Double Staining Technique for Study Ultrastructure of Citrus hystrix DC Leaves

Abstract

Kaffir lime (Citrus hystrix DC) leaves is part of Rutaceae family. It is medicinal herb that native various parts of Southeast Asia. Kaffir lime leaves are widely used as part of many food ingredients due to it is unique fragrant. The aroma derived from the oil gland that distributed on leaf surface. In addition, Kaffir lime leaves extract have been reported that there are bioactive properties such as antileukemic, antioxidant, antimicrobial and anti-inflammatory. In this report, we used light microscope and double staining technique using toluidine blue and basic fuchsin to study ultrastructure of leaves. The semi-thin section of leaves were stained using 5 conditions. Also, double staining technique help distinguish and enhance visualization of tissue. The studied showed that when compared 3 condition of double staining, the optimum combination was obtained as 1 minute of 1% toluidine blue O in 2.5% sodium carbonate and 2 minute of 2.5% basic fuchsin in 50% ethanol. Toluidine blue O clearly showed chloroplast and xylem while basic fuchsin excellently stained idioblast cells and phloem. Our study that the staining technique could apply to routine research and also used as a preliminary monitoring for study ultrastructure of the other plants.