An improved technique for clearing and staining plant tissues for detection of nematodes.

Numerous procedures have been used to stain and clear nematode-infected plant tissues (7). Staining with acid fuchsin and destaining with lactophenol (4,7) has been the most widely used technique. More rap id means, of clearing and staining roots include autoclaving (1), exposure to saturated chloral hydrate (6), and root macera t ion after staining (3). However, extreme care is required for these methods. Researchers working with mycorrhizal ftmgi found potassium hydroxide (KOH), nitric acid, hydrogen peroxide (H202), or sodium hypochlorite (NaOC1) to be effective in clearing roots (2,5), depending on the type of tissue to be cleared. Alkal ine H:O., was the best bleaching agent for pigmented roots (5). Excessive bleaching with NaOCI often resulted in poor staining of tissues. A modified acid-fuchsin staining-destaining wocedure utilizing NaOC1 as a prestaining t rea tment for nematode-infected plant tissues is described herein. Roots are washed, chopped into 1-2-cm segments, aml placed in a 150-ml beaker with 50 ml tap water. Twen t y milli l i ters of chlorine bleach (5.25,.o/.0 NaOC1) are added to give 1.5% NaOC1, and the root tissue pieces are allowed to remain in this solution for 4 ntiu with occasional a~ ta t ion . Following the NaOCI treatment, the root segments are rinsed in runn ing water (30-45 sec) and allowed to soak in tap water for 15 min to remove residual NaOC1. T h e material is then drained and t ransferred to a beaker containing 30 ml of water to which has been added 1 ml of stain (3.5 g acid fuchsin, 250 ml acetic acid, and 750 ml distilled water). Th i s solution is then heated to boil ing for about 30 sec. After cooling to room temperature , excess stain is removed by rinsing in runn ing water. T h e root, mater ia l is then placed in 20-30 ml of glycerin acidified with a few drops of 5N HC1, heated to boiling, and