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

264 Histopathology of Seed-Borne InfectionsMany of the chemicals used for clearing tissues, e.g., sodium hydroxide (5%)and lactic acid (9%), are corrosive. Safety precautions must be taken while usingthem. A lab coat, protective gloves, and protective glasses should be used (Mathurand Kongsdal, 2003).10.4.2 FREEHAND SECTIONSSections, cut by razor or by means of a sliding microtome and handled loosely, notattached to the slide by means of an adhesive, are in this category. The material maybe fresh or fixed, but it should be fairly rigid. Hard or woody components of seedare usually cut freehand. Dry seeds are cut after softening.The difficulty lies in holding the seed tightly to avoid bending when the razorstrikes the seed. This can be achieved by holding seed in the pith, any soft but rigidplant material, or by embedding directly (without dehydration and infiltration) inparaffin wax or soap.Sections may be stained with cotton blue and mounted in lactophenol or stainedwith safranin and fast green and mounted in glycerin jelly or polyvinyl alcohol(PVA).10.4.3 MICROTOMYUsually paraffin-embedded seeds and seed parts are used for histopathological investigationsof seeds infected by microorganisms other than viruses. Resin-embeddedpieces of components of seeds and ultratrome cut sections are required for TEM forthe study of viruses. A brief account of the paraffin method is given below. Forultramicrotomy and TEM and SEM techniques, the readers are advised to consultGlauret (1974), Aldrich and Todd (1986), Robards and Wilson (1993), and Neergaard(1997).10.4.3.1 Fixing and StorageBuds, flowers, developing seeds as such, and mature dry seeds after water boilingmay be fixed in FAA (formalin-acetic acid-alcohol) for 24 to 48 hours. For betterfixation, dehydration, and infiltration, seeds may be cut longitudinally on one side,exposing the internal soft components. Wash and store in 70% ethyl alcohol.10.4.3.2 DehydrationThe tertiary butyl alcohol (TBA) series is most satisfactory, although other dehydratingseries, e.g., alcohol-xylol and chloroform, have also been used. TBA doesnot cause excessive hardening of the material. The composition of TBA proposedby Johansen (1940) is widely used (Table 10.1).Keep soft materials for 2 to 4 hours in each solution. Seeds may be kept for 12hours in each grade.After a 100% alcohol solution, give three changes with pure TBA. Keep thematerial for 6 hours in the first two changes and overnight in the last.