Apart from a few natural pigments such as melanin, the cells and other elements making up most specimens are colorless. In order to reveal structural detail using brightfield microscopy, some form of staining is required. With this method, cell nuclei are stained blue, and cytoplasm and many extra-cellular components in shades of pink.
However, sometimes additional information is required to provide a full differential diagnosis, and this requires furthermore specialized staining techniques. These methods can all be applied to paraffin sections, and in most cases, the slides produced are completely stable and can be kept for many years. After staining, the sections are covered with a glass coverslip and are then sent to a pathologist who will view them under a microscope to make an appropriate diagnosis and prepare a report.
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Please upgrade to a modern browser. To use the quotation basket you have to enable JavaScript in you Web browser. Apply for self-reported educational credits. Preparation options Because of the microscopy requirements, options for preparing specimens are limited to: Whole-mounts, where an entire organism or structure is small enough or thin enough to be placed directly onto a microscope slide e.
Sections, where specimens are supported in some way so that very thin slices can be cut from them, mounted on slides, and stained. Section preparation. Figure 1: A diagnostic section being prepared with a cryostat microtome. The section, which has been cut from snap-frozen tissue, is being picked up onto a warm slide where it will be immediately fixed and stained. For this reason, a stage micrometer should be used to calibrate the ocular micrometer.
This is a special microscope slide with a printed scale showing absolute distances. The ocular micrometer can be calibrated using this tool by comparing the distance between marks on the ocular micrometer, and marks on the stage micrometer, using various objectives.
In this section, we will describe the steps for basic operation of an optical microscope. These steps can be thought of as general for any of the specimen preparation techniques described here.
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Conduct Lifestyle Grants Academia. Quote Laboratory Techniques , Protocols , Science. Introduction In most types of microscopy, the most complicated and sensitive aspect of the analysis is the preparation of specimens. Staining Since microorganisms are mostly transparent, staining can be very helpful in visualizing them, including their internal structures. Smear preparation For most staining procedures, the first step involves the preparation of a specimen by making a smear.
There are a number of possible variations on the procedure used for smear preparation, but in general, the following steps can be used : Gather materials, and prepare the work area and any slides you plan to use. Clear off an area of the lab bench and lay down a paper towel in the work area. Prepare any slides you plan to use. Clean the slides using soap and water, and thoroughly rinse and dry them, taking care to either blow dry or wipe with a lint-free cloth to avoid forming water spots.
Draw circles using a wax pencil on the slides. These circles will define the target areas on the slide for the specimens, and also help to confine the specimens to these areas. It is also helpful to mark the slide with any description of the samples for example, lab notebook page numbers, the date, or your initials. Pre-clean an inoculation loop using a bunsen burner flame, and allow it to cool.
A sterile disposable tool can be used in place of an inoculation loop, for example, a swab. If the samples are from a solid medium an agar culture, for example , place drops of water in the wax circle using the inoculation loop.
For broth liquid cultures, this step is not needed. Gather a small portion of the sample onto the loop, and place it onto the slide in the wax circle area. If the sample is solid, gently mix it into the water drop using the transfer tool. If the sample is coming from a liquid broth, it is advisable to mix the broth using a vortex mixer or other device prior to sampling. Allow the sample to air dry. This will prevent splattering of the specimen in the next step. Heat the slide by passing it over a bunsen burner briefly times, specimen side up, or by placing it into a microincinerator.
If a loop was used, re-sterilize it by holding it in the bunsen burner flame until glowing red. A picture of a finished bacterial smear is shown in Figure 1. Types of Stains There are a number of staining techniques which have been developed over many years for the accurate imaging of specific organisms and cellular structures.
Simple Stain This is a technique that only uses a single stain, and is useful for simply visualizing cells, to determine characteristics like size, shape, and count. Negative Stain A negative stain works in the opposite way.
Nigrosin is a stain that can be used in this way. Gram Stain Gram staining is a more complex, multi-step technique, but is a powerful method for classifying bacteria. Therefore, the most complete guidelines for the staining procedure usually include advice to: Use relatively young cultures Prepare light, thin films If possible, repeat the same smearing and Gram staining procedure on two additional control samples- one that is known to be Gram-negative, the other known to be Gram-positive.
Acid-fast staining Acid-fast staining is a similar method to Gram staining, but adapted for organisms with significantly nonpolar and impenetrable cell walls. Flagella stain Some microorganisms have one or more small, thin appendages that are used to move the organism around in a liquid. An alternative method for visualizing such small structures is the use of electron microscopy.
Lab Procedures for Staining As discussed above, there are many staining procedures that can be used, all with their own chemistry and step-by-step procedure. Make a smear specimen as described in the previous section. When preparing an unknown specimen, prepare a known gram-positive and gram-negative specimen along side the unknown sample and carry through the staining procedures on those smears also. Place a few drops of crystal violet stain onto the smear, to completely cover it.
Let the slide sit for or 1 minute, then rinse with water. The rinsing can be done by dipping the slide into a beaker of water or multiple beakers if needed. Rinse the smear with the decolorizer ethanol or an acetone-ethanol mixture for roughly 5 seconds, then rinse with water.
Be very careful in this step to not overexpose the smear by rinsing with decolorizer for too long. This can have the effect of removing stain from the gram-positive organisms. This can be accomplished by holding the slide at an angle, and letting the decolorizer flow over the stain until the decolorizer flowing off the slide is clear.
At this point, quickly dip the slide into water. Expose the smear to safranin or fuchsin for 1 minute, then rinse with water. Allow the slide to dry completely in air, or blot it dry. A hanging drop mount can be made using the following procedure: Holding it by its edges, apply a small amount of petroleum jelly to the corners of a cover slip. Using an inoculation loop, transfer a small amount of a liquid sample in the middle of the coverslip. Place a well side, with the concave side down, over the coverslip, so that the sample droplet is centered in the well and the petroleum jelly forms a seal between the slide and cover slip.
Flip the assembly over so the coverslip is on top. Hemocytometers A hemocytometer is a special type of microscope slide that can be used for quantitative counting and sizing of cells. Ocular and Stage Micrometers An ocular micrometer is an optical element that is placed in the eyepiece of a microscope, which superimposes marks of known pitch onto the magnified image the pitch is the distance between marks.
Operation of the Optical Microscope In this section, we will describe the steps for basic operation of an optical microscope. Develop a plan and set of goals for the analysis, or simply write the questions you hope to answer. Moreover, staining allows scientists count the number of cells of a particular type within a certain biomass. Twenty or more different types of stains exist, and each one has its purpose.
The main purpose of staining is to highlight cells and parts of cells. Over 20 different types of stains exist, and the type of stain you use depends on what you are looking for. The choice of stain depends what you are looking for.
Not all stains are suitable for living cells, but those that are include Bismarck brown, toluene red, Nile blue and Nile red, and certain fluorescents used to highlight DNA. Some stains highlight spores, some detect lipids and proteins, and some change color in the presence of starches. The purpose of the examination determines the best type of stain to use.
It's an acidic fluorescent dye that turns red when it contacts red blood cells, cytoplasm and cell membranes. It's also used to test blood marrow.
In some cases, an investigator may use more than one stain. For example, hematoxylin is a stain that turns cell nuclei blue. When used in conjunction with eosin, which turns the other parts of the cell red or pink, it provides a stronger contrast and makes the nuclei easier to differentiate.
PAP smears and blood marrow samples are easier to examine when these two stains are used together.
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