Histology

Thank you for joining session 2, Histology. To get your mind really working, we have included the slides used as well as other tasks to gauge your understanding. Please complete the task in order.

This should take you about 1 hour but you can stop and come back to it later if you need to. You can submit your work via the Google Form at the bottom of the page.

TASK 1: Introduction to Session 2

Above is a 10-15 minute PowerPoint introduction to Session 2. Please download the slides, and in the top panel press 'Slide Show' and then 'From Beginning'. The slides will progress automatically, with a pause at the end of each section for you to review your thoughts and jot down any notes. Once you've viewed and listened to the slides, please move on to the next task.

What happens next?

Once a tissue specimen has been processed by a histology lab, and transferred onto a glass slide, it needs to be appropriately stained for microscopic evaluation. This is because unstained tissue lacks contrast: all of the fixed materials have a similar refractive index and a similar color. If you viewed an unstained tissue section under the microscope, everything would appear a uniform dull grey color.

The staining process therefore makes use of various dyes that stain particular cell components within tissues, so that you can distinguish different cell parts from each other.

Haematoxylin and Eosin

For routine examination, haematoxylin and eosin (H&E) is the stain of choice. This stain produces colors different tissue structures, which would otherwise be transparent, so that you can get a detailed view of the tissue.

As its name suggests, H&E stain makes use of a combination of two dyes – haematoxylin and eosin. The two stains were independently introduced in 1865 and 1875, respectively, by Böhmer and Fischer. In 1876, Wissowzky described their use in combination as a tissue staining method for staining different materials different colors.

Despite its simplicity, this stain has stood the test of time. Even now, over a century later, H&E remains the most frequently used tissue stain worldwide.

The Science Behind H&E

Staining does not produce color randomly; instead, the dyes exploit differences in the chemistry of the tissue to differentially color various components. Ionic bonding is the most important type of bonding that occurs in histologic staining techniques. It involves electrostatic attraction between ions of opposite charge, one of which is fixed in the tissue, and the second of which is in the dye.

Haematoxylin alone is not technically a dye, and will not directly stain tissues. It therefore needs to be used in combination with a “mordant” – a compound that helps it link to the tissue. The mordant used is typically a metal cation, such as aluminium. Haematoxylin in complex with aluminium salts is cationic and acts as a basic dye. It is positively charged and can react with negatively charged, basophilic cell components, such as nucleic acids in the nucleus. These stain blue as a result.

Eosin is anionic and acts as an acidic dye. It is negatively charged and can react with positively charged, acidophilic components in the tissue, such as amino groups in proteins in the cytoplasm. These stain pink as a result.

TASK 4: Setting up a Microscope

Have a go at virtually setting up a microscope. We will be using Labster for the virtual content.

• Assemble the light microscope and discover how the key components help to magnify an image up to 1,000 times.

• In this simulation, you will learn how to use a light microscope to analyze an intestine tissue sample.

• You will discover why biological samples need to be processed before they can be imaged and what the applications and limitations of light microscopy in biology are.

• Your mission is to decide whether the Mallory staining can be used in the experiment your colleagues in the lab are proposing.

TASK 6: What have you learned today?

Have a look through the following slides to remind yourself of all that you've learnt. I've also included some information on what research Brookes is doing that uses histology!