ABSTRACT
There is a considerable body of information of a technical and review nature on negative staining elsewhere in the literature (Bremner et al., 1992; Harris and Horne, 1991, 1994; Hayat and Miller, 1990; Holzenburg, 1988; Horne, 1991; Nermut, 1991; Spiess et al., 1987; Valentine and Horne, 1962); therefore, throughout this handbook I shall attempt to avoid undue repetition and present the current laboratory approaches with which I am personally familiar and have achieved some technical success. After considering the range of procedures necessary for the production of carbon and carbon-plastic support films (sometimes termed substrates) emphasis will be placed upon the droplet negative staining technique (Harris and Agutter, 1970; Harris and Horne, 1991). I was introduced to this approach by Ennio Lucio Benedetti in his laboratory, at the start of my doctoral studies in 1966 (Anderson, 1966; Benedetti and Emmelot, 1965, 1968) and with minor variations it is still useful for many biological applications. Secondary to this approach, but with perhaps considerably greater scope for further technical development, are the floating method and the negative staining–carbon film (NS–CF) procedures. Actually, both of these are ‘floating’ variants of the negative staining technique and have their roots in the mica-carbon film production procedure (Section 3.1), using the inherent property that a freshly released floating layer of carbon has a strong adsorptive and stabilization capacity for proteins and viruses (Valentine et al., 1966). When developed further by Ivonne Pasquali-Ronchetti and Bob Horne in the early 1970s by spreading virus suspensions in the presence of ammonium molybdate on a mica surface before carbon coating, this led to the production of ordered arrays of viruses attached to the carbon film, which subsequently could be negatively stained (for details see Section 3.3); thus the proposal of the rather verbose terminology ‘negative staining-carbon film technique’. My own interest in the NS–CF procedure for the production of ordered arrays and 2-D crystals of protein molecules commenced soon after the initial description of this approach (Horne and Pasquali-Ronchetti, 1974) and continues strongly to this day (Harris, 1991; Harris and Holzenburg, 1989, 1995).
