PREPARING WET MOUNTS

Once the smear is dry, heat fix the microorganisms to the slide hy attaching a clothespin to a short side of the slide and passing the slide through an open flame. 

Transfer a small amount of the microorganism with an inoculating needle. Mix with the water on the slide and spread over a large area. 

Allow the smear to dry at room temperature. Do not expose the smear to heat from a flame. 

Frequently, a wine microbiologist does not have time to isolate microorganisms from a sample. Therefore, it is easier to examine a sample under a wet mount using a microscope equipped with phase-contrast. Normally, a minimal population of 10 cells per milliliter is necessary to be seen microscopically. As such, several microscopic fields should be examined and/or samples should be centrifuged to concentrate microorganisms prior to microscopic evaluation. 

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Prepare wet mounts as described above, and examine them. 

Prepare wet mounts using drops of the test fluids and observe under high power. 

Microscopic investigation for the presence of blas-tospores or pseudohyphae saline wet mount has a sensitivity of 40% to 50%, while a potassium hydroxide (KOH) preparation has a sensitivity of 50% to 70%.7... 

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... 

A solution of buffered methylene blue (pH 3.6) may be used as a vital stain for the examination of fresh specimens for protozoa. The wet mount is prepared as described above, with buffered methylene blue substituted as diluent and 5 to 10 min allowed for the dye to become incorporated in the organisms before examination. Organisms become overstained in 20 to 30 min. 

A funnel with a clamped rubber tube on the stem is placed in a ring stand. A circular mesh screen is placed across the funnel approximately one-third from the top, a portion of coarse fabric such as muslin is placed on the screen, and feces is added. Tap water at 37°C is added so that the water just touches the feces. Let the specimen stand 1 h, remove 2 ml of fluid from the stem, and centrifuge the sample at 300 x g for 3 min. Prepare a wet mount of sediment, and examine it for larvae. 

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. 

At 24 and 48 h, remove 1 drop of liquid from the lowest point of the overlay, and prepare a wet mount. 

Prepare four or five similar wet mounts and examine them as described above. To. each slide, immediately add 2 drops of 1% acetic acid solution and mix it well (microfilariae will be killed and straightened). Allow the slide to air dry. 

A motility test is usefrd because B. anthracis is a nomnotile bacterium. Two motihty tests available are the wet mount and motihty medium variety. In a wet-mount preparation, organisms with Brownian movement or no movement will support the presence of B. anthracis. The presence of B. anthracis in a motdity medium preparation would be a single line of growth along the original inoculum stab (CDC, ASM, APHL, 2002). 

Direct examination of tissue or body fluids believed to be infected can provide simple, rapid information to the clinician. Microscopic examination of wet-mount specimen preparations can provide valuable information regarding potential pathogens. Applications of this procedure with or without staining preparations include direct examination of sputum, bronchial aspirates, scrapings of mucosal lesions, and urinary sediment. The Gram stain is one of the... 

Prepare a wet mount slide for DIC microscopy. Melt 2% agarose in H O by microwaving, and place a 1-cm diameter drop in the center of a microscope slide. 

Further, microscopic observation is complicated by the fact that molds easily fragment when disturbed. Thus, preparation of wet mounts, as used in bacterial and yeast identification, is of minimal value. 

Mix, prepare a wet mount, and examine microscopically. Where quantification is necessary, a counting slide may be utilized. 

The coating of silicone rubber on the porous sublayer is done in the following way. The wet polyethersulfone membrane prepared above is dried in a desiccator for about a week. The membrane is then kept in an oven with forced air circulation at 60 C for 1 h. The dry polyethersulfone membrane so prepared is mounted at the bottom of a cylindrical test cell that can hold 50 ml of silicone in a hexane solution in the feed side chamber. The latter solution is prepared by dissolving 5 g of polydimethylsiloxane, 0.25 g of tetraethyl orthosilicate, and 0.25 g of dibutyltin dilaurate in 100 ml of hexane. The solution is left in the test cell for 1 h. The hexane solution is then poured out of the test cell, and the cell is kept upside down for 30 min so that any residual solution is drained and a thin silicone layer is formed after hexane evaporation. The silicone layer is subjected to cross-linking by keeping the cell in an oven at 60 C for I h. The coating can be repeated to form a multilayer. The silicone-coated membrane, together with the test cell, can be used for the permeation experiment immediately. 

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. 

Wolford s stain is used to monitor viable yeast by preparing a wet mount (Section 12.5) that is examined using a microscope (McDonald, 1963). Viable cells reject the stain and will appear clear, whereas dead cells absorb Wolford s stain and appear deep red or reddish-purple. 

Wet fluorescent magnetic particle inspection of the remaining journal fillets on this crankshaft revealed the presence of a number of secondary cracks in the same orientation and position as the failure initiation site. A metallographic specimen, including the matching halves of the fracture from the initiation site in the fillet of main journal, was cut, mounted, polished and etched in 2% nital, in preparation for examination with an optical microscope. The polished and etched specimen is shown in Figure 7.44. 

Except for wet specimens, samples of test or control articles, and specially prepared material (e.g., histochemical, electron microscopic, blood mounts, teratological preparation, and uteri from dominant lethal mutagenesis... 

The Portable Unit was designed to demonstrate the performance of the Shirco Infrared Incinerator in many thermal treatment applications. The construction details and process functions of the trailer-mounted incinerator are identical to a full-scale infrared incinerator. The system consists of a feed preparation system, an infrared primary chamber, a gas-fired secondary chamber, a wet gas scrubber, an exhaust system, heating element power centers (HEPC), and data acquisition and control systems. All equipment is enclosed within a 45-foot trailer. A schematic representation of the Portable Unit is shown in Figure 1. 

Samples for SAXS were prepared in sealed vials as reported elsewhere (2). Two Kapton (polyimide) films (7.5 pm thick) were buffed in the y-direction with an ethanol-wetted tissue paper. About 100 mg of sample were molten at 55°C to a liquid crystal and transferred to a tense, fixed Kapton film. Before cooling to below 35 °C, another Kapton film was put over the sample and both tense films were gently rubbed against each other at -0.5 mm/s in the y-direction for -10 s. Within three minutes the sandwiched sample was mounted in a hot copper block with a hole for the beam, and annealed at 58 1 °C for 30 min prior to the start of a 22-h exposure, during which annealing was continued. 

Though the previous methods allow for long term storage, fresh smear-slide preparation arguably retains the most fidelity to in vivo systems as the tissue is not processed in any way prior to observation. In smear-slide preparation, samples are harvest from the embryos at different stages. Each sample is then immediately smeared on a slide wetted with PBS (PH 7.4) buffer then mounted with a cover slip. This method is best observed by polarization microscopy, which can be conducted immediately following the sample smear preparations. 

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