Clinical and Morphological Diagnosis and Treatment of Sexually Transmitted Infections - Yakimova T. P. 2007

Methods for Obtaining Biomaterial for Cytological Examination
Methods for Staining Cytological Preparations

The most common Methods for staining cytologic preparations include staining with a 1% methylene blue solution, the Nocht, Pappenheim, and Romanowsky-Giemsa methods.

At the same time, preparations are quite frequently stained using Alekseev's, Leishman's, and other Procedures.

PAPPENHEIM'S STAINING METHOD

Pappenheim's staining method is generally considered the optimal approach in laboratory practice. This technique requires May-Grunwald fixative-stain, as well as stock 1% aqueous solutions of azure II and eosin.

To perform this staining, air-dried smears are fixed with May-Grunwald fixative-stain for 3 minutes. After 3 minutes, without discarding the stain, an equal volume of distilled Water is gently poured onto the slide. One minute later, the staining solution is drained off the slide, and without rinsing the smear, the working azure-eosin mixture is applied for 10-30 minutes, depending on the empirically determined exposure time for the specific batch of stain. Only after the final staining step are the preparations rinsed with distilled or tap water. The optimal exposure time for staining is selected experimentally for each new batch of stain.

To prepare the azure-eosin mixture as a working solution, add 1-4 drops of stock eosin solution and 1-4 drops of stock azure II solution to 1 ml of distilled water at pH 6.8. The volume of water is adjusted According to the number of smears to be examined. Each cytologic preparation requires approximately 3-4 ml of freshly prepared stain.

Ready-to-use azure-eosin mixtures are commercially known as Romanowsky-Giemsa stain. However, this stain is unstable during storage and tends to form a precipitate. Therefore, stock staining solutions are prepared separately in the laboratory. To prepare the azure II solution, 1 g of azure II is dissolved in 1 l of distilled water, which is boiled immediately before preparing the stain. The second solution is prepared from eosin. For this, 1 g of sodium or potassium eosin is dissolved in 1 liter of freshly prepared distilled water. Each stain is stored in a separate container, and these solutions remain stable indefinitely. Before staining cytologic preparations, working solutions are prepared from the stock 1% azure II and eosin solutions. Unlike the stock solutions, which have a long shelf life, the working solution can only be used on the day the staining mixture is prepared. The working solution is mixed in the following proportions: 40 ml of azure solution, 30 ml of eosin solution, and 70 ml of distilled water. The staining time for cytologic preparations ranges from 10 to 30 minutes, depending on the material and the titer of the stains. Ready-made stains are commercially available and commonly used in laboratory practice. The working dilution of the stains is prepared on the day the smears are stained, utilizing a 1:10 dilution of the dye in distilled water.

LEISHMAN'S STAINING METHOD

The air-dried smear is fixed in Leishman fixative for 3 minutes. The fixative is then drained, the smear on the Glass slide is rinsed with running tap water, and subsequently stained with Romanowsky-Giemsa stain for 10-20 minutes. The ready-to-use Romanowsky stain is diluted with water at a ratio of 15 drops of the stock staining solution per 10 ml of distilled water at pH 6.8. Using water with a different pH level can lead to poor, undesirable, or in some cases entirely unusable staining results for cytologic analysis.

Preparation of Leishman fixative: 2.5 g of dry Leishman stain powder is dissolved in 1 l of methyl alcohol and left in a tightly stoppered glass container for 3 days with occasional agitation. After 3 days, the solution is filtered and transferred to another vessel. The resulting solution is stable.

ALEKSEEV'S STAINING METHOD — RAPID STAINING PROTOCOL

The first rapid staining method was developed by N.G. Alekseev.

Apply 5-10 drops of ready-to-use Romanowsky-Giemsa staining solution directly onto the dried smears, distributing it evenly across the glass slide. After 30 seconds, add twice the volume of distilled water preheated to 50-60°C and leave for 3-5 minutes. Pour off the water, rinse the smears with running tap water, blot dry gently with filter paper, and examine under a Microscope.

The second rapid staining method by N.G. Alekseev.

Immerse the air-dried smear into Leishman fixative for 30 seconds. Without draining the fixative, pour a hot Romanowsky-Giemsa staining solution (preheated to 50-60°C) directly onto the preparation for 3 minutes. Rinse the stain off with ordinary running tap water. Dry the preparation carefully by blotting it with filter paper. The total staining time is 4 minutes.

To prepare the fixative, dissolve 4 g of dry Leishman stain in 1 l of methanol. To prepare the working stain, add 1.5 drops of Romanowsky-Giemsa stain to 1 ml of neutral distilled water at pH 6.8-7.0.

T.P. YAKIMOVA'S RAPID STAINING METHOD

Freshly prepared, mildly air-dried smears are dipped into 96% alcohol and immediately removed without any exposure time. With gentle agitation, the smears are rinsed several times in a large vessel containing an ample amount of tap water, after which Ehrlich's hematoxylin stain is applied to the microscope slide for 1-2-3 minutes. After 1-2-3 minutes, the stain is drained, the smear is rinsed with running water, blotted with filter paper, and examined microscopically. The staining duration of 1-2-3 minutes depends on the required examination time for the native preparation.

PREPARATION OF EHRLICH'S HEMATOXYLIN STAIN

In a wide-necked vessel, combine 2 g of hematoxylin, 100 ml of absolute alcohol, 100 ml of glycerol, 100 ml of distilled water, and 10 ml of glacial acetic acid; mix thoroughly and add an excess of pulverized ammonium alum (3 g). Cover the vessel with gauze and leave it exposed to light to allow the stain to mature—approximately 14 days—with occasional swirling. Once matured, the stain acquires a red-purple hue. Store the stain in a dark cabinet in a tightly stoppered container.

Class="center">EHRLICH'S Staining Procedure

GRAM STAINING

The foundational dye of this method is crystal violet, which certain Bacteria retain after alcohol decolorization, staining blue, while others fail to retain it and stain red following decolorization. In the first instance, bacteria stained blue are designated as Gram-positive, whereas In the second instance, bacteria stained red are termed Gram-negative. Additionally, gram-variable bacteria exist, which can stain either dark blue or pinkish-red.

GRAM STAINING PROCEDURE

The air-dried smear is fixed over a burner flame for a few seconds. Small pieces of filter paper are placed onto the fixed smear and saturated with a carbol crystal violet solution for 1–2 minutes. After 1–2 minutes, remove the filter paper with forceps and, without washing off the stain, flood the slide with Lugol's iodine solution for 1 minute. After 1 minute, pour off the Lugol's solution and apply a few drops of 96% alcohol to the slide for 0.5–1 minute, gently rocking the slide back and forth while the alcohol acts on the smear. Following these steps, rinse the smear thoroughly with running tap water and counterstain with an aqueous solution of Ziehl's fuchsin for 1–2 minutes. After staining, rinse the slide again with running tap water, air-dry it, and proceed to Cell/15.html">Microscopy. Strict adherence to the staining protocol is essential, as under-decolorization or over-decolorization of the smear with alcohol will lead to false staining results.

Microscopic examination of all smears is carried out under two optical settings: initially at low magnification using a x10 objective, followed by high magnification using an x90 or x100 oil immersion objective, combined with an x7 eyepiece.

METHYLENE BLUE STAINING

Staining reagent: a 1% aqueous solution of methylene blue. To prepare the solution, dissolve 1 g of methylene blue dye in 100 mL of distilled water and filter.

For staining, the air-dried smear is fixed in 96% ethanol for 3 minutes. Once fixed, the slide is allowed to dry, and a 1% methylene blue solution is applied for 1–10 minutes, depending on The Nature and amount of the sample. Most commonly, the preparation is stained for 1 minute. After staining, pour off the dye and rinse the slide under a stream of running tap water. Allow the preparation to dry, then examine it under low magnification using an x10 objective and x7 eyepiece, followed by microscopy with an x90 oil immersion objective.