ultramicroscopists with the appropriate technical skills [11].

### **Author details**

The sudden appearance of vesicular lesions or respiratory illness in farm animals may be

Exotic infections in several animal species have also been identified by electron microscopy [20]. Coronavirus particles were detected in ferrets with clinical status of diarrhea [105].

Several outbreaks of viral diseases were detected in wild animals using electron microscopy techniques. An enteric coronavirus was detected in capybaras [106] and the presence of

Electron microscopy plays a fundamental role in assisting veterinary clinics and hospitals,

The immediate results of examinations of electron microscopy allows the rapid introduction of therapeutic, preventive and control measures in breeding sites and plan strategies for

In the event of new outbreaks caused by infectious agents, electron microscopy allows to assess

The introduction of electron microscopy techniques in diagnostic routine during outbreaks in farm animals allows to helping determine the risk areas at the site to be studied, collaborating

Another important use of electron microscopy is the identification of an unknown virus that has been isolated in tissue culture [20] or those viral agents particularly difficult to cultivate [3] and when alternative standard diagnostic methods fail to produce reasonable results [26].

The obtained electron micrographs are widely used to illustrate practically any text scientific

Future applications in this area include negative staining and cryo-negative staining, cryopreparation methods of vitreous sections (CEMOVIS) and digital images for transmission electron microscopy that can be processed by software programs may contribute to the

The electron microscope is an expensive (costs about 600,000 euros), sophisticated, and extremely efficient equipment for rapid veterinary diagnosis, the annual cost spent on

The main requirement of an Electron Microscopy Laboratory, however, is the need for highly trained operators with knowledge and skills to handle the equipment, prepare the sample, and

The preparation of a negative staining sample amounts to less than 0.5 euros, which is much

The cost to the user is around \$ 16.50 per sample processed by this technique in our laboratory.

papers, monographs, atlases and books in cell biology, anatomy, and pathology [95].

improvement of veterinary diagnostic by electron microscopy [57,101,108,109].

maintenance, should not be considered a disadvantage.

realize the diagnosis accurately [6,12,26,43,110].

less than the costs for alternative molecular tests [6].

fighting infection, avoiding unnecessary loss of animals and economic damages.

the possibility of developing specific vaccine for the protection of the creations.

in this way with the National Agribusiness, giving a base for health programs.

The gain in time is an important factor in the control of infections [1,107].

evidence of an emerging disease, a possible zoonosis or an agriterrorist act [19].

poxvirus and paramyxovirus was confirmed in wild birds [32, 98].

Ecological Parks, Zoos, and breeding farms.

342 The Transmission Electron Microscope – Theory and Applications

M.H.B. Catroxo\* and A.M.C.R.P.F. Martins

\*Address all correspondence to: catroxo@biologico.sp.gov.br

Electron Microscopy Laboratory - Research and Development Center in Animal Health - Biological Institute of São Paulo, São Paulo,SP, Brazil

### **References**


[24] Bozzola JJ, Russel LD. Electron microscopy: principles and techniques for biologists. 2nd ed. Jones & Bartlett: Boston, 1999, 670p.

[10] Harris KM, Perry E, Bourne J, Feinberg M, Ostroff L, Hurlburt J. Uniform serial sec‐ tioning for transmission electron microscopy. J.Neurosci. 2006; 26:12101–12103. [11] Curry A, Appleton H, Dowsett B. Application of transmission electron microscopy to the clinical study of viral and bacterial infections: present and future. Micron. 2006;

[12] Schramlova J, Arientova, S Hulinska, D. The role of electron microscopy in the rapid

[13] Fenner F, Bachmann, P A. Gibbs, E. P. J.; Murphy, F.A.; Studdert, M. J. & White, D.

[14] Fields BN, Knipe DM, Howley PM, editors. Fields virology. 3rd ed. Lippincott-Raven:

[15] Hayat M A, Miller S E. Negative staining. Mc. Graw-Hill Publ. Company: New York,

[16] Kapikian AZ, Wyatt RG, Dolin R. Visualization by immune electron microscopy of a 27 nm particle associated with acute infectious non-bacterial gastroenteritis*.* J. Virol.

[17] Plummer FA, Hammond GW, Forward K, Sekla L, Thompson RN, Jones B, Kidd IM, Anderson MJ. An erythema infectiosum-like illness caused by human parvovirus. N.

[18] Hazelton PR, Aoki FY, Hammond GW, Coombs KM, Dawood M. Identification of a proposed novel agent of viral gastroenteritis. 18th Annual Meeting of the American Society for Virology, Amherst, MA. American Society for Microbiology: Washington,

[19] Hazelton PR, Gelderblom HR. Electron microscopy for rapid diagnosis of infectious

[20] Goldsmith CS, Miller SE. Modern uses of electron microscopy for detection of virus‐

[21] Goldsmith CS, Tatti KM, Ksiazek TG, Rollin PE, Comer JA, Lee WW, Rota PA, Bank‐ amp B, Bellini WJ, Zaki SR. Ultrastructural characterization of SARS coronavirus.

[22] Goldsmith CS, Ksiazek TG, Rollin PE, Comer JA, Nicholson WL, Teresa CT, Erdman P DD, Bellini WJ, Harcourt BH, Rota PA, Bhatnagar J, Bowen MD, Erickson BR, McMullan LK, Nichol ST, Shieh W-J, Paddock CD, Zaki SR. Cell culture and electron microscopy for identifying viruses in diseases of unknown cause. Emerg. Infect. Dis.

[23] Madeley C R. Electron microscopy and virus diagnosis. J. Clin.Pathol. 1997;

agents in emergent situations. Emerg. Infect. Dis. 2003; 9:294-303.

diagnosis of viral infections – review. Folia Microbiol. 2010; 55(1):88-101.

O. Virologia Veterinária. Acribia: Zaragoza, 1992, 691p.

37: 91–106.

344 The Transmission Electron Microscope – Theory and Applications

Philadelphia, 1996.

1972; 10:1075-1081.

1999, pp.10-14.

2013; 19:864-869.

50*:*454-456.

Engl. J. Med.1985; 313:74-79.

es. Clin. Microbiol. Rev. 2009; 22:552-563.

Emerg. Infect. Dis. 2004; 10:320-326.

1990, 253 p.


[54] Morgan C, Rifkind RA, Hsu KC, Holden M, Seegal BC, Rose HM. Electron micro‐ scopic localization of intracellular viral antigen by the use of ferritin-conjugated anti‐ body. Virol. 1961; 14(2):292-296.

[39] Gerna G, Passarini N, Sarasini A, Battaglia M. Characterization of human rotavirus strains by solid-phase immune electron microscopy. J. Infect. Dis.1985; 152:1143-1151.

[40] Gerna G, Sarasini A, Passarini N, Torsellini M, Parea M, Battaglia M. Comparative evaluation of a commercial enzyme-linked immunoassay and solid-phase immune electron microscopy for rotavirus in stool specimens. J. Clin. Microbiol. 1987;

[41] Humphrey CD, Cook EH Jr, Bradley DW. Identification of enterically transmitted vi‐ rus particles by solid phase immune electron microscopy. J. Virol. Meth. 1990;

[42] Lewis DC. Three serotypes of Norwalk-like virus demonstrated by solidphase im‐

[43] Field AM. Diagnostic virology using electron microscopic techniques. Advan. Virus

[44] Almeida JD, Waterson AP. Immune complexes in hepatitis. Lancet, 1969; 2:983-986.

[45] Anderson N, Doane FW. Specific identification of enteroviruses by immuno-electron microscopy using a serum-in-agar diffusion method. Can. J. Microbiol.

[46] Milne RG, Luisoni E. Rapid high-resolution immune electron microscopy of plant vi‐

[47] Lee TW, Megson B, Kurtz J B. Enterovirus typing by immune electron microscopy. J.

[48] Chenchev I, Kazachka D, Martinov S. Using the immunoelectron microscope meth‐ ods for diagnosing the herpesvirus infection in horses. Biotechnol. & Biotechnol.

[49] Milne RG. Immunoelectron-microscopy for virus identification. In: Electron micro‐ scopy of plant pathogens. Mendgen K, Lesemann (Eds.). Springer-Verlag: New York,

[50] Wright DM. Immunoelectron microscope techniques in plant virus diagnosis*.* Meth.

[51] Knutton S. Electron microscopical methods in adhesion. Methods Enzymol.1995;

[52] Almeida JD, Skelly J, Howard CR, Zuckerman S. The use of markers in immune elec‐

[53] Berthiaume L, Alain R, McLaughlin B, Payment P, Trepanier P. Rapid detection of human viruses by a simple indirect immune electron microscopy technique using

tron microscopy. J. Virol. Methods. 1981; 2(3):169-174.

ferritin-labelled antibodies. J. Virol. Methods. 1981; 2(6):367-373.

mune electron microscopy. J. Med. Virol.1990; 30:77-81.

25:1137-1139.

346 The Transmission Electron Microscope – Theory and Applications

29:177-188.

Res.1982; 27:2-69.

1973;19:585-589.

ruses. Virol.1975; 68:270-274.

Equip. 1997; 11(1-2):65-68.

Mol. Biol. 2005; 295:193-206.

1990.

253:145-158.

Med. Microbiol.1996; 44:151-153.


[81] Narang HK. Ruthenium red and lanthanum nitrate a possible tracer and negative stain for scrapie "particles"? Acta Neuropath. (Berl.). 1974; 29:37-43.

[67] Morgan C, Rifkind RA, Rose HM. The use of ferritin-conjugated antibodies in elec‐ tron microscopic studies of influenza and vaccinia viruses. Cold Spring Harb Symp.

[68] Breese SS Jr, Stone SS, De Boer CJ Hess WR. Electron microscopy of the interaction of African swine fever virus with ferritin-conjugated antibody. Virol.1967; 31(3):

[69] Meiselman N, Kohn A, Danon DJ. Electron microscopic study of penetration of New‐ castle disease virus into cells leading to formation of polykaryocytes. J. Cell. Sci. 1967;

[70] Anderson KL. Cationized ferritin as a stain for electron microscopic observation ob

[71] Neyrolles O, Brenner C, Prevost MC, Fontaine T, Montagnier L, Blanchard A. Identi‐ fication of two glycosylated components of *Mycoplasma penetrans*: a surface-exposed capsular polysaccharide and a glycolipid fraction. Microbiol. 1998; 144(5):1247-1255.

[72] Ubertini T, Wilkie BN, Noronha F. Use of horseradish peroxidase-labeled antibody for light and electron microscope localization of reovirus antigen. Appl. Microbiol.

[73] Shabo AL, Vetricciani JC, Kirschstein RL. Identification of herpes simplex and vacci‐ nia viruses in corneal cell cultures with immunoperoxidase: a light and electron mi‐

[74] Chasey D. Investigation of immunoperoxidase-labelled rotavirus in tissue culture by

[75] Marsh M, Griffiths G, Dean GE, Mellman I, Helenius A. Three-dimensional structure

[76] Martell D, Deerinck TJ. Sancak Y, Poulos TL, Mootha VK, Sosinsky GE, Ellisman MH, Ting AY. Engineered ascorbate peroxidase as a genetically encoded reporter for

[77] Bayer ME, Bayer MH. Lanthanide accumulation in the periplasmic space of Escheri‐

[78] Holmes IH, Watson DH. An electron microscope study of the attachment and pene‐

[79] Grimley PM, Berezesky IK, Friedman RM. Cytoplasmic structures associated with an arbovirus infection: loci of viral ribonucleic acid synthesis. J. Virol. 1968; 2(11):

[80] Lambertenghi G, Harven E, Sato T, Tennant JR. Electron microscope study of a BALB/c leukemia virus in cell culture systems. Cancer Res.1972; 32:1108-1116.

of endosomes in BHK-21 cells. Proc. Natl. Acad. Sci. 1986; 83*:*2899-2903.

bacterial ultrastructure. Biotech. Histochem. 1998; 73(5):278-288.

croscopic study. Invest. Ophthalmol. 1973; 12(11):839-847.

electron microscopy. Nat. Biotechnol. 2012; 30:1143-148.

tration of herpes virus in BHK21 cells. Virol. 1963; 21(1):112-123.

chia coli B. J. Bacteriol. 1991; 173(1):141-149.

light and electron microscopy. J. Gen. Virol.1980; 50(1):195-200.

Quant Biol.1962; 27:57-65.

348 The Transmission Electron Microscope – Theory and Applications

508-513.

2:71-76.

1971; 21(3):534-538.

1326-1338.


[97] Bersano JG, Catroxo MHB, Villalobos EMC, Leme MCM, Martins AMCRPF, Peixoto Z MP, Portugal MASC, Monteiro RM, Ogata RA, Curi NA. Varíola suína: Estudo so‐ bre a ocorrência de surtos nos Estados de São Paulo e Tocantins, Brasil. Arq. Inst. Bi‐

[98] Catroxo MHB, Martins AMCPF, Petrella S, Milanelo L, Aschar M, Souza F, Nastari BDB, Souza RB. Avian paramyxoviruses. Detection by transmission electron micro‐

[99] Pacheco RC, Moraes-Filho J, Marcili A, Richtzenhain LJ, Matias P J, Szabo MP, Ca‐ troxo MHB, Bouyer DH, Labruna MB. *Rickettsia monteiroi* sp. nov., Infecting the Tick *Amblyomma incisum* in Brazil. Appl. Environ. Microbiol. Appl. 2011; 77(15):5207-5211.

[100] Bleck CKE, Merz A, Gutierrez MG. Comparison of different methods for thin section EM analysis of *Mycobacterium smegmatis*. J. Microsc. 2010; 237(pt 1):23-38.

[101] Mielanczyk L, Matysiak N, Michalski M, Buldak R, Wojnicz R. Closer to the native state. Critical evaluation of cryo-techniques for Transmission Electron Microscopy:

preparation of biological samples. Folia Histochem. Cytobiol. 2014; 52(1):1-17. [102] Marshall JA, Catton MG. Specimen collection for electron microscopy. Emerg. Infect.

[103] Jindal N, Mor SK, Goyal SM. Enteric viruses in turkey enteritis. Virus Dis. 2014; 25(2):

[104] Madeley CR. Diagnosing smallpox in possible bioterrorist attack. Lancet. 2003;

[105] Gregori F, Catroxo MHB, Lopes VS, Ruiz VLA, Brandão PE. Occurrence of ferret en‐ teric coronavirus in Brazil (preliminary report). Braz. J. Vet. Res. Anim. Sci. 2010;

[106] Catroxo MHB, Miranda LB, Lavorenti A, Petrella S, Melo N A, Martins AMCPRF. Detection of coronavirus in capybaras (*Hydrochoeris hydrochaeris*) by transmission

[107] Gelderblom HR. Electron microscopy in diagnostic virology. BIOforum Int. 2001;

[108] Hurbain I, Sachset M. The future is cold: cryo-preparation methods for transmission

[109] Harris JR, De Carlo S. Negative staining and cryo-negative staining: applications in

[110] Vale FF, Correia AC, Matos B, Nunes JFM, Matos APA. Applications of transmission electron microscopy to virus detection and identification. Formatex. 2010;128-136.

electron microscopy of cells. Biol. Cell. 2011; 103:405-420.

biology and medicine. Methods Mol. Biol. 2014; 1117:215-258.

electron microscopy in São Paulo, Brazil. Int. J. Morphol. 2010; 28(2):549-555.

scopy techniques. Int. J. Morphol. 2012; 30(2):723-730.

ol. 2003; 70(3):269-278.

350 The Transmission Electron Microscope – Theory and Applications

Dis. 1999; 5:842.

173-185.

361*:*97-98.

47(2):156-158.

5:64-67.

## *Edited by Khan Maaz*

This book The Transmission Electron Microscope abundantly illustrates necessary insight and guidance of this powerful and versatile material characterization technique with complete figures and thorough explanations. The second edition of the book presents deep understanding of new techniques from introduction to advance levels, covering in-situ transmission electron microscopy, electron and focused ion beam microscopy, and biological diagnostic through TEM. The chapters cover all major aspects of transmission electron microscopy and their uses in material characterization with special emphasis on both the theoretical and experimental aspects of modern electron microscopy techniques. It is believed that this book will provide a solid foundation of electron microscopy to the students, scientists, and engineers working in the field of material science and condensed matter physics.

The Transmission Electron Microscope - Theory and Applications

The Transmission Electron

Microscope

Theory and Applications

*Edited by Khan Maaz*

Photo by ffly / DollarPhoto