Histopathology of Seed-Borne Infections - Applied Research Center ...

Microtechniques in Seed Histopathology 265TABLE 10.1Composition of Tertiary Butyl Alcohol SeriesReagentsAlcohol Percentage50 70 85 95 100Distilled water 50 30 15 — —95% ethyl alcohol 40 50 50 45 —Tertiary butyl alcohol 10 20 35 55 75100% ethyl alcohol — — — — 2510.4.3.3 InfiltrationInfiltration is the process of transfer from TBA to the paraffin wax in tissue. It is animportant but slow process. The authors prefer to carry out the process initiallyunder a bulb (60 W). Add flakes of paraffin wax to a vial. Gradually increase theamount by adding more flakes every 2 hours. Continue until the amount of mixturehas doubled, leave overnight, drain half of the fluid, add melted paraffin wax, andkeep for 6 or more hours. Repeat the process twice and transfer the vials into theoven at 60°C. Drain the fluid and change with melted wax. Make changes every 6or more hours until traces of TBA are removed.10.4.3.4 EmbeddingEmbed the infiltrated material in melted wax. Arrange the material in proper order(for details, see Johansen, 1940).10.4.3.5 Softening of Embedded MaterialThis step is very important for microtomy of seeds. The following protocol isrecommended:• Cut block into small individual blocks with one seed per block.• Trim individual blocks, particularly on the cut side of the seed leavingabout 1 mm of the paraffin wax covering it.• Immerse blocks in 1% aqueous solution of sodium lauryl sulphate for24 hours.• Wash with water.• Transfer to a mixture of glycerin and acetic acid (1:1) for 1 to 4 weeksdepending on the size and hardness of seed.• Wash thoroughly with water and store.10.4.3.6 Sectioning and Mounting of RibbonsMicrotome sections can be cut using any rotary microtome. For more details consultJohansen (1940).