The determination of the concentration of infectious phage particles is fundamental to many protocols in phage biology, genetics, and molecular biology. In this chapter the classical overlay protocol is described.

The determination of the concentration of infectious phage particles is fundamental to many protocols in phage biology, genetics, and molecular biology. Described here is a drop plaque assay, which, being simpler, faster and more efficient than either the classical overlay or direct plating methods, enhances efficiency in processing large numbers of samples.

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Verotoxin 1 is an Escherichia coli-derived subunit toxin that specifically binds to the glycolipid globotriosylceramide and is cytotoxic for cells that contain this plasma membrane glycolipid. Glycolipid incorporation experiments have now been performed using human lymphoid cells of the B lineage that lack this receptor, to conclusively demonstrate that globotriosylceramide alone is a functional receptor for this toxin. Globotriosylceramide incorporated into the membrane of toxin-resistant cells provides intracellular access to verotoxin by receptor-mediated endocytosis. Protein synthesis is then inhibited and globotriosylceramide-containing cells are killed.Verotoxin (VT) is an Escherichia coli elaborated subunit toxin (1, 2) highly homologous with Shiga toxin (from Shigella dysenteriae type 1) and, therefore, also referred to as Shiga-like toxin or SLT (2, 3). At least two variants of VT have been described, termed VT1 and VT2 (4) [or SLTI and SLTII (5)]. Research interest has focused on these toxins since they have been strongly implicated as the causative agent of the renal damage seen in the hemolytic uremic syndrome (HUS) (6, 7). Evidence for the presence of VT has been found in nearly 90% of patients with HUS in this hospital over the last 5 years.A4 HUS is usually preceded by hemorrhagic colitis (6) and similar gastrointestinal symptoms are obtained when purified VT is administered to rabbits (8).Most cells are entirely resistant to VT cytopathology. HeLa (9), Vero (6), and Daudi human lymphoma cells (10) have, however, been shown to be highly susceptible. It is possible that the basis of such differential sensitivity resides at the level of the cell surface toxin receptor.VT1 (11), VT2 (12), and Shiga toxin (9, 13) have been shown to bind specifically to the neutral glycolipid globotriosylceramide (galactose al-4galactose,81-4glucosylcera-mide; Gb3). We have proposed (10) MATERIALS AND METHODS Gb3 and globotetraosylceramide (Gb4) were purified from human renal tissue by a modification of the procedure as described by Strasberg et al. (14). The chloroform/methanol tissue extract was first applied on a Bio-Sil A (Bio-Rad) silica column in chloroform. The column was extensively washed with chloroform and neutral glycolipids were eluted with acetone/methanol, 9:1 (vol/vol). The neutral glycolipid fraction was then applied on a second Bio-Sil A column in chloroform/methanol, 98:2 (vol/vol). Glycolipids were then resolved with a linear solvent gradient comprising equal weights of chloroform/methanol, 15:1 (vol/vol), to chloroform/methanol, 4:1 (vol/vol). Digalactosyldiacylglycerol (DGDG) was purchased from Supelco (Ind), galactosylceramide (GC) was from Sigma (Ill), and phosphatidylethanolamine (PE) and phosphatidylserine (PS) were from Avanti. VT1 was purified as described (1). Rabbit anti-VT antibodies were kindly provided by S. Richardson (Department of Microbiology, Hospital for Sick Children). Monoclonal antibody against the B subunit of VT1 (13-C4) was a generous gift from A. O'Brien (Unifor...