Journal zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA of Vimlogical Methods, 1 I (1985) 23-27 Elsevier JVM 00383 23 zyxwvu THE USE OF POLYLYSINE DURING NEGATIVE STAINING OF VIRAL SUSPENSIONS ORTRUD MONIKA BARTH Departamento de Virologia. Instituto Oswaldo Crur. Caixa Postal 926, 20000, Rio de Janeiro, Brazit (Accepted 29 November 1984) The use of 0.1% aqueous solution of polylysine (poly-L-lysine) is proposed as a prior step to negative staining of viral or particle suspensions. Particles spread better on films precoated with polylysine than with other substances used for the same purpose. This applies particularly to samples from sucrose or CsCl gradients. The use of negative contrast substances is discussed. polylysine negative contrast viral suspensions INTRODUCTION Negative staining of particles or macromolecules in suspension (Brenner and Horne, 1959) is a routine technique for rapid diagnosis and recognition using trans- mission electron microscopy. Variations of this technique have been reviewed by Horne (1965), Nermut (1973) and Holt and Beveridge (1982). Some specific observa- tions were published by Jokelainen et al. (1970) for hepatitis B, Beveridge and Murray (1976) for bacteria and cellular membranes, Boublik and Hellmann (1978) for ribo- somes, Mrena (1980) for microsomes and Barth (1984) for some vertebrate viruses. The greatest source of failure in the use of negative staining for diagnostic purposes lies in the preparation of the viral suspensions. Precipitation of particles in a suspen- sion, followed by serial centrifugation and ultra-centrifugation, cleans the material for observation, but with each step of purification, the preparation of electron micro- scope grids becomes more difficult. The reason for this, is an electron charge repulsion between the formvar-carbon coated copper grid and the drop of the suspension. Different substances such as bovine serum albumin (BSA), bacitracin, alcohol or freshly carbon-coated grids have been used to improve particle adhesion. Mazia et al. (1975) used polylysine (or poly-L-lysine) to create a positively charged support for cells. We have used the same approach to improve the adhesion of virus particles. This technique proved particularly useful for the adsorption of highly purified virus suspensions and has been adopted as a routine procedure. 0166-0934/85/$03.30 0 1985 Elsevier Science Publishers B.V. (Biomedical Division)