**2.6 Some important indices**

The microscopic hair examination also made use of certain indices that have a significant value in species characterization. The indices find valuable use in statistical analysis. Three indices are commonly used and they are defined as follows:


#### **2.7 Methods to study hair characteristics**

### *2.7.1 Preliminary examination*

Hair samples need to be initially examined for their color, texture, thickness, etc. before microscopic examination. The thickness can be measured in microns using a oculometer on a light microscope.

### *2.7.2 Examination of cuticle*

## *2.7.2.1 Light microscopy–based method*

The almost transparent and very thin layer of the cuticle cannot be appreciated under a transmitted light microscope by using a whole mount of hair. Only the cortex and medulla are visible in the whole mount. Therefore, to view the cuticular structure of hair, a "cast" of hair has to be made and viewed under a microscope. The suitable method is to prepare a cast of the hair. In the case of the hair "scale cast" method, a hair is placed on the surface of a suitable material such that the surface structure of the hair gets reproduced as a three-dimensional cast. This cast can be viewed under a light microscope to observe the cuticular structure of the hair. About 10–20% solution of gelatin in distilled water or 50% solution of polyvinyl acetate in distilled water is used for the preparation of scale casts [28]. A fine and uniform film of the casting media is made on a clean microscopic glass slide with the help of a glass rod or a flat brush in a single stroke along the length of the slide surface. The slide is then placed on a horizontal surface and hair samples are placed one by one on the slide with the help of tweezers. The casting media is allowed to dry for about 20–30 minutes and the hair are removed by plucking gently, leaving behind the three-dimensional cast of the hair surface, which can be used to study the scale patterns, margins and shapes. The cast is viewed under the microscope at a magnification of 100× to 400× depending upon the thickness of the hair. **Figure 1** depicts the scale pattern of the Indian Bison (*Bos gaurus*) under microscope 400× magnification.

#### *2.7.2.2 SEM-based method*

Scanning electron microscopy (SEM) is also a recommended method to study the cuticle of hair as it offers resolution much higher than light microscopy. In this method, the hair samples are initially coated with a thin film of gold or palladium under a very low pressure (10–6 Torr) to make the surface conducting. These hair samples coated with a very fine film of gold or palladium are then viewed under

**37**

**Figure 2.**

*represent the area where xylene has not infiltrated).*

*Forensic Analysis in Wildlife Crime Cases: Microscopy, DNA Profiling and Isotope Analysis*

an electron microscope, with the help of an electron beam. The method also has added advantage of studying the elemental profile of the hair if the SEM is coupled with energy dispersive X-ray analysis (SEM-EDXA) or wavelength dispersive X-ray

*The cuticle of Indian bison (Bos gaurus) under microscope 400× magnification.*

Medulla can be visualized in the whole mount of hair. However, it is not usually possible to observe the fine structural details of the medulla because of the air filled in vacuoles of the medulla. Hence, it appears as a dark central region when viewed under a microscope. For a proper appreciation of the fine structure of the medulla, the air vacuoles need to be infiltrated with a solvent like xylene. To achieve this, the hair samples are cut into small pieces (0.5 cm to 1.0 cm in length) with a razor blade and immersed in xylene (preferably overnight). These hairpieces after overnight treatment with xylene can be mounted directly on a glass slide in DPX or Canada balsam and viewed under a light microscope. **Figure 2** depicts the Medulla of Serow (*Capricornis sumatraensis*) under microscope 400× magnification. From **Figure 2**, the extent of infiltration by xylene can be clearly appreciated, as medulla

*Medulla of Serow (Capricornis sumatraensis) under microscope 400× magnification (note black globules* 

*DOI: http://dx.doi.org/10.5772/intechopen.98252*

analysis (SEM-WDXA).

**Figure 1.**

*2.7.3 Examination of medulla*

*Forensic Analysis in Wildlife Crime Cases: Microscopy, DNA Profiling and Isotope Analysis DOI: http://dx.doi.org/10.5772/intechopen.98252*

#### **Figure 1.**

*Forensic Analysis - Scientific and Medical Techniques and Evidence under the Microscope*

The microscopic hair examination also made use of certain indices that have a significant value in species characterization. The indices find valuable use in statistical analysis. Three indices are commonly used and they are defined as follows:

a.Scale Index: Ratio of the free proximo-distal length of the scale to the diameter

b.Scale Count Index: Number of scales per unit (1 mm) length of the hair shaft

Hair samples need to be initially examined for their color, texture, thickness, etc. before microscopic examination. The thickness can be measured in microns using a

The almost transparent and very thin layer of the cuticle cannot be appreciated under a transmitted light microscope by using a whole mount of hair. Only the cortex and medulla are visible in the whole mount. Therefore, to view the cuticular structure of hair, a "cast" of hair has to be made and viewed under a microscope. The suitable method is to prepare a cast of the hair. In the case of the hair "scale cast" method, a hair is placed on the surface of a suitable material such that the surface structure of the hair gets reproduced as a three-dimensional cast. This cast can be viewed under a light microscope to observe the cuticular structure of the hair. About 10–20% solution of gelatin in distilled water or 50% solution of polyvinyl acetate in distilled water is used for the preparation of scale casts [28]. A fine and uniform film of the casting media is made on a clean microscopic glass slide with the help of a glass rod or a flat brush in a single stroke along the length of the slide surface. The slide is then placed on a horizontal surface and hair samples are placed one by one on the slide with the help of tweezers. The casting media is allowed to dry for about 20–30 minutes and the hair are removed by plucking gently, leaving behind the three-dimensional cast of the hair surface, which can be used to study the scale patterns, margins and shapes. The cast is viewed under the microscope at a magnification of 100× to 400× depending upon the thickness of the hair. **Figure 1** depicts the scale pattern of the Indian Bison (*Bos gaurus*) under microscope 400× magnification.

Scanning electron microscopy (SEM) is also a recommended method to study the cuticle of hair as it offers resolution much higher than light microscopy. In this method, the hair samples are initially coated with a thin film of gold or palladium under a very low pressure (10–6 Torr) to make the surface conducting. These hair samples coated with a very fine film of gold or palladium are then viewed under

c.Medullary Index: Ratio of the medullary thickness to the hair thickness

**2.6 Some important indices**

of the hair shaft

(diameter)

*2.7.1 Preliminary examination*

*2.7.2 Examination of cuticle*

*2.7.2.2 SEM-based method*

oculometer on a light microscope.

*2.7.2.1 Light microscopy–based method*

**2.7 Methods to study hair characteristics**

**36**

*The cuticle of Indian bison (Bos gaurus) under microscope 400× magnification.*

an electron microscope, with the help of an electron beam. The method also has added advantage of studying the elemental profile of the hair if the SEM is coupled with energy dispersive X-ray analysis (SEM-EDXA) or wavelength dispersive X-ray analysis (SEM-WDXA).

#### *2.7.3 Examination of medulla*

Medulla can be visualized in the whole mount of hair. However, it is not usually possible to observe the fine structural details of the medulla because of the air filled in vacuoles of the medulla. Hence, it appears as a dark central region when viewed under a microscope. For a proper appreciation of the fine structure of the medulla, the air vacuoles need to be infiltrated with a solvent like xylene. To achieve this, the hair samples are cut into small pieces (0.5 cm to 1.0 cm in length) with a razor blade and immersed in xylene (preferably overnight). These hairpieces after overnight treatment with xylene can be mounted directly on a glass slide in DPX or Canada balsam and viewed under a light microscope. **Figure 2** depicts the Medulla of Serow (*Capricornis sumatraensis*) under microscope 400× magnification. From **Figure 2**, the extent of infiltration by xylene can be clearly appreciated, as medulla

#### **Figure 2.**

*Medulla of Serow (Capricornis sumatraensis) under microscope 400× magnification (note black globules represent the area where xylene has not infiltrated).*

in that part is clearly visible, whereas in the part where xylene has not infiltrated, the medulla shows dark globules. The dark globules can be cleared by increasing the time of keeping the hair cuttings under xylene.

The following four observations can be made:

