An Improved Technique for Clearing and Staining Plant Tissues for Detection of Nematodes D. W. BYBD, JR., T. K1RKPATRICK, and K. R. BARKER1 Journal of Nematology 15(1):142-143. 1983. Numerous procedures have been used to cooled. The root segments can then be stain and clear nematode-infected plant pressed between glass plates or microscope tissues (7). Staining with acid fuchsin and slides for observation. destaining with lactophenol (4,7) has been The NaOCl-acid fuchsin-glycerin tech- the most widely used technique. More rapid nique has given good results with soybean means, of clearing and staining roots in- roots infected with Hetc'rodera glycines clude autoclaving (1), exposure to saturated lchinohe (Fig. l-A) aud cotton roots in- chloral hydrate (6), and root maceration fected with Meloidogyne incognita Kofoid after staining (3). However, extreme care gc White (Chitwood) (Fig. l-B). This tech- is required for these methods. nique results in high quality specimens in Researchers working with mycorrhizal young, succnlent root tissue. In older or ftmgi found potassium hydroxide (KOH), more ligneous roots such as cotton or corn, nitric acid, hydrogen peroxide (H202), or some di/ficulty in clearing or destaining sodium hypochlorite (NaOC1) to be effec- may be encountered. Two methods have tive in clearing roots (2,5), depending on given satisfactory results with older cotton the type of tissue to be cleared. Alkaline roots infected with M. incognita: i) chlorine H:O., was the best bleaching agent for pig- bleach pretreatment can he increased to 30 mented roots (5). Excessive bleaching with ml per 50 ml of tap water (2.1% NaOC1), NaOCI often resulted in poor staining of or the pretreatment exposure time may be tissues. increased to 5-6 rain; and ii) roots may be A modified acid-fuchsin staining-destain- soaked in 30°//0 H.,O~ for approximately 1 ing wocedure utilizing NaOC1 as a pre- h, rinsed, and stained. staining treatment for nematode-infected Phillips and Hayman (5) found that plant tissues is described herein. cotton roots heated in 10% KOH prior to Roots are washed, chopped into 1-2-cm treatment with alkaline H.,O 2 (10 min-I segments, aml placed in a 150-ml beaker h) allowed rapid and relatively easy detec- with 50 ml tap water. Twenty milliliters tion of fungal infections. However, we of chlorine bleach (5.25,.o/.0 NaOC1) are fouml that extreme care must be taken in added to give 1.5% NaOC1, and the root the use of KOH in nematode-infected roots tissue pieces are allowed to remain in this or the nematodes may be destroyed. Ade- solution for 4 ntiu with occasional a~tation. qtmte root clearing was achieved with a 1-h Following the NaOCI treatment, the root exposure to reagent grade H202 (30%). segments are rinsed in running water (30-- Use of either clearing agent (H202 or 45 sec) and allowed to soak in tap water NaOCI), when combined with glycerin for for 15 min to remove residual NaOC1. The destaining, eliminates exposure to. toxic material is then drained and transferred phenols. This method also minimizes the to a beaker containing 30 ml of water to time required for specimen preparation. which has been added 1 ml of stain (3.5 g When acidified glycerin is used, destained acid fuchsin, 250 ml acetic acid, and 750 root material can be stored for several ml distilled water). This solution is then months with very little change in the ini- heated to boiling for about 30 sec. tial contrast between nematodes and root After cooling to room temperature, ex- tissue. cess stain is removed by rinsing in running water. The root, material is then placed LITERATURE CITED in 20-30 ml of glycerin acidified with a few 1. Ah'es, L. M., and G. B. Bergeson. 1966. A drops of 5N HC1, heated to boiling, and quick destaining procedure for showing contrast between nematodes and root tissue. Plant Dis. Rept. 51:511. Received for publication 26 April 1982. ~Department of Plant Pathology. North Carolina State 2. Bevege, D. I. 1968. A rapid technique for University, Raleigh, NC 27650. clearing tannins and staining intact root for de- 142 Staining technique: Byrd et al. 143

Fig. 1. Nematode-infected plant roots stained with NaOCl-acid fuchsin-glycerin technique. A) Soybean roots infected with Heterodera glycines. B) Cotton roots infected with Meloidogyne incognita. tection of mycorrhizas caused by Endogone spp., parasitic and vcsicular-arbuscular mycorrhizal and some records of infection in Australian plants. fungi for rapid assessment of infection. Trans. Br. Trans. Br. Mycol. Soc. 51:808-810. Mycol. Soc. 55:158-161). 3. Marks, R. J., and L. A. McKenna. 1981. A 6. Southards, C. J. 1965. The efficiency of soil simple procedure for the efficient estimation of assays in estimating nematode diseases of certain potato cyst nematode larvae in lactophenol-treated crop plants. Ph.D. thesis, North Carolina State rof~ts. Ann. Appl. Biol. 97:323-324. University, Raleigh. 4. McBeth, C. W., A. L. Taylor, and A. L. 7. ~mthey, J. F., editor. 1970. Laboratory meth- Smith. 1941. Note on staining nematodes in root ods for work with plant and soil nematodes. Tech. tissues. Proc. Helminthol. Soc. Wash. 8:26. Bull. Minis. Agric. Fish. & Food No. 2. 5th ed. 5. Phillips, J. M., and D. S. Hayman. 1970. Im- London: Her Majesty's Stationery Office. proved procedures for clearing roots and staining