Advanced Slide-Making

Introduction

How can microscopic organisms be preserved permanently? How are microscope slides prepared? Make slides of organisms from your environment.

Concepts

• Histology

• Clearing
• Fixing
• Mounting

Background

Histology is the study of the structure and chemical composition of animal and plant tissues as related to their function. Histology and microscopes are closely intertwined. Histologists use microscopes and staining techniques to investigate the nature of cellular arrangements and structures. Unfortunately, no biological specimen, dead or alive, can be studied under a microscope for very long before natural processes begin to destroy the tissue of the dead specimen or kill the organism. Fresh tissues tend to dry out and shrink, and bacteria begin breaking down the delicate structures of life almost immediately.

The art and science of histology involves trying to permanently preserve the qualities viewed in the living specimen. This task is often very difficult and sometimes even impossible. Experimental histology involves systematically treating specimens with incremental changes in procedure until a desired visual outcome is achieved. Specimens are typically put through a “standard” procedure in an attempt to preserve the specimen. Through the experimental process, the best preserving procedure for different specimens is found. The steps include some or all of the following:

1. Fixation/Clearing

In the process of fixation, water in the specimen’s tissue is replaced with a chemical that destroys all life, including decaying bacteria. The fixative (fixing fluid) serves a dual purpose. It enters all of the cells of the tissue and kills all of the bacteria. The substitution of one fluid (fixative) for another (cell cytoplasm) maintains the fluid pressure within the cells and thus keeps the tissue cells plump. This is a delicate part of the procedure—if fluid pressure is not maintained, the tissue will collapse and become a visual mess. For this reason, fixing is often done by a slow replacement process. A specimen is soaked in ever-increasing concentrations of the fixative as the cell’s fluids are replaced by the fixative fluid. It is also the reason why soft-bodied organisms are more difficult to “fix” than hard-bodied organisms.

Cellular proteins are not always soluble in fixing agents and may precipitate to form deposits on the cell walls giving the tissue a plaster-like appearance. In a large specimen, different organs can take fixatives at different rates and thus certain organs can be highlighted by leaving a specimen in a fixative for varying amounts of time.

Neither water nor most fixatives mix with the compounds used to permanently mount a specimen to a slide. Thus, the fixative must be “cleared” from the fixed specimen and the fixative replaced with clearing fluids prior to permanent mounting. So-called clearing fluids, which are compatible with mounting materials, are used to replace the fixative fluids in the cells.

2. Imbedding/Sectioning

If large specimens are to be cut into thin sections for viewing from various angles, the specimen is imbedded into wax or plastic. The wax/plastic block holds the specimen firmly in place as the block and specimen are sliced as one unit. The wax/plastic section then contains the slice of tissue in a thin transparent sheet. With the use of a microtome, slices can be made 1–150 microns (10^–6 m) thick for light microscopy and 30–60 nanometers (10^–9 m) thick for electron microscopy.

3. Staining/Mounting

Prior to mounting, specimens are often treated with various solutions of metallic salts (stains, dyes). These dyes tend to accumulate on certain structures and can greatly enhance the visual acuity of the final mounted specimen.

For light microscopy, excess stain and moisture are removed and a coverslip is permanently “glued” to the top of the mounted specimen. A mounting media having a refractive index close to that of glass is usually used. Mounting is usually done on a glass slide or a metal grid depending upon the ultimate viewing device. Mounting materials (glues) are used to hold the specimen firmly in place. Mounting fluids are usually soluble in clearing fluids and thus eventually impregnate all the cells of the specimen. When the fluid hardens in and around the cells, the mount becomes permanent.

Mounting specimens is not an exact science. Several similar specimens treated the same way are likely to end up looking slightly different. Patience and perseverance are important traits of a histologist. Permanently mounted, clear, instructive slides of interesting specimens, however, make all the effort worthwhile.

Materials

Safety Precautions

Acetone, xylenes, Canada balsam and isopropyl alcohol are fire risks and are slightly toxic by ingestion and inhalation. Fast green solution is moderately toxic by ingestion. Exercise extreme care when using razor blades. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron when preparing slides. Please consult current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Fast green and methylene blue solutions may be disposed of according to Flinn Suggested Disposal Method #26b. Isopropyl alcohol and acetone solutions may be disposed of according to Flinn Suggested Disposal Method #18a. Xylenes and Canada balsam solutions may be disposed of according to Flinn Suggested Disposal Method #18b.

Procedure

1. Secure specimens for mounting. Small ants or other hard-bodied insects work well for a first slide preparation.
2. Use forceps to carefully place the dead specimen into a microcentrifuge tube. If the specimen will not fit into the tube, use a smaller organism or take only part of the organism (e.g., head, leg).
3. Use a pipet to cover the specimen with 70% isopropyl alcohol. Let the specimen soak in the alcohol for at least one hour. (overnight if it exceeds the available class time). Larger insects may take as long as six hours to “fix” in alcohol. The volume of the alcohol should be at least 10 times the volume of the specimen.
4. Use a pipet to remove most of the 70% isopropyl alcohol from around the specimen. Be careful not to “suck” the specimen into the pipet!
5. Use a forceps to carefully remove the specimen from the tube, and place it on a dry paper towel. Let the specimen air dry for 1–2 minutes. While the specimen is drying, dry the inside of the microcentrifuge tube with a rolled-up paper towel.
6. Replace the specimen into the tube and use a pipet to cover it with 100% isopropyl alcohol. Let the specimen soak for two hours or more—overnight if it exceeds available laboratory time.
7. Remove the 100% isopropyl alcohol using a pipet. Remove the specimen using a forceps and let it air dry on a paper towel. Dry the tube with a paper towel.
8. Replace the specimen into the tube and cover it with a mixture of equal amounts of xylene and 100% isopropyl alcohol. Soak the specimen overnight.
9. Remove the solution using a pipet. Remove the specimen using a forceps and allow it to air dry while cleaning the microcentrifuge tube.
10. Replace the specimen into the tube and cover it with 100% xylene solution. Allow the specimen to soak in the xylene for at least one hour.
11. To make the slide, carefully remove the specimen from the tube and place it in the depression of a well slide. Center the specimen in the well.
12. Add a drop or two of Canada balsam solution on top of the specimen. Add a glass coverslip to the top of the specimen, being careful not to trap air bubbles under the coverslip. Let the Canada balsam resin set and harden before moving or viewing the slide.
13. After the balsam has hardened, examine the slide carefully using the low power of your microscope. How successful was the fixing and clearing process? What is visible in your specimen? Share your slide with others. Try additional slides/techniques as directed by your instructor.