Microscopic Examination of Stained Urinary Sediment
The Sternheimer-Malbin (S-M) stain is named for the two American workers who developed and pioneered its use. For convenience, it is frequently called the "S-M stain." The descriptions of the urinary structures that follow are made with reference to this stain only.
a. Stock Solution A. Stock solution A is made by dissolving 3.0 g of crystal violet (gentian violet) in 20.0 mL of 95 percent ethyl alcohol, adding 0.8 g of ammonium oxalate, and diluting to a final volume of 80.0 mL with distilled water. The reagent may be stored indefinitely.
b. Stock Solution B. Stock solution B is composed of 0.25 g of safranin dissolved in 10.0 mL of 95 percent ethyl alcohol and diluted to a final volume of 100.0 mL with distilled water. It also is good indefinitely.
c. Working Solution C. Working solution C, which must be replaced every 3 weeks, is made by mixing 3 parts of solution A and 95 parts of solution B. The solution is filtered and stored in a dropper bottle. It is important to filter solution C every 3 days during use so that particles of precipitated stain do not interfere with microscopic examination.
All basic ingredients of the S-M stain are also components of the gram stain for bacteria and can be found in any medical laboratory. It is basically a general stain for most organized structures. In addition, it is both convenient and economical since the chemicals are readily available. The preparation of the urinary sediment is the same as for a routine analysis of unstained sediment, and all the structures have the same basic recognizable features as unstained structures. The S-M staining procedure is not intended to replace any method for the identification of elements found in urinary sediment. Instead, the real value of the staining technique is its use as an aid in making a more rapid and accurate analysis of the urinary sediment.
A 10 mL to 15 mL sample of well-mixed urine is placed in a standard conical centrifuge tube. The urine is centrifuged at 1500 rpm for five minutes. The supernatant urine is discarded, and the sediment is resuspended by vigorous "finger-flicking." The staining procedure is quite simple. A drop of stain is added for each estimated drop of sediment. The sediment and stain are mixed by "finger-flicking" and then examined under the microscope, using a cover slip.
3-22. WHITE BLOOD CELLS
White blood cells are characterized by lobulated nuclei and relatively scant cytoplasm. The nuclei of granulocytes (granular white blood cells; neutrophils, eosinophils, and basophils) stain either pale blue or dark red to purple.
a. Pale-Staining Variety. The pale-staining variety is often larger than the dark- staining variety. Occasionally these granulocytes may be small with a glassy appearance and with an indistinct nucleus. However, they usually appear swollen and variable in shape. The nucleus is typically multilobulated or divided into four separated nuclei; it stains a very light blue. The cytoplasm of such cells contains granules, which show brownian movement if the specific gravity of the urine is not too low. As this brownian movement causes a constant variation of reflected light, the pale-staining white blood cells are often termed "glitter cells." Previously these cells were regarded as specific for pyelonephritis. However, it has been shown that glitter cells can be seen in almost any active urinary tract infection. Apparently pale-staining white blood cells are still living and are unable to bind dye molecules.
b. Dark-Staining Variety. Dark-staining white blood cells represent inert forms that have undergone autolysis and thus have binding sites available to take up the dye. These cells have dense, purple nuclei. Granules in the cytoplasm are either not evident or are characterized by a purple granularity. They are generally uniform in size and occur commonly in lower urinary tract infections with renal involvement.
3-23. EPITHELIAL CELLS
a. Renal Epithelial Cells. Renal epithelial cells are only slightly larger than white blood cells. They have a very thin rim of cytoplasm and a round nucleus with a dark band of chromatin at the periphery. The cytoplasm stains an orange-purple color.
b. Bladder Epithelial Cells (Caudate Cells). Bladder epithelial cells are frequently boat-shaped (navicular cells), and some appear to have tails. The cells with tails are often called caudate cells. These cells also have a round nucleus. However, they have more cytoplasm than the renal cell, and the cytoplasm is distinctively pale blue with occasional inclusions.
c. Squamous Epithelial Cells. Squamous epithelial cells have small, dark purple, pyknotic (thickened, shrunken) nuclei, and extensive pale purple cytoplasm. They frequently occur in sheets. It is not possible to differentiate squamous cells by their site of origin.
Waxy Casts. Waxy casts represent the ultimate stage in cellular degeneration. Their typical features are a homogeneous "ground glass" appearance, indentations, and angulation. The ends are sharp as if they were broken off. They stain pale pink or may not stain at all.
b. Hyaline Casts. Hyaline casts stain pale pink to light purple and have a homogeneous matrix. These casts are much more readily observed with the S-M stain than without any stain. However, at times these casts may not stain at all.
c. Granular Casts. The individual cells, which originally composed the coarsely granular cast have lost their integrity and demonstrate indistinguishable cell margins. The granules stain deep purple. The finely granular casts have fine granules, which stain a lighter purple and the hyaline matrix is light pink.
d. Red Blood Cell Casts. Red blood cell casts appear as hyaline casts with unstained or pale lavender red blood cells in a pale pink hyaline matrix.
3-25. MISCELLANEOUS STRUCTURES
Crystals. Crystals have the same general appearance when they are stained as when they are unstained. However, it should be noted that improper filtration can result in a precipitate of the stain which can be confused with various types of crystals. Therefore, it is crucial that the staining solution be filtered properly before using it.
b. Spermatozoa. Spermatozoa appear as usual except for the heads, which stain purple or blue. For this reason, they can be confused with other structures when the tail is not attached.
c. Trichomonas. They stain a pale blue with a purple nucleus.
d. Bacteria. Bacteria vary in color when stained due to their great diversity.
e. Identification of Double Refractile Fat Bodies. Refractile fat bodies often occur together with fatty casts. Therefore, one must be quite careful in identifying fat bodies. Staining and the use of polarized light facilitate the examination for fat bodies, particularly those containing cholesterol. Under polarized light, the double refractile bodies stand out against a dark background. They also manifest the distinctive Maltese cross pattern whereby the body appears to be divided into four quadrants. This technique also highlights hair and clothing particles and crystals; however, these structures do not exhibit the Maltese cross pattern. Likewise, neutral fat (triglyceride) does not exhibit the Maltese cross form.