In many labs, the bulk of processing is carried out overnight. At present, there is considerable pressure on laboratories to use processors capable of rapid processing in an effort to improve workflow and reduce turnaround times. After processing, the specimens are placed in an embedding centre where they are removed from their cassettes and placed in wax-filled molds.
At this stage, specimens are carefully orientated because this will determine the plane through which the section will be cut and ultimately may decide whether an abnormal area will be visible under the microscope. The cassette in which the tissue has been processed carries the specimen identification details, and it is now placed on top of the mold and is attached by adding further wax.
The cassette, now filled with wax and forming part of the block, provides a stable base for clamping in the microtome. The block containing the specimen is now ready for section cutting. This makes handling easier. After thorough drying, they are ready for staining. 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.
The content, including webinars, training presentations and related materials is intended to provide general information regarding particular subjects of interest to health care professionals and is not intended to be, and should not be construed as, medical, regulatory or legal advice.
Any links contained in the content which provides access to third party resources or content is provided for convenience only. For the use of any product, the applicable product documentation, including information guides, inserts and operation manuals should be consulted. Leica Biosystems and the editors hereby disclaim any liability arising directly or indirectly from the use of the content, including from any drugs, devices, techniques or procedures described in the content.
All rights reserved. Geoffrey Rolls is a Histology Consultant with decades of experience in the field. We are looking for more great writers to feature here. Send us a submission and we'll be in touch! Get more Knowledge Pathway content like this delivered directly to your inbox. Unsubscribe at any time. Back Leica SM R. Back View All. Featured Products. Learn more. Reagent Alcohol, Isopropyl Alcohol Learn more. It's New. Service Back Aperio Training.
All Shop. You won't be able to use the quotation basket until you enable cookies in your Web browser. A drop of cells is spread on a slide and viewed without fixation. The stain is a suspension of carbon, found in India ink or nigrosin. The carbon particles are negatively-charged, as is the cell membrane. The background looks black or sepia colored and the cells remain clear, since they repel the dye.
Some positively charged inclusion bodies, such as sulfur, may stain. This stain gives accurate information on cell morphology and capsule presence because the cells are not fixed. Cell size appears slightly larger because any extracellular coatings or secretions on the outside of the cell membrane also do not stain.
Negative stains are useful for rapid determination of the presence of Cryptococcus neformans , the causative agent of cryptococcisis, in cerebral spinal fluid.
This technique is also used when you stain for endospores and capsules. Just as in preparing a smear, you only need a small amount of organism. It is also important not use too much nigrosin.
If it is too thick, the background will have a cracked appearance similar to mud puddles drying in the sun. You want to get a light film.
Your instructor will demonstrate this technique for you. Nigrosin comes off the slide and onto your oil immersion lens very easily. Be sure to thoroughly clean your oil lens when you are finished. Then clean it again. Once it dries on the lens it is very difficult to remove and will impair your ability and the other micro students using that scope to see clearly out of the lens.
The Gram stain is the most common differential stain used in microbiology. Differential stains use more than one dye. The unique cellular components of the bacteria will determine how they will react to the different dyes. The Gram stain procedure has been basically unchanged since it was first developed in Almost all bacteria can be divided into two groups, Gram negative or Gram positive. A few bacteria are gram variable.
Trichomonas , Strongyloides , some fungi, and some protozoa cysts also have a Gram reaction. Very small bacteria or bacteria without a cell wall, such as Treponema , Mycoplasma , Chlamydia , or Rickettsia do not have a gram reaction. The characterization of any new bacteria must include their gram reaction. Typically a differential stain has four components; the primary stain, a mordant that sets the stain, a decolorizing agent to remove the primary stain, and a counter stain.
In the Gram stain, the primary stain is crystal violet. This gives the cell an intense purple color. The mordant, iodine, forms a complex with the crystal violet inside the cell wall. Gram positive cells will retain the dye complex and remain purple. The dye rinses out in gram negative cells. The large iodine-crystal violet complex is retained within the cell walls of gram positive cells because of the molecular structure of the many layers of peptidoglycan in the cell wall.
There are lots of cross-linked teichoic acids and the iodine-dye complex cannot physically get out. There is also less lipid in the membrane and the decolorizing agent cannot get to it as well.
Gram negative cells have an outer membrane and only one layer of peptidoglycan, with more lipid. The crystal violet dye is easily washed out.
The accuracy of the Gram stain is dependent on the integrity of the bacterial cell wall. There are a variety of things that can influence the cell wall integrity; old cells i. Under these conditions, gram positive cells will come out as gram-negative. If you de-colorize too long, Gram-positive cells will look like Gram-negative cells. Conversely, if you do not decolorize enough, Gram-negatives will look like Gram-positives. The only way you can trust your results it to always run a known Gram-positive and a known Gram-negative on the same slide.
If they stain as predicted you can be pretty sure the result of your unknown sample is reliable. The Gram staining takes practice to get right. Do not expect to get a good Gram stain on your first try. It is a good idea to hold your slide with a clothespin; your gloves will get pretty psychedelic as will everything you touch!
The Congo Red Capsule stain is a modification of the nigrosin negative stain you may have done previously. The bacteria take up the congo red dye and the background is stained then with acid fuchsin dye. The capsule or slime layers, highly hydrated polymers, exclude both dyes.
The background will appear blue, the bacterial cells will appear pink, and the clear halos are the capsules. Clinically, the capsules of some highly pathogenic bacteria i.
In the second step, the specimen is exposed to an acid-alcohol solution. Only cells with a sufficiently impenetrable cell wall will retain the dye in this step, and these cells are said to be acid fast. A second counter-stain can then be used to re-stain the decolorized organisms, similar to the Gram staining procedure. Some microorganisms have one or more small, thin appendages that are used to move the organism around in a liquid.
Therefore, special staining procedures are necessary to visualize these structures using optical microscopy. One such procedure uses a simple basic stain dissolved in ethyl alcohol. As with other stains for flagella, the technique requires rigorously cleaned microscope slides and otherwise very careful, detail-oriented technique. As discussed above, there are many staining procedures that can be used, all with their own chemistry and step-by-step procedure.
As an example of a common procedure, the following steps can be used for Gram staining. The simplest type of wet mount method is to place a drop of liquid containing the microorganism onto a slide, then gently placing a coverslip over the drop. This method is susceptible to drying, however.
A more complex but stabler type of wet mount is the hanging drop. In this method, a liquid droplet with the microorganism is hanging down below the coverslip, with the sample sealed from atmosphere using a ring of wax or petroleum jelly. An illustration of a hanging drop mount is shown in Figure 3. Figure 3 : hanging drop mount side view. Analysis of hanging drop samples will sometimes involve judgements of cell motility, or the ability of the cells to self-propel.
Even non-motile or dead cells can move in liquid, due to convective flow of the liquid, or by Brownian motion. So any analysis of cell motility should be done by carefully observing cell movement over time, to ensure any movement is true motility. A hemocytometer is a special type of microscope slide that can be used for quantitative counting and sizing of cells.
The principle of the hemocytometer is that it consists of chambers that are marked into regions of known volume. The counting chamber, and an example of the marked regions, are shown in Figure 4.
By counting the numbers of cells within one or more known volumes, the concentration of cells can be determined cells per unit volume. A schematic view from the side and top of a hemocytometer is shown in Figure 4. The device itself, also shown in Figure 4, is a thick microscope slide, with an indented well in the center that serves as the counting chamber, and deeper depressions to capture any overflow of sample liquid. Besides the special slide, a coverslip that is thicker than the standard must be used, so that the surface tension of the liquid under analysis does not deform it.
Figure 4 : a schematic of the side view of a hemocytometer, b photo of a commercial hemocytometer slide, and c example layout of the etched lines in the counting chamber. Determining cell concentration using a hemocytometer involves first thoroughly agitating the sample to ensure it is fully mixed, then making the proper dilution.
Because the technique relies on visually counting cells, overlapping or agglomerated cells can artificially reduce the measured concentration. So a high dilution must be used if the original sample is very concentrated.
After diluting and placing the sample into the hemocytometer, the next step is to count the cells in a given set of demarcated regions. The analyst must also be systematic in whether or not to count cells that lie on a demarcation line. For example, one system is to count cells that line only on the lower or left side of a given area.
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. Ocular micrometers are a subset of a type of optical element known as a reticle; examples of other types of reticles are shown in Figure 5. Figure 5 : various types of reticles for optical microscopes, including ocular micrometers 2nd and 4th reticles. By knowing the pitch and the total magnification, the analyst can estimate distances in the field of view, and use that to estimate the sizes or other dimensions of microorganisms, or any other specimen under analysis.
However, this approach is often imprecise since the exact magnification and pitch are not known. 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. As an Amazon Associate Conductscience Inc earns revenue from qualifying purchases The modern pipette has had a colorful history as a standard tool in the. Stereotaxic Accesories. 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.
0コメント