microtome Archives - 3DBiology.com

One of the many steps involved in the procedure of microtome sectioning is the preparation of paraffin wax embedded section blocks. So, what is a paraffin section block? And why is paraffin wax used in microtome sectioning instead of other materials? In this article I will describe the use of paraffin wax in sectioning and the reasons why it is the chosen material. Paraffin wax in microtome sectioning is meant to provide structural support to the tissue sample so that it can smoothly undergo the process of slicing by the microtome without any morphological or microscopic damage to the sample. The presence of a wax support also makes the handling of these sections easier and they last longer. Before getting into paraffin, lets discuss microtomy briefly. Microtome sectioning is an important part of any biological lab. Using microtome sectioning a specimen from a larger sample is obtained for microscopic evaluation. It’s a long procedure, and usually requires a lot of practice and expertise to prepare good specimens. Once the sample is sliced and placed on the slide, the paraffin wax is removed with the help of xylene and alcohol. Following dewaxing, the sample is ready for staining, mounting, and observation. Therefore, paraffin wax is not a permanent constituent of the sample, or the slide, and is strictly meant for providing support for the sample while sectioning. Microtomy is a crucial part of a biological lab, as it is the first step in the preparation of a slide. Let’s explore a little more and see what exactly microtomy is. What Is Microtomy? Microtomy is the procedure of obtaining thin sections (or slices) of a tissue sample using a microtome. A microtome is a device that slices the block of paraffin containing the sample. A thin and evenly sliced sample is essential to create a good microscopic slide, as a thin sample would enable the examiner to understand the microscopic details better. Therefore, it is essential to master the art of preparing a microtome section for anyone who works in a biological lab. The steps of microtomy, in brief, is discussed below: The sample is obtained. This can be a human organ tissue obtained via biopsy, animal tissue, or some other biological sample. The sample is then fixed— very commonly with a 4-10% formalin, or some other fixative like Buoin’s fluid (preferred in animal samples), Cornoy’s fluid, etc. This step is important as this prevents any post-mortem changes in the sample. An ideal fixative is one that doesn’t cause any morphological, nuclear, or cytological changes in the sample either. The tissue sample is then washed under running water for about 30 mins and then dehydrated with the help of alcohol. Proper dehydration of the sample is essential for proper sectioning, and so to check for any remnant of water molecules, xylene is used. Xylene is a great indicator for water as it turns milky on reacting with water. If there are no water molecules, the sample can be taken to the next step. If water is still present, the process of dehydration is repeated. Now the sample is ready for the preparation of the section block, and this is done using molten paraffin wax. The paraffin wax section block, once prepared, is put in the microtome and sliced to obtain the specimen. You can see how a microtome is used in this video: Advantages of Paraffin Paraffin tissue blocks have several advantages over frozen sections. They are easier to store, which can be done in room temperature (as long as its below 80F), and are not heavy like glass slides. They can be stored for many years. Another advantage of paraffin is that you can utilize larger tissue sections, such as brain slices of an entire human brain hemisphere. Another advantage is paraffin blocks maintain tissue morphology better than frozen sections. They are more stable and damage to tissue is less likely, a very important factor when tissue is scarce such as from human subjects. How To Prepare A Microtome Section Block Using Paraffin Wax? Once the sample is fixed, washed, and dehydrated, it is ready for section block preparation. For this, paraffin wax is melted (melting point 61 degrees Celsius) and poured into a small mold formed using two L-shaped molds. Since two L-shaped molds are used, the size of the block can be adjusted according to the size of the sample. The molten paraffin wax is then poured into this mold. This is followed by the placement of the sample in the molten paraffin wax as well. The paraffin wax is then allowed to cool down and harden up. Once complete wax is hard, the mold is removed, and a paraffin wax section block is obtained. Before placing this block in the microtome, the paraffin wax must be trimmed well at the edges so that the block can fit better in the device. The block is then sliced, put in a water bath (to remove any wrinkles in the sections), and eventually on the slide. One drop of egg albumin is smeared on the slide before the specimen is put on it. The egg albumin acts as an adhesive that prevents the washing out of the specimen while staining the slide. Once the specimen is safely put on the slide, the paraffin wax is removed using xylene and alcohol. This procedure is known as deparaffinization. It then proceeds to the steps of staining. How To Get Good Quality Paraffin-Embedded Tissue Section To obtain a good quality section, a few things must be kept in mind: Once you have the paraffin block, the first step would be to make sure that the edges of the block are smooth. If it’s not even, it should be smoothed out using a blade. This is done to ensure the sample block fits well in the machine. An uneven paraffin wax block will wobble during the process of slicing the sample, and therefore will create an uneven specimen. Additionally, the extra wax on the front of the block must also be removed using the microtome itself. This is done to expose the sample— which will ensure the slice contains a good amount of specimen in it. The second basic thing to keep in mind is that the blade in the microtome must be sharp. If the blade is not sharp enough, the paraffin wax will crumple while it’s being sliced, and proper sectioning wouldn’t be possible. The section block must be cooled down before slicing. This can be done by either putting the block in cold water for 24 hours, or by keeping the block on ice for some time. This is also done to ensure that the specimen slice doesn’t crumble while slicing. In addition to these basic precautions, you also want to keep in mind that different tissues may require different kinds of handling. For example, Fatty tissue must be dried for a couple of hours after slicing. Fatty tissues are generally difficult to work with as they are not easy to penetrate. Hard tissues, like bone and uterine tissue, should be treated with 1% acid alcohol to soften the specimen. If this step is not done, the microtome might not be able to slice the sample properly and evenly. Microtomy section preparation is a skill that everyone who works in a biological lab must know. However, it can be tricky and may need a lot of practice to create good paraffin section blocks and to be able to slice them well to get a perfect specimen. Things to remember about paraffin wax The paraffin wax is meant to provide support to the sample so that the slicing of the sample is easier. The wax must be cooled down before slicing to ensure that a good quality specimen is obtained. Also, the paraffin wax must be removed once the specimen is safely placed on the slide, as aqueous stains may not be able to penetrate wax. After staining of the slide, mounting of the slide is done using a mounting medium to preserve the specimen. You can read about how you can prepare your own mounting medium here: How to make a microscope slide mounting medium.