Water is ubiquitous in histology laboratories. It is used in water baths, floatation baths, and in most of the automated instruments present in the laboratory (stainers, tissue processors, etc.). Poor quality water may generate hard deposits or bacterial biofilms which may interfere with the proper functioning of these instruments. In addition; since water is the main component in many of the reagents prepared in the histology laboratory, water contaminants may interfere with the quality of the final slides.
Many different stains are used in histology, and each has unique properties. Unfortunately, there is no precise description in the literature of the specific contaminants that may interfere with each and every type of stain. Therefore, most histotechnologists rely on serendipity and experience when troubleshooting staining problems. In addition, histological procedures refer to the use of tap, deionized or distilled water, making it confusing as to which type of water is preferred.
The quality of the water may impact both the reagent quality and the staining process. Poor quality water may cause the precipitation of the stain (as in the case of silver stains), or may reduce its chemical stability. Staining reactions are based on a variety of basic chemical reactions, such as acid-base chemistry or oxido-reduction. Chemical contaminants that can interfere with these reactions may therefore alter the quality of the staining. The final staining may be lighter, or conversely, there may be a higher background or non-specific binding. The false color or specificity may also be obtained.
Using purified water can reliably reduce the risks of water contaminants interfering with the staining process, as well as ensure the proper functioning of stainers and tissue processors.
The following water contaminants may have effects on histology:
Chlorine, often present in tap water, can have a bleaching effect on various types of stains.
Ions, such as calcium and various metals, are known to interfere with a variety of stains (silver stain, etc.). For instance, an AgCl precipitate will form if the chloride level is too high in water.
Organics: if the water contains large amounts of organic molecules and other nutrients, bacteria and molds may grow in water baths and tissue floatation baths, inside stainer and tissue processor tubings, and in solutions, which may generate artifacts on slides.
Bacteria: the water used in water baths and to prepare solutions should be devoid of bacterial contamination. Bacteria and their by-products may adhere to the tissue sections and generate artifacts on slides.
Particles: the water used in water baths and to prepare solutions should be devoid of particles. Particulate material may adhere to the tissue sections and generate artifacts on slides. They may also form deposits inside instrument lines and alter their functioning.
Hardness and silica: water hardness and silica may form deposits inside the lines of stainers and tissue processors and alter their functioning. These water contaminants may also be deposited on tissue and slides.
pH: although pH is not stricto sensu a water contaminant, it can change due to the presence of different water contaminants. Some stains are highly sensitive to pH, such as alcian blue or trichrome. By reducing the levels of ions and organic acids present in water as much as possible, one can keep the pH of water in the vicinity of 7.
Examples illustrating the impact of water quality on histology
Hematoxylin and Eosin (H&E) Stain The Hematoxylin and Eosin (H&E) stain is the fundamental stain for all pathology tissue samples. In areas where tap water is acidic or fluctuates, it is difficult to reliably perform the bluing step using tap water. Tap water also may contain elements that can destain hematoxylin, such as iron (which acts as a mordant), chlorine (which bleaches the stain) and sulfur (which can influence the pH).
Photomicrographs of tonsil tissue stained with H&E, 10X. Rinsing steps and Scott’s tap water substitute prepared with (A) tap water or (B) Elix® purified water. Pictures courtesy of E. Macrea and W. Lange, Southwest Skin Pathology Services, Tucson, Arizona, U.S.A.
When substituting Elix® purified water for tap water, the quality of the H&E staining was comparable to that of the staining done with tap water. Using purified water minimizes the risk of tap water contaminant interference with the staining and provides consistency without compromising staining quality.
Special Stains Silver stains are well known for their sensitivity to water contaminants. Grocott’s Methenamine Silver (GMS) stain was used on sections of lung tissue infected with Pneumocystis carinii (Pneumocystis jiroveci).
(1) Various types of water were used to prepare the solutions of methenamine silver nitrate: Elix purified water, distilled water or deionized water (purified with ion-exchange resins).
Photomicrographs of lung tissue with Pneumocystis carinii, stained with GMS, 20X. GMS staining was done with solutions of methenamine silver nitrate prepared with (C) Elix® purified water, (D) distilled water, or (E) deionized water. Pictures courtesy of E. Macrea and W. Lange, Southwest Skin Pathology Services, Tucson, Arizona, U.S.A.
Water purified with the Elix® system (C) gave satisfactory results: good staining of P. carinii and no background staining. 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. Water purified with an Elix® system can therefore be substituted for the distilled or deionized water called for in the GMS stain procedure.
(2) Elix® purified water spiked with various types of common water contaminants was used to prepare the solutions of methenamine silver nitrate.
Photomicrographs of lung tissue with Pneumocystis carinii, stained with GMS, 20X. GMS staining was done with solutions of methenamine silver nitrate prepared with Elix® purified water and one of the following contaminants: potassium chromium sulfate (F), sodium silicate (G), endotoxin (H). Pictures courtesy of E. Macrea and W. Lange, Southwest Skin Pathology Services, Tucson, Arizona, U.S.A.
Potassium chromium sulfate severely reduced the intensity of the P. carnii staining, when compared to solutions prepared with Elix® purified water. This was also observed when other metals were added to the Elix® purified water. On the other hand, sodium silicate and endotoxins suppressed the P. carnii staining and led to excessive background staining. This study confirmed that GMS staining, which is known to be very sensitive to water quality, can be affected by a variety of water contaminants.
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 in place of distilled or deionized water and gives similar results, while reducing the risk of interferences due to water contaminants and day-to-day variations in water quality.