DWI WINARNI Departemen Biologi Fakultas Sains dan Teknologi Universitas Airlangga Surabaya

Pembuatan sayatan: metode parafin Peralatan yang diperlukan 1.Mikrotom, pisau mikrotom, holder 2.Water bath 3.Hot plate atau drying oven 4.Kuas 5.Scalpel 6.Tissue atau lap bersih 7.Gelas obyek bebas lemak 8.Slide rack atau slide tray 9.Perekat cupis 10.Es batu

 Setting the waterbath  Setting the microtome  Trimming the tissue block  Attach the trimmed tissue block on to holder  Cutting sections  Floating out sections  Picking up sections and adhere on to slide  Drying sections

A. Pita hasil irisan tidak membentuk pita lurus tetapi membelok (curved) Cause 1.Leading and trailing edges of block not parallel 2.Knife blunt in one area 3.Surplus area of wax at one side 4.Tissue varying in consistency Remedy 1.Trim with sharp scalpel 2.Sharpen or use different part of knife 3.Trim away excess wax 4.Reorientate block by turning throuh 90o. Cool block with ice.

B. Alternate section thick and thin Cause 1.Wax too soft for tissue 2.Block or knife loose 3.Insufficient clearance angel 4.Mechanism of microtome faulty Remedy 1.Cool block with ice or re-embed in higher MP wax 2.Tighten block and knife 3.Slightly increase clearance angel 4.Check for any obvious faults

C. Sections won’t join to form a ribbon CauseRemedy 1.Wax too hard for sectioning condition 2.Debris on knife edge 3.Knife angle too steep or too shallow 1.Re-embed tissue in lower MP wax 2.Clean with xylene moistened cloth 3.Adjust to optimal angle

C. Sections become attached to block on return stroke Cause 1.Insufficient clearance angle 2.Wax debris on knife edge 3.Debris on block edge 4.Static electrical charge on ribbon Remedy 1.Increase clearance angel 2.Clean with xylene moistened cloth 3.Trim edge with sharp scalpel 4.Earth microtome to water tap. Humidify air with moist bath near the knife. Place bunsen burner near knife to ionise air D. Areas of tissue in block not present in sections CauseRemedy 1.Incomplete impregnation of tissue 2.Wax block becoming detached from wood 1.Return tissue to vacuum impregnating bath for a few hours or reprocess 2.Re-attach with hot spatula

C. Sections expand and disintegrate on water surface Cause 1.Poor impregnation of tissue 2.Water temperature too high Remedy 1.Return tissue to vacuum impregnation bath for a few hours 2.Cool D. Sections roll on into a tight coil instead of remaining flat on knife CauseRemedy 1.Knife blunt 2.Too little rake angle 3.Section thickness too great for wax in use 1.Sharpen or change knife with the new one 2.Resharpen 3.Reduce section thickness or use slightly higher MP wax. Breathe on block as sections are being cut

Affixing adhering tissue sections on slide  Most of tissue sections will adhere to the slides without the use of any adhesive, because that sections are initially dried on to the slides,  Adhesive is used to prevent sections detach from slide during staining or histochemical methods using alkaline solutions for tissue sections of “soft” tissue (brain, spinal cord, blood clot, decalcified tissue) and any tissue which have been fixed in strong protein-coagulant fluid