PREPARING SMEARS

In order to see a yeast or bacterium under a microscope, sometimes it is necessary apply a stain such as methylene blue (yeast) or to perform a Gram stain (bacteria). Before staining, a slide smear of the microorganism must be prepared. Whereas dye can be added to wet mounts (Section 12.5), smears rely on preparations that are dried. 

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Prepare smears, heat-fix and stain by any conventional method (e.g. Wright s stain), and then examine under oil immersion. 

Poor preparation of the substrate can result in loss of adhesion, pitting, roughness, lower corrosion resistance, smears, and stains. Because electroplating takes place at the exact molecular surface of a work, it is important that the substrate surface be absolutely clean and receptive to the plating. In the effort to get the substrate into this condition, several separate steps may be required, and it is in these cleaning steps that most of the problems associated with plating arise. 

Klatsch-praparat, n. (Micros.) a smear preparation made by pressing the material between two cover glasses and sliding them apart, -rose, /. corn poppy, klauben, v.t. pick, sort (cap., by hand). 

In the first LDMS-based detection of malaria in human subjects (unpublished), lOOpl P. falciparum or P. v/vax-infected blood samples, grouped into three different parasitemia ranges—low (10-150 parasites/pl), mid (2 x 103 parasites/pl), and high (25 x 103-60 x 103 parasites/pl)—have been examined using both sample preparation protocols. Parasitemia levels in these samples were previously determined independently for each sample by optical microscopy examination of blood smears. The LDMS data clearly indicate that... 

Direct wet mounts are prepared by placing a small drop of 0.85% saline toward one end of a glass slide (2 by 3 in. [ca. 5 by 7.5 cm]) and a small drop of appropriate iodine solution (see below) toward the other end. With an applicator stick, a small portion of specimen (1 to 2 mg) is thoroughly mixed in each diluent, and a no. 1 cover slip (22 mm) is added. The density of fecal material should be such that newspaper print can be read with difficulty through the smear. The material should not overflow the edges of the cover slip. Grit or debris may prevent the cover slip from seating and may be... 

Permanent stains of fecal smears are most needed for the detection and identification of protozoan trophozoites, but they are also used for the identification of cysts. Wet mounts of fresh feces, even with stains such as methylene blue, are not as sensitive for trophozoites and therefore do not substitute for permanent stains. It is sometimes difficult to identify cysts which are detected in wet mounts thus, for each specimen, regardless of consistency, it may be worthwhile to fix a portion in PVA fixative or to prepare two fecal films fixed in Schaudinn fixative so that permanent stains can be performed if needed. Permanent stains also provide a permanent record and are easily referred to consultants if there are questions on identification. 

Preparation of smears, (i) Unpreserved specimens with Schaudinn fixative. 

To prepare thin, uniform smears, place a drop of saline on a glass slide (1 by 3 in. [ca. 2.5 by 7.5 cm]). With an applicator stick, transfer a small, representative portion of the specimen to the drop of saline, and mix the two. Spread the solution into a film by rolling the applicator stick along the surface. Remove any lumps. 

The following procedure is useful for staining Nocardia species as well as Cryptosporidium species. This procedure may be used on fresh or Formalinfixed material or on material from concentration procedures. If the specimen is liquid, centrifuge it, and use the sediment to prepare a smear. 

Permanent stains can be prepared by the fixation of sediment in PVA fixative, with the subsequent preparation of smears and staining. 

Measurements show some variation depending upon the staining solution used and the method of application. In dried and fixed smears, the cell wall and slime layer do not stain with weakly staining dyes such as methylene blue but do stain with the intensely staining pararosaniline, new fuchsin, crystal violet, and methyl violet. The great majority of bacteria have been measured in fixed and stained preparations. In some instances dried, negatively stained smears have been used. Therefore, the method employed should be specified when measurements of bacteria are reported otherwise the results will be of doubtful v alue. 

Robinow prepared wet smears of Escherichia coli. Slides were fixed in osmic acid vapor, dried, and immersed in normal HC1 for about 9 min. at S3 to SS°C., then washed and stained in 1 20 Giemsa solution for 10 to 60 min., depending on the staining properties of the specimen. 

The goal of our investigations was to characterise the morphology of the sample, and to determine the size and location of the PTFE and silicone oil phases by different methods [46,47], For phase characterization using Raman microscopy, no special sample preparation was necessary. For FTIR imaging, microtomed sections (5 pm in thickness) had to be prepared by cutting the sample with a diamond knife at — 80°C ("cryo-microtomy") to prevent smearing and to obtain flat surfaces. 

Careful attention should be paid to sample preparation, however. The materials investigated may be changed by the preparation procedure (e.g., smearing during microtomy of soft materials at room temperature is avoided when using cryo-microtomy) or during the measurement (radiation damage, contact with ATR crystal, etc.). 

Shidham VB, Chang C-C, Rao RN, et al. Immunostaining of cytology smears a comparative study to identify the most suitable method of smear preparation and fixation with reference to commonly used immunomarkers. Diagn. Cytopathol. 2003 29 217-221. 

Liu J, Farhood A. Immunostaining for thyroid transcription factor-1 on fine-needle aspiration specimens of lung tumors a comparison of direct smears and cell block preparations. Cancer 2004 102 109-114. 

Shidham VB, Lindholm PF, Kajdacsy-Balla A, et al. Methods of cytologic smear preparation and fixation. Effect on the immunoreactivity of commonly used anti-cytokeratin antibody AE1/AE3. Acta Cytol. 2000 44 1015-1022. 

Even though cell block is the first choice for ICC, there is not always sufficient cell sample for cell block preparation. Therefore, it is necessary to develop techniques that allow multiple immunostaining on limited smears. 

Brown G, Tao L. Restoration of broken cytology slides and creation of multiple slides from a single smear preparation. Acta Cytol. 1992 36 259-268. 

Jimenez-Joseph D, Gangi M. Application of diatex compound in cytology use in preparing multiple slides from a single routine smear. Acta Cytol. 1986 30 446—447. 

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