Preparation of Plant tissues for histological study

Infiltration step This step aim To adding the paraffin wax gradually and reduce the clearing solution. To prepare the tissue for the embedding step.

To do this: add the crystals of paraffin wax to the tissue gradually inlet the tissue infiltrate completely. For example add one small piece to the xylene from previous step then don’t add another pieces inlet the first one is full dissolved. Make sure that the lid of the tube is removed while you are adding the wax. And incubate the tube in 50 ˚c. this step take approximately h.

Preparation of paper boat 1.Prepare paper that has 8cm length and 4 cm width using ruler. 2.From both side, fold 1cm from paper edge which parallel to the length of previous paper. 3.From both side of edge parallel the width of paper fold 2.5 cm. 4.Fold one end of each side corner together. 5.Now you are ready to pour the wax material with sample.

Exercise lab Complete the previous experiment of prepare plant permanent slide. Make your own paper boat.

Embedding Embedding is a process in which the tissue is surrounded with a medium to support it during sectioning. The most important step during the embedding process is arranging the specimens in such a way that the desired orientation can easily be obtained during sectioning.

One can also prepare paper boats or use simple aluminum dishes for embedding as long as solidified wax blocks can be removed easily. Paper boats are easy to prepare, inexpensive, and different sizes can be made to suite one’s need. Once removed from the oven, wax begins to solidify at room temperature, especially for paraffin wax. One needs to work fast in order to arrange all the specimens properly in a desirable orientation before solidification of wax.

Once embedding is complete, the wax molds are allowed to solidify at room temperature before storing them either at room temperature or in a box with a desiccant in a refrigerator.

Paraffin wax Paraffin wax is a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils. It is about two thirds the density and slightly more elastic than dried protein. Paraffin wax is traditionally marketed by its melting points which range from 39°C to 68°C.

Features of Paraffin wax The properties of paraffin wax are improved for histological purposes by the inclusion of substances added alone or in combination to the wax: – Improve ribboning. – Increase hardness. – Decrease melting point – Improve adhesion between specimen and wax.

Embed Specimens Carefully Avoid under-filling the cassette as this can allow unstable clamping in the microtome and lead to cutting “thick then thin” sections and other problems. Avoid over-filling cassettes as this can interfere with the correct alignment of the block face for sectioning. Any excess wax on the outside of a cassette should be removed before clamping to ensure the block is firmly held during sectioning. Specimen orientation is very important.

The wax is clear of clearing agent. No dust particles must be present. Immediately after tissue embedding, the wax must be rapidly cooled to reduce the wax crystal size

Protocol of Embedding 1.Transfer one vial with tissues, a pre-warmed mold, spatula, and forceps from the oven to a hot plate (or slide warmer) which was previously set at 60 °C. 2.Carefully swirl the vial and pour some samples into the mold, so that only one third of the mold is filled.

3.Use the pre-warmed spatula and/or forceps (which can be rewarmed using a Bunsen burner or an alcohol burner) to orient the samples at the bottom of the mold depending on the desired plane of sectioning. 4.Once the samples are in place, transfer the partially full mold onto a flat coldsurface and let the bottom layer of wax solidify for a few seconds. Gently fill the whole block with more molten wax.

5.Place the mold on a tray containing water and ice for about 30 min to allow there maining wax to solidify completely. Store the molds in the fridge until further use. 6.Repeat Steps 2–5 with the remaining vials. 7.Remove the block from the mould. 8.Cross check block, label and worksheet.

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