MICROSCOPICAL TECHNIQUES PREPARATIONS

Preparations by hand samples into the stem of elder (Sambucus nigra), by a blade generally cross sections fast, simple, but too thick sections Leaf surface casts remove the epiderm with a needle epiderm cells connect to each other very closely, but to the middle part of the leaf very loosely after removing: staining with a microscopic stain Leaf clearing study of the dead organelles (e.g. crystals, cell wall, vascular tissues) boiling of the leaf in 10% ethanol:KOH, 5% H 2 O 2, distilled water, 96% ethanol staining

Leaf clearing: Cut the leaf

Leaf parts Boiling

Cleared leaf with the vascular tissue elements and crystals crystals

CONSERVATION Solution of Strassburger-Flemming: 100% ethanol : glycerol : distilled water (1:1:1) Fixed/conserved sample Solutions for dehydration (remove excess water) acetone 70 % acetone 90 % acetone 100 % xylol I. xylol II. Samples in bags

PREPARING PARAFFIN BLOCKS Solid paraffin in the thermostat Sample bags in the dissolved paraffin (56 °C)

Pour dissolved paraffin into the block forms (moulds) Put the plant samples into the moulds

Remove the solid block from the mould with the sample Trimming: cut unnecessary parts of the block Ready-to-cut block

Sectioning by rotation microtome Block with sample ← 1-20 μm thick

STAINING OF PREPARATIONS Special histochemical stains: phloroglucin with HCl:lignin rutenium red:cell walls with pectins Sudan III.:lipids, essential oils, wax, chutin, suberin Fe(III)Cl 3 :gallic acids, flavonoids Dragendorff reagent:alkaloids (red) Lugol solution (KI): amyloplasts (blue), aleurone (brown) Completed preparation Staining dishes with the samples + Canada balsam + cover glass

Evaluation of ready preparations (e.g. morphological measurements with softwers)

Evaluation of ready preparations (e.g. morphological measurements with softwers)

Morphological evaluation with Image Tool 3.0 program