Chapter 1

Question 1

Haematoxylin and Eosin (H&E)

Answer 1

The combination of the two dyes, haematoxylin (blue) and eosin (red), is the most useful stain for the examination of biologi- cal material; it is simple to perform, reliable, inexpensive and informative. Cell nuclei stain blue (depending on section thick- ness and the formulation of haematoxylin used), and most components of the cell cytoplasm stain pink/red. Most of the micrographs in this book are stained with H&E, particularly in the Practical Histology sections.

Question 2

Van Gieson Method

Answer 2

The simple van gieson method stains collagen pinkish-red and muscle yellow (see Fig. 10.21); it is commonly used in com- bination with a stain for elastic fibres. The elastic van gieson (EVg) stain is valuable for demonstrating and differentiating the common support cell fibres, particularly elastic fibres, which stain brown-black, and collagen fibres, which stain pinkish- red; muscle is stained yellow (see Fig. 10.21).

Question 3

Trichrome Methods

Answer 3

The trichrome methods employ a mixture of three dyes to stain different components in different colours. There are many trichrome methods, and they can be used to demonstrate general architecture, to emphasize support fibres, or to distinguish support fibres from muscle fibres. An important use of a trichrome method is the demonstration of the cellular, osteoid and mineralized components of bone in non-decalcified bone embedded in acrylic resin (see Figs 13.17a, 13.19b).

Question 4

Periodic Acid–Schiff (PAS) Method

Answer 4

The widely used PAs method has many applications, particularly in the demonstration of various carbohydrates, either alone (e.g. glycogen; Fig 1.5) or combined with other molecules, such as proteins (e.g. glycoproteins), which are stained magenta. it can therefore be used to delineate basement membranes (see Fig. 4.12a) and some neutral mucins secreted by various secretory epithelial cells. The mucous cells of the stomach are strongly PAs-positive.

Question 5

Alcian Blue Method

Answer 5

The Alcian blue dye method is used mainly to demonstrate acidic mucins secreted by some epithelial cells (see Fig. 11.44b), and can be combined with the PAs reaction to distinguish between acidic and neutral epithelial mucins. Through control of pH or other variables in the staining solution, the Alcian blue method can be used to demon- strate the extracellular glycosaminoglycan matrix (see Fig. 4.14d) of support cells.

Question 6

Transmission electron microscopy

Answer 6

In transmission electron microscopy, the electrons in a vacuum chamber pass through a very thin section of fixed tissue, some components of which absorb all the elec­ trons (‘electron dense’), whereas others allow the passage of all electrons through the other side of the tissue sec­ tions (‘electron lucent’). Some tissue elements allow only a percentage of electrons through, the remainder being absorbed by the tissue. The electrons that pass through strike a phosphorescent screen, allowing direct vision of the image, or a photographic plate, which renders the image as a permanent record in black, white and various shades of grey. The natural variations of electron density and electron lucency of the tissue com­ ponents are emphasized by the use of ‘stains’, such as osmium tetroxide, which has an affinity for lipid compo­ nents and renders them more electron dense, and other solutions of heavy metal salts.

Question 7

SEM

Answer 7

Scanning electron microscopy uses solid pieces of tissue rather than ultrathin tissue sections, and allows percep­ tion of three­dimensional views of the surface of cells, tissues and subcellular structures.
A small piece of fixed tissue is dried and coated in gold. An electron beam then scans the specimen and electrons produced from the surface are used to recon­ struct a fine three­dimensional representation of the surface (see Figs 7.2b, 7.14b, 11.11, 11.12b, 11.39d).
If living cells are frozen and then fractured, there is a tendency for the fractures to open cells along mem­ branes and distinct planes, which can then be studied using the electron microscope. This technique of ‘cryo­ fracture’ provides information about the surface features of cell membranes.

Question 8

light microscopy

Answer 8

In the light microscope, the illuminating system com­
prises a low­voltage electric lamp, with an adjustable condenser lens which focuses and concentrates the light into the plane of the object. After passing through the specimen, the light passes into the objective lens, the function of which is to collect the light rays and form a magnified intermediate image within the body tube above the objective lens. The projector lens in the micro­ scope eyepiece further magnifies the intermediate image and presents the retina of the eye with a magnified virtual image, which appears to the microscopist to be in the plane of the tissue specimen.