If Triton X-100 is omitted there is a greater tendency for the solution to break down resulting in a fine blue precipitate forming in the Coplin jar and sometimes on the slides and tissue sections. Triton X-100 is added to the potassium ferrocyanide solution because it helps to stabilize the hydrochloric acid-potassium ferrocyanide solution. Therefore, we decided to use Pearl ’ s method routinely because it is more cost effective. The staining results on a variety of tissues, including plastic bone marrow sections, were almost identical in this study. Because most texts on histologic technic indicate the use of Pearl ’ s method we decided to do a comparative study of the two methods. The potassium ferrocyanide and hydrochloric acid solutions used in this method are much stronger that those used in Pearl ’ s method. Dilute hydrochloric acid liberates loosely bound ferric iron from protein.įor many years we used Gomori ’ s method for demonstrating ferric iron. The principle of Pearl ’ s Prussian blue reaction is that potassium ferrocyanide will form ferric ferrocyanide (Prussian blue) with reactive ferric salts in an acid solution. Treating a tissue section with 10% sulfuric acid overnight will usually remove this pigment. (attached to water molecules) through water chemistry, scale deposits which are in the water will precipitate (come out of solution). This yellow-brown pigment is insoluble in alkalis and water but is soluble in acid even after fixation. It may be present in tissues in certain pathologic conditions such as hemochromatosis. Hemosiderin is a breakdown product of hemoglobin and is thought to be composed of ferric iron and protein. Cool, filter, add a few grains of thymol as a preservative. Prepare just before use and discard after use.ĭissolve 0.1 gm nuclear fast red in 100 ml of 5% solution of aluminum sulfate Hydrochloric Acid-Potassium Ferrocyanide SolutionĢ% hydrochloric acid - 20.0 mlġ% potassium ferrocyanide - 20.0 ml Potassium ferrocyanide - 1.0 gmġ0% Triton X-100 - 2.0 ml Triton X-100 - 10.0 mlĭistilled water - 90.0 mlĪdd a few grains of thymol to prevent the growth of fungi. Hydrochloric acid, concentrated - 2.0 mlĭistilled water - 98.0 ml Allow the slide to dry at room temperature and examine under microscope.TECHNIQUE: Paraffin sections cut at 5 μ m.Without disturbing the slide, flood the distilled water and wash until the thinner parts of the film are pinkish red.A metallic sheen (or green ‘scum’) should appear on the slide if mixing is appropriate. Add approximately equal amount of buffered water (pH 6.5).The undiluted stain fixes and partially stains the smear. Cover the blood film with undiluted staining solution.Place the air-dried smear on the slide staining rack, smear side facing upwards.Prepare a film of blood or bone marrow on a microscopic slide and allow to air dry.Potassium dihydrogen phosphate, anhydrous = 0.663 gmĭisodium hydrogen phosphate, anhydrous = 0.256 gm The dye may be purchased as a powder which is then mixed to methanol or a ready-made solution may be obtained. Alternatively, prepare another Gram-stained smear. The neutral components of the cells are stained by both components of the dye, producing variable colors. If an excess of precipitated stain is observed, decolorize and restain slide. Methylene blue stains acidic cellular components such as nucleic acid and basophilic granules in varying shades of blue. Eosin stains the basic components such as hemoglobin and eosinophilic granules an orange to pink color. When diluted in buffered water, ionization occurs. Eosin Y is an acidic anionic dye and methylene blue is basic cationic dye. However, fixation helps to reduce water artefact that can occur on humid days or with aged stain. Following this treatment, sections can be restained to obtain clean images.

As the Wright stain is methanol based, it doesn’t require a fixation step prior to staining. The alkali treatment also extracts stain from the tissue itself.

Wright’s stain is a polychromatic stain consisting of a mixture of Eosin and Methylene blue. The stain distinguishes easily between the blood cells and hence became widely used for performing differential WBC counts and evaluate the morphology of blood cells. Wright’s stain is named for James Homer Wright, who devised the stain in 1902 based on a modification of Romanowsky stain. It is also used for staining bone marrow aspirates, urine samples and to demonstrate malarial parasites in blood smears. Wright’s stain is a type of Romanowsky stain, which is commonly used in hematology laboratory for the routine staining of peripheral blood smears.

Stain precipitate