If you’re a medical student, chances are that you’ve looked at histology slides of different organs during your histology practical sessions. Have you ever wondered how these histology slides are made? Read on for a brief overview of the process of preparing a histology slide for microscopic evaluation.
1. Tissue fixation
Slide preparation begins with fixation of the tissue sample. Fixation is a crucial step in tissue preparation, and its main purpose is to prevent tissue autolysis and putrefaction. For best results, biological tissue samples should be transferred into fixative immediately after collection. Although there are many types of fixative, most specimens are fixed in 10% neutral buffered formalin. The optimum formalin-to-specimen volume ratio should be at least 10:1. This will allow most tissues to become adequately fixed within 24-48 hours. After fixation, specimens are trimmed using a scalpel to enable them to fit into an appropriately labelled tissue cassette.
2. Tissue processing
Processing of tissues requires the following steps:
- Dehydration is the first step, which involves immersing your specimen in increasing concentrations of alcohol to remove the water and formalin from the tissue.
- Clearing is the next step, in which an organic solvent such as xylene is used to remove the alcohol and allow infiltration with paraffin wax.
- Embedding is the final step, where specimens are infiltrated with the embedding agent – usually paraffin wax. The tissue is then embedded in paraffin wax, creating what is now referred to as the “block”. Once the block solidifies upon cooling, it provides a support matrix that allows for very thin sectioning.
The tissue block is now ready to be cut into sections that can be placed on a slide. Blocks are chilled on a refrigerated plate or ice tray for at least 10 minutes before sectioning. A microtome is an instrument used to slice extremely thin tissue sections. Most tissue blocks are cut at around 4- 5 µm. Once cut, the tissue sections are carefully transferred to a warm water bath to allow them to flatten out. Here they are allowed to float on the surface, and can then be scooped up onto a slide placed under the water level. Slides are then allowed to dry upright at 37oC for a few hours to gently melt the excess paraffin wax and fix the section to the slide.
Most cells are transparent, and appear almost colourless when unstained. Histochemical stains are therefore used to provide contrast to tissue sections, making tissue structures more visible. The standard histochemical stain used for routine purposes is the haematoxylin and eosin (H&E) stain. Following staining, a glass coverslip is mounted over the tissue specimen on the slide, using optical grade resin, to help protect the specimen.
And there you have it in a nutshell – how to prepare a stained histological section for microscopic assessment. Please spare a thought for all the hard work and skill that it takes to prepare slides for teaching histology to medical students. So do the right thing and handle your slides with care!
For those of you who want to know more, this website has more detailed information on slide preparation.