Robley C. Williams, Dean Fraser
1953 Journal of Bacteriology  
The development of a new freeze-drying technique for the preparation of biological specimens for electron microscopy (Williams, 1953a) has led to the reexamination of the Escherichia coli phages T3 and T7 and to the observations that these supposedly spherical, tailless phages have short, stubby tails and a generally hexagonal cross-section (Fraser and Williams, 1953) . These findings, coupled with observations on a few other viruses (Williams, 1953b) which indicate that the sizes previously
more » ... sizes previously reported might be substantially erroneous, have suggested the desirability of a complete electron microscope reexamination of the T-phages. In the present woork all seven of the phages have been prepared under substantially identical conditions, examined biologically to establish their identity and viability in the final stages of purification, and photographed in the electron microscope following both air-drying and freeze-drying. The morphological details of the frozen-dried specimens are different significantly from those wlhich have been observed using previous techniques. NIETHODS Cultures. The original stocks of the T-phages were obtained from M\. Delbriuck. For purposes of the present work each type of phage was grown in a synthetic medium containing glycerol and "casamino acids" as carbon sources and the usual assortment of inorganic salts (Adams, 1950) . The only differences in the conditions of growth weere necessitated by the requirement of T5 for extra calcium in the medium and the finding that the plaque titer of T5 seenms to drop rapidly if aeration of the culture is prolonged after most of the bacteria have lysed. The phages were purified by differential centrifugation. It was found that the speeds which are required for T3 and T7 (15,000 rpm for one hour in the 40rotor of the Spinco _Model L ultracentiifuge) are 1 Aided by a grant from the National Foundation for Infantile Paralvsis. excessive for the rest of the set and result in the loss of the tails (and viability) of a large proportion of the phages. In general the minimum speed which will form a pellet should be used. We have found that one hour at 10,000 rpm for T2, T4, and T6, 11,000 rpm for TI, and 9,000 rpm for T5 is satisfactory. The high speed cycles were alternated with low speed cycles of 5,000 rpm for five minutes. In all cases the phages were resuspended in a minimum volume of their original supernatant fluid and the mixture was diluted slowly with whatever solution was desired. Generally we have usedI an isotonic ammonium acetate buffer for air-dried preparations and an isotonic ammonium benzoate buffer for frozen-dried preparations, but have foundI that at least some of the phages, as described later, can be resuspended in redistilled water provided that care is taken to dilute slowly. This is not safe with T5. The final purified suspensions were plated for viability in all cases-we can generally account for 80 to 90 per cent of the original titer-and were examined for identity by such biological tests as were indicated for the phage in question; i.e., the observation of the usual properties of host range and plaque type where applicable (Adams, 1950) , and quantitative inactivation Nith antisera to distinguish T3 from T7, to distinguish the even-numbered phages from each other, and to identify TI and T5. Preparation for electron miiicroscopy. Specimen mounts of both air-dried and frozen-dried viruses were l)repared for electron microscopy. The material to be air-dried, suspended in either isotonic ammonium acetate oIr in distilled water, was deposited upon the collodion-filmed microscope grids in the forim of minute droplets (Backus and Williams, 1950) from a Vaponefrin Nebulizer. The material for freeze-drying was suspended in either isotonic ammonium benzoate or in (listilled water and was frozen-dried by the method described by W\=illiams (1953a). Ther e were no visible differences between particles wlhich had been suspended in distilled water or in the applropriate isotonic buffer. 458 on May 7, 2020 by guest
doi:10.1128/jb.66.4.458-464.1953 fatcat:2u4rwfoa25ag3eqo5ypbzwkmta