The Role of Water Quality in Histology Procedures

Water is used in histology labs in several ways. Not only it is the key component of many of the reagents prepared in the laboratory, but is also used for tissue floatation baths, tissue processors and water baths.

Water purity is however sometimes taken for granted and its potential impact on experimental outcomes overlooked.

Water Contaminants

Tap water may contain several compounds apart from water molecules.

• Inorganic ions: chlorides, nitrates, sulfates, sodium, calcium, iron, etc.
• Organic molecules: humic acids, phenols, tannins, pesticide residues, etc.
• Particles and colloids
• Microorganisms and their by-products
• Dissolved gases

These compounds may interfere with the final slide quality as water is the key component in many of the reagents prepared in the histology laboratory.

Water Purification

pH and tap water composition may vary based on the location and season. However it is essential to obtain reliable and reproducible results. By using purified water, not only the risk of contaminant interference with the quality of the final slides is reduced, but experimental variability is also reduced.

Stain Sensitivity to Water Quality

Ions are removed from water via ion-exchange resins by the deionization process. Even though this process is very efficient at removing charged molecules, neutral organic molecules are not removed. Consequently deionized water may be free of ions but may contain some organic contaminants.

Distillation

A wide range of contaminants are removed by distillation. The water contained in a boiler is heated, the vapor produced is condensed and the purified water is collected in a receiving flask. This process may be repeated (double-distillation). During this process, it is expected that contaminants initially present (ions, organics, particles and bacteria) will not distill and will remain in the boiler. Figure 1 shows the schematic representation of purification technologies included in the Elix UV water purification system.

Figure 1. Schematic representation of the purification technologies included in the Elix^® UV water purification system

Advanced Water Purification Technologies

Distillation has for a long time been considered the "gold standard" of water purification, but today newer purification technologies have proven their efficacy and robustness.

These include the following:

• Reverse osmosis - a membrane-based technology that removes a large spectrum of contaminants.
• Electrodeionization - a self-regenerating process and is comprised of ion-exchange resins, semi-permeable membranes and an electrical current.
• Ultraviolet light at 254nm inactivates bacteria.

Elix^® UV water purification systems combine these three purification technologies and typically deliver water with low levels of organic contaminants (Total Organic Carbon, or TOC, < 30 ppb in-line), low levels of ions (resistivity > 5 MΩ-cm) and low levels of bacteria.

Practical Illustrations

A study was performed to assess the suitability of water purified with the Elix^® UV water purification system when performing commonly used staining procedures, and to investigate which common water contaminants might have an effect on these procedures.

Hematoxylin and Eosin (H&E) stain

The basic stain for all pathology tissue samples is the H&E stain. Hematoxylin stains cell nuclei in blue; eosin stains the cytoplasm and most connective tissue various shades of red, pink or orange.

The H&E staining was performed using a Leica Multistainer™ Workstation (Leica Microsystems, Bannockburn, IL). A regressive staining was performed. This stain procedure employs running tap water for the rinse steps performed before and after the hematoxylin, differentiation and bluing steps.

In the present study, all the rinsing steps, as well as the preparation of the bluing solution (Scott's tap water substitute, Surgipath Medical Industries, Richmond, IL), were performed entirely with either tap water or water purified using an Elix^® 5 UV system (EMD Millipore Corporation, Billerica, MA).

When substituting tap water for Elix^® purified water, the quality of the H&E staining was comparable to that of the staining done with tap water as shown in Figure 2. Using purified water brings down the risk of contaminant interference with the staining and provides consistency without compromising staining quality.

Figure 2. Photomicrographs of tonsil tissue stained with H&E. Rinsing steps and Scott's tap water substitute prepared with tap water (A) or Elix^® purified water (B). Pictures courtesy of E. Macrea and W. Lange.

Grocott's Methenamine Silver (GMS) stain

Silver stains are highly sensitive to water contaminants. The GMS stain is commonly used to demonstrate the presence of fungus, yeast and Pneumocystis. Most fungi are considerably large and their cell walls are rich in polysaccharides. The GMS stain uses chromic acid to oxidize the polysaccharides to formaldehydes, which can then react with silver ions.

For the experiment, sections of lung tissue infected with Pneumocystis carinii (Pneumocystis jiroveci) were mounted on Superfrost^® slides (Erie Scientific Co., Portsmouth, NH), and a commercial GMS kit was used (Grocott's Method for Fungi, Poly Scientific R&D Corp, Bay Shore, NY). Solutions of methenamine silver nitrate were prepared with Elix^® purified water, distilled water, or deionized water.

Water purified with the Elix^® system (C) gave satisfactory results: good staining of P. carinii and no background staining as shown in Figure 3. Distilled water (D) lightened the staining of the organisms, but did not contribute to background staining. Deionized water (E) appeared to intensify background staining as well as staining of P. carinii.

Additional methenamine silver nitrate solutions were prepared with Elix^® water and one of the following contaminants in order to assess their impact on staining: potassium chromium sulfate (1 mg/l, or ppm), cupric sulfate (1 ppm), nickel sulfate (1 ppm), ferrous sulfate (1 ppm), sodium hypochlorite (4 ppm), sodium triphosphate (100 ppm), sodium silicate (10 ppm), endotoxin (1000 EU/ml), humic acid (1 ppm).

The remainder of the procedure was the same for all slides. All slides were prepared in duplicate and were evaluated using identical lots of all applicable reagents for each set of slides.

Figure 3. Photomicrographs of GMS staining using solutions of methenamine silver nitrate prepared with Elix^® purified water (C), distilled water (D), or deionized water (E). Pictures courtesy of E. Macrea and W. Lange.

All the metal sulfates tested brought down the intensity of the P. carnii staining, when compared to solutions prepared with Elix^® purified water as shown in Figure 4. Copper and iron also led to the staining of elastic fibers.

Figure 4. Photomicrographs of GMS staining using solutions of methenamine silver nitrate prepared with Elix^® purified water and one of the following contaminants: potassium chromium sulfate (F), cupric sulfate (G), nickel sulfate (H), ferrous sulfate (I), sodium hypochlorite (J), sodium triphosphate (K), sodium silicate (L), endotoxin (M), humic acid (N). Pictures courtesy of E. Macrea and W. Lange.

Conclusion

Water purified with a combination of reverse osmosis, electrodeionization and ultraviolet light is suitable for a wide array of histology experiments, from commonly used and robust procedures, such as H&E staining, to more delicate ones, such as silver staining.

It can be used instead of deionized or distilled water and provides similar results while bringing down the risk of interferences due to water contaminants. Purified water produced by Elix^® systems can be used throughout the laboratory.

These water purification systems are easy to use and maintain and they consume considerably less energy and water than conventional distillation equipment.

Using a water purification system provides constant and reliable water quality, which is important at a time when an increasing number of histological procedures are automated and quality management systems are being implemented in histology laboratories.

This information has been sourced, reviewed and adapted from materials provided by EMD Millipore - Lab Water Business Unit.

For more information on this source, please visit EMD Millipore - Lab Water Business Unit.