Histologic Plant Tissue Preparation Techniques. 5 - Embedding Step

Bot 213 Iram siddique & Alanoud Alfagham
Histologic plant tissue preparation techniques.

5 - Embedding step
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.
Embedding step
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. Disposable
embedding molds of various sizes are available commercially. 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. If a paper boat is used, practice by embedding
a few specimens at first; with improving skills, more specimens can be embedded at one time.
Overall, polyester wax solidifies slower than paraffin 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.
A. 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
1
traditionally marketed by its melting points which range from 39°C to

68°C.
• 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
A. 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. A magnifying lens can be used at this stage to help with
the orientation.
4. Once the samples are in place, transfer the partially full mold onto a flat cold surface 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 the remaining
wax to solidify completely. Store the molds in the fridge until further use.
6. Repeat Steps 2–5 with the remaining vials.
2
7. Remove the block from the mould.

8. Cross check block, label and worksheet.
Types of mold.
3
Exercise lab
1. Complete the previous experiment of prepare plant
permanent slide.
2. Make your own paper boat.

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Bot 213 Iram siddique & Alanoud Alfagham

Histologic plant tissue preparation techniques.

Preparation of paper boat.

traditionally marketed by its melting points which range from 39°C to

A. Embed Specimens Carefully

• The wax is clear of clearing agent.

• No dust particles must be present.

7. Remove the block from the mould.

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