This article incorporates, in modified form, material from the not-yet-published Illustrated Guide to Forensics Investigations: Uncover Evidence in Your Home, Lab, or Basement.
Historically, hair has been considered class evidence because a hair specimen could not be identified with certainty as having originated from a particular person. A forensic scientist who studied the morphology (shape, form, and structure) of a hair specimen could testify as to the gross physical characteristics of the hair (color, degree of curl, etc.), its internal and external structural characteristics, the likely somatic region from which the hair originated (scalp, beard, pubic, axillary, etc.), and–at least for scalp hair, and often for pubic hair, the probable race of the person from whom the hair originated. But all of those are class characteristics rather than individual characteristics, so the most the forensic scientist can state based on morphological examination is that a hair specimen is “consistent with” or “similar in all respect to” another specimen.
That changed with the advent of forensic DNA testing. Initially, DNA testing of hair focused on nuclear DNA, which can identify an individual unambiguously but which is present only in the follicle that surrounds the root of the hair (see Figure 6-4). For this reason, many people still believe, incorrectly, that DNA testing of hair requires a hair specimen that has been pulled out by the root and includes the follicle.
Figure 6-4. Major structural components of hair
In fact, DNA tests can be done on a hair specimen that includes only the shaft, including specimens that have been cut rather than pulled from the scalp. Although the shaft does not contain nuclear DNA, it does contain mitochondrial DNA (mtDNA). Unlike nuclear DNA, mtDNA is not unique to an individual because it is inherited directly from the individual’s mother rather than from both parents. Technically, that means that mtDNA can provide only class evidence rather than individual characteristic evidence, but an mtDNA match greatly reduces the size of the class in question. Despite this limitation, mtDNA tests are frequently done on hair specimens, because mtDNA testing is faster and cheaper than nuclear DNA testing.
Despite the availability of DNA testing, morphological classification of hair specimens remains an important part of the work of any forensics lab. Relative to morphological examination, DNA testing is very expensive and time consuming. Accordingly, morphological examination is used to do preliminary screening of hair specimens. Morphological examination can definitively rule out matches between some specimens, so only specimens for which a match cannot be ruled out morphologically need be submitted for DNA testing.
This preliminary morphological screening is done in two phases:
Macroscopic examination consists of observing specimens with the naked eye and at low magnification with a stereo microscope or magnifier to note such characteristics as color, length, and degree of curl. Ideally, this examination should be done by both reflected and transmitted light, although often only reflected light is used. The purpose of this examination is preliminary screening of a questioned specimen against known specimens. For example, if the questioned hair specimen is red, all knowns that are not red can immediately be eliminated from consideration.
Macroscopic examination may eliminate none, some, or all of the known specimens from consideration. The remaining specimens–those that are consistent macroscopically with the questioned specimen–can then be subjected to additional testing, including microscopic examination and possibly DNA testing.
Characteristics such as degree of curl are best observed with unmounted specimens at low magnification. After examining the unmounted specimens, you may if you wish dry mount the specimens, either simply by covering them with a coverslip or by securing them to the slide with two drops of nail polish or other adhesive, one on either side of the cover slip.
Unmounted or dry-mounted specimens are best for observing the external characteristics of the specimens. Later in the lab session, we’ll wet-mount specimens to view their internal structures.
Microscopic examination consists of observing specimens with a compound or comparison microscope at medium to high magnification (typically, 100X to 400X or more) to note fine structural details. For this phase, the specimen is wet-mounted using a mounting fluid with a refractive index close to 1.52, that of keratin, the protein which is the chief structural component of hair. Wet mounting reveals internal structure that is invisible with a dry mount. Additional knowns can often be eliminated based upon observed microscopic characteristics, leaving fewer specimens that require DNA analysis.
In this session, we’ll examine the gross morphology of human scalp hair under low magnification. We’ll then wet-mount the specimens and observe the fine morphology under medium to high magnification.
Required Equipment and Supplies
- goggles, gloves, and protective clothing
- stereo microscope, loupe, or magnifier
- compound microscope (100X and 400X, with ocular micrometer)
- microscope slides and coverslips (as required)
- disposable pipettes (as required)
- ruler (graduated in mm)
- forceps or tweezers
- mounting fluid (see Substitutions and Modifications)
- human scalp hair specimens (see Substitutions and Modifications)
All of the specialty lab equipment and chemicals needed for this and other lab sessions are available individually from the Maker Shed or other laboratory supplies vendors. Maker Shed also offers customized laboratory kits at special prices, and a wide selection of microscopes and microscope accessories.
Although none of the activities in this lab session present any significant risks, as a matter of good practice you should always wear splash goggles, gloves, and protective clothing when working in the lab, if only to avoid contaminating specimens. Obviously, you may need to work without goggles when using a microscope or magnifier to examine specimens.
Substitutions and Modifications
- For temporary wet mounts, you can use distilled water (RI ~ 1.33), although glycerol (RI ~ 1.47), castor oil (RI ~ 1.48), or clove oil (RI ~ 1.54) is a closer match for the refractive index of the keratin that makes up the hair. Permount, Canada balsam, or a similar mounting fluid matches the RI of keratin very closely, and can be used to make permanent mounts.
- The most convenient source of human scalp hair specimens is, of course, yourself. Obtain at least half a dozen specimens from different areas of your scalp and label each with the source (crown, side, front, back, etc.) At least one or two of those specimens should be plucked, so that you can observe the root structure. Other specimens can be cut. You can also obtain undifferentiated specimens from your hair brush.
This lab has three parts. In Part I, we’ll do a macroscopic examination of each specimen. In Part II, we’ll wet-mount the specimens. In Part III, we’ll do a microscopic examination of each wet-mounted specimen.
Part I – Macroscopic examination
- Examine each of your specimens, by eye and with the stereo microscope or other low-magnification optical aid, unmounted and, optionally, dry-mounted, by both reflected and transmitted light. (You can use your compound microscope at its lowest magnification, usually 20X or 40X.)
- Record the following information about each specimen in your lab notebook or Table 6-2: source, somatic region, color, uniformity of color from root to tip, length, degree of curl (straight, wavy, slightly curled, tightly curled, kinky, etc.), description of proximal (root) end (presence or absence of root, etc.); description of distal (tip) end (tapered, square cut, angle cut, split end, etc.)
Table 6-2. Morphology of Human Scalp Hair – observed macroscropic data
Part II – Wet-mount specimens
Wet-mounting is used to prepare specimens for microscopic examination. Using a mounting fluid with a refractive index close to that of the hair reveals the medulla, cortical fusi, pigment granules, and other internal structure of the hair. You can use distilled water, glycerol (glycerin), castor oil, or clove oil to make a temporary wet mount. For a permanent or semi-permanent wet mount, use Permount, Canada balsam, Melt Mount, or a similar mounting fluid.
The traditional method of wet-mounting a hair specimen in a loop is sometimes called the figure-8 mount or infinity mount because the mounted specimen often resembles the numeral eight or an infinity symbol. To wet-mount your specimens, proceed as follows:
- Label a microscope slide with a description of the specimen.
- Place a drop or two of mounting fluid in the center of the microscope slide.
- Using forceps, place the center of the specimen in the mounting fluid, and loop back the ends until they adhere to the mounting fluid, as shown in Figure 6-5.
- Carefully place a coverslip over the specimen and press it into place, making sure no air bubbles are trapped beneath it. If there are air bubbles that can’t be removed with gentle pressure on the coverslip, use a disposable pipette to add another small drop of mounting fluid at the edge of the coverslip, which should be drawn under the coverslip. Slight additional pressure should dislodge the air bubbles.
- Use the disposable pipette or the corner of a paper towel to remove any excess mounting fluid from around the coverslip. Be careful not to draw off so much mounting fluid that it is pulled from under the coverslip.
- Repeat steps 1 through 5 for each of the remaining specimens.
Figure 6-5. Barbara wet-mounting a hair specimen
Storing Wet Mounts
Always store wet-mounted specimens flat to prevent the coverslip from shifting. Specimens wet-mounted with distilled water remain usable for at least a few hours and perhaps overnight or longer. Specimens wet-mounted with glycerol, oils, or other less volatile mounting fluids may remain usable for several weeks or longer. Specimens wet-mounted with Canada balsam, Melt Mount, and similar permanent or semi-permanent mountants may remain usable indefinitely if they are stored properly.
Part III – Microscopic Examination
As useful as macroscopic examination is for preliminary screening, examining a specimen at low magnification by reflected light reveals little about the internal structural features of the specimen. In the microscopic examination, we’ll examine each specimen by transmitted light at medium (100X) and high (400X) magnification. We’ll observe, measure, and record the following microscopic characteristics about each specimen:
Note the shape of the hair shaft as round, oval, oblate, triangular, or other. Use the ocular micrometer to measure the minimum and maximum diameters of the main body of the hair shaft. (Human hair ranges from about 20 Âµm to 175 Âµm in diameter; the diameter may vary substantially between somatic regions and even within the same somatic region from hair to hair.) Note any special appearance of the shaft, such as whether it is split, undulated, invaginated, buckled, shouldered, or convoluted.
Although we’ve already recorded the color based on macroscopic examination, microscopic examination often reveals more detail. Note the color of the hair as colorless, blond, red, brown, black, or other, by both reflected and transmitted light. A sharp change in color near the proximal (root) end of the hair indicates that the hair is dyed, as does coloration that is relatively evenly distributed throughout the cortex. Note if the hair appears to have been bleached (overall yellowish tinge to the cortex), dyed, or otherwise treated.
Note the presence of pigment bodies (absent, few, abundant) and the size of the individual pigment bodies as small, medium, or large (this obviously requires comparison with other specimens or with a reference book). Also note the degree of aggregation (uniformly distributed, patches, streaks, clumps) and the size of these aggregates (small, medium, or large). Describe the density of the pigment bodies as opaque, heavy, medium, light, or sparse, and their distribution as uniform, peripheral (clustering near the cuticle), one-sided, or otherwise. Pigment bodies are also called pigment granules.
Note the absence or presence of the medulla. If present, note whether the medulla is continuous, broken, or fragmentary. Note its appearance as opaque, translucent, amorphous, or cellular. Note any other characteristics, such as a split, division, doubling, or twist. Use the ocular micrometer to measure the width of the medulla and use that to calculate the medullar index (MI).
The medullar index (MI) is simply the ratio of the diameters of the medulla and the shaft. For example, if medulla diameter is 15μm and shaft diameter 100 μm, record the MI as 0.15. MI is one easy way to discriminate human hair from animal hair. Human hair has an MI of 0.35 or lower (usually much lower) while animal hair has a high MI value.
Note the texture of the cortex as coarse, medium, or fine. If the cortex has an unusual appearance (e.g., cellular or striated), note that as well.
Cortical fusi are tiny air bubbles in the cortex, which appear black under the microscope. Cortical fusi, if present, are most common near the root of human hair, although they may be found anywhere within the cortex, and are typically larger than pigment granules. Note the size, shape, abundance, and distribution of cortical fusi.
Ovoid bodies are large (much larger than pigment bodies and larger than most cortical fusi) round or oval bodies with sharp, regular edges. Ovoid bodies are more commonly found in cattle, dog, and other non-human hair, but are sometimes found in human hair. Note the size, abundance, and distribution of any ovoid bodies visible in the specimen.
Note the absence or presence of the cuticle. If the cuticle is present, note the appearance of the outer cuticle margin (exterior surface of the hair) as flat, smooth, cracked, or serrated. If scale detail is visible at high magnification, note the appearance of the scales. Note the appearance of the inner cuticle margin (where the cuticle touches the cortex) as distinct, diffuse, or otherwise.
Note the presence or absence of a root on the proximal end. If a root is present, describe its appearance. Note the appearance of the distal end (tip) as tapered (natural or razor-cut), scissors- or clipper-cut (square or angled), split, frayed, abraded, crushed, broken, or otherwise.
Complete your microscopic examination of the hair specimen as follows:
- Place the wet-mounted specimen on the microscope stage and examine it at 100X, working from one end to the opposite end. (It doesn’t matter if you start from the proximal or distal end, but be consistent.) Record your observations in your lab notebook or Table 6-3.
- Increase magnification to 400X, and examine the fine internal structure of the hair, noting any details that were not visible at 100X.
- If you have the necessary equipment, shoot at least three images of the hair specimen: proximal tip, distal tip, and main body. Record the details, including the specimen number, by image filename for each image.
- Repeat steps 1 through 3 for each of your hair specimens.
Figure 6-6. A human scalp hair at 100X
Table 6-3. Morphology of Human Head Hair – observed microscopic data
|example||round, smooth shaft w/ uniform diameter ~ 45 Âµm Â± ~ 2Âµm; uniformly red by reflected light w/ evenly distributed small pigment bodies; amorphous medulla with frequent small breaks; MI ~ 0.2; fine homogeneous cortex; few, small, random cortical fusi near proximal end; no ovoid bodies visible; cuticle present, w/ smooth outer margin w/ no scale pattern visible and diffuse inner margin|
Q1: Which two somatic regions yield hairs that are most significant forensically? Why?
Q2: What are the advantages and limitations of nuclear DNA testing versus mitochondrial DNA testing?
Q3: What is the primary consideration in choosing a mounting fluid for a hair specimen, and why?
Q4: What two characteristics suggest that a hair specimen has been dyed?