SCPA603 Embedding and Sectioning

Transcription

1 SCPA603 Embedding and Sectioning Associate Professor Dr. Wannee Jiraungkoorskul Department of Pathobiology, Faculty of Science, Mahidol University Tel: , 1

2 Objectives The student must be able to: Describe the purpose of embedding and sectioning processes. Describe the propose and how to use rotary microtome, water bath, adhesive, warmer in the sectioning process. Describe problems and troubleshooting in the process. 2

3 Embedding It is done by transferring the tissue which has been cleared of the alcohol to a mold filled with molten wax and is allowed to cool and solidify. After solidification, a wax block is obtained which is then sectioned to obtain ribbons. 3

4 Embedding process is very important, because the tissues must be aligned, or oriented, properly in the block of paraffin. Such plastics are Embedding Methyl methacrylate is very hard and good for embedding undecalcified bone. Glycol methacrylate has the most widespread use since it is the easiest to work with. Araldite is about the same as methacrylate, but requires a more complex embedding process. Epon is routinely used for electron microscopy where very thin sections are required. 4

5 Hints: Specimen orientation Embedding Proper pressure force applied to the entire specimen during to the entire specimen during orientation and initial solidification to obtain flat tissue Hard tissue such as bone, can will section more easily if they are embedded diagonally. Tissue with wall, such as cyst, gall bladder, and GIT must be embedded on the edge. 5

6 Troubleshooting in Embedding Incorrect orientation: Section may be incorrectly oriented at the embedding station if the correct method was not indicated. Prevention: Marking the side of tissue to be embedded facing up with ink. 6

7 Troubleshooting in Embedding Tissue carryover: Small pieces or fragments of tissue may be carried from 1 tissue to the next at the embedding table, resulting in cross-contamination. Prevention: Carefully clean forceps used at the embedding table between specimens. Open only the cassette with the tissue to be embedded 7

8 Troubleshooting in Embedding Tissue not embedded at the same lavel: if the tissue is not properly flattened by pressing it down uniformly when it is placed in the embedding mold, or multiple tissues to be placed in the same mold are embedded at different levels. Prevention: Press the tissue uniformly Keep the paraffin molten enough to get all pieces embedded at athe same level. Work very fast when embedding multiple pieces. 8

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11 Remove tissue from cassette 11

12 Cool and flatten as required Fill mold with wax and orientate tissue 12

13 Mold and ring, fill more wax and put on cold plate, finally remove from mold 13

14 Section cutting is the procedure in which the blocks which have been prepared are cut or sectioned and thin strips of varying thickness are prepared. The instrument by which this is done is called as a microtome. Rotary microtome is the most commonly used. In this the block holder moves up and down while the knife remains fixed. It is suitable for cutting of small tissues and serial sections can be taken on it. Sectioning 14

15 It is important to have a properly fixed and embedded block or much artefact can be introduced in the sectioning. Common artefacts include tearing, ripping, venetian blinds, holes, folding, etc. Knives Thin like a disposable razor blade Thick metal knife (but expensive) Glass knife can section down to about 1 micron Diamond knife for electron microscopy (1/4 micron and very expensive) Sectioning 15

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17 Principle of cutting on a rotary microtome 17

18 Tissue floating bath It is a thermostatically controlled water bath with the inside colored black. It is maintained at a temperature maintained 5-6 degree below the melting point of paraffin wax. 18

19 Paraffin sections cut at each turn of the flywheel 19

20 Paraffin block placed in holder and excess wax trimmed Ribbon of paraffin sections 20

21 Sections floated onto a water bath 21

22 Sectioning Microtomes have a mechanism for advancing the block across the knife (for most paraffin embedded tissues at 6 to 8 microns). Once sections are cut, they are floated on a warm water bath that helps remove wrinkles. Then they are picked up on a glass microscopic slide. then placed in a warm oven for about 15 minutes to help the section adhere to the slide. 22

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24 Troubleshooting in Sectioning Crooked ribbons: Result when the horizontal edges of the block are not parallel. If the lower block edge is not parallel to the knife edge. Prevent: The upper and lower edge should be parallel The lower block edge is parallel to the knife edge 24

25 Troubleshooting in Sectioning Block face unevenly sectioned: Occur when the block holder is not parallel to the blade. One side of the block is exhausted while attempting to get a complete section of the block face. Prevent: Ensure at the beginning of the sectioning that the block holder is adjusted so that the block face and the blade are perfectly parallel. 25

26 Troubleshooting in Sectioning Vertical scratches: Caused by defect in the blade edge, calcium, bone or hard material in the specimen. Prevent: Ensure at the beginning of the sectioning that the block holder is adjusted so that the block face and the blade are perfectly parallel. 26

27 Troubleshooting in Sectioning Holes in the section: Occur when block is faced too aggressively. The specimen is either excessively dehydrated or improperly processed. Prevent: Ensure to chill the block with ice before cutting and discard ribbons until the hole disappear. Facing the block less aggressively, with smaller micrometer advances of the block for each section removed. 27

28 Troubleshooting in Sectioning Failure of ribbon to form: Commonly caused by dull blade. Could result from too hard paraffin, too much blade tilt Prevent: Paraffin with lower melting point. Decrease blade tilt Change room temperature 28

29 Chatter or microscopic vibration: Commonly caused by over dehydration of lack of moisture in the tissue. It could also result from dull blade or too much blade tilt which cause the section to be scraped rather than cut. Or cutting too rapidly. Prevent: Proper processing Troubleshooting in Sectioning Restore moisture by facing the block down on an ice tray Decrease the blade tilt Decrease cutting speed; one wheel per second is considered reasonable speed 29

30 To obtain a section from tissues which are often brittle after processing. This is often the case with tissues which contain a lot of blood or colloid such as spleen or thyroid. This section stained H & E shows a deliciously brittle blood clot which are always difficult to cut. They often end up like this broken and curled. Apply cotton wool or gauze soaked in warm water to the surface of the block for a few seconds. Then try to cut a section. If that doesn t work. Soak the block in warm, soapy water for a few minutes. Place the block on ice for a few seconds. Then cut a section again 30

31 Wrinkles appearing in sections floated on a flotation bath. This can be caused by the Flotation bath being too cold. If the water is too cold the wax surrounding the section will not expand and flatten. Ideally the flotation bath should be kept at temperature about 5 C below the melting point of the wax being used. 31

32 This is a section of uterus stained Haematoxylin and Eosin and showing thick and thin areas. The thick area, of course, stains more darkly than the thin area. One reason for this is a loose knife or loose block. So always ensure that the knife is held firmly in place and also the block and remember a loose knife can destroy a block. 32

33 References Suvarna KS, Layton C. Bancroft JD. Bancroft s Theory and Practice of Histological Techniques. 7 th edition. Churchill Livingstone Elsevier: Canada p. 33