Simple Method for Plating Escherichia coli Bacteriophages Forming Very Small Plaques or No Plaques under Standard Conditions

Abstract

The use of low concentrations (optimally 2.5 to 3.5 g/ml, depending on top agar thickness) of ampicillin in the bottom agar of the plate allows for formation of highly visible plaques of bacteriophages which otherwise form extremely small plaques or no plaques on Escherichia coli lawns. Using this method, we were able to obtain plaques of newly isolated bacteriophages, propagated after induction of prophages present in six E. coli O157:H ؊ strains which did not form plaques when standard plating procedures were employed.In the second half of 20th century, bacteriophages were among the most important models in molecular biology. However, at the end of that century, after learning principles of molecular biology using bacteriophage models, these viruses became considered less attractive research subjects due to development of sophisticated systems for studying eukaryotic cells. Nevertheless, recent years may be recognized as a new era in bacteriophage biology. This is because of several unexpected findings, which demonstrated that viruses infecting bacterial cells may be very important not only in basic biological studies but also in medicine and biotechnology (for reviews, see references 7 and 38). The breakthroughs included discoveries that many toxins (including the vast majority of verotoxins) produced by bacteria pathogenic to animals and humans are encoded in bacteriophage genomes (20, 37); that bacteriophages can be used in vaccination and in specific kinds of therapy (10); that bacteriophages can be extremely dangerous for bioprocesses based on bacterial activities in biotechnological factories (25, 35) but also may be employed in genetic engineering and biotechnology in a very sophisticated manner (10, 28); and finally that they are the most abundant creatures worldwide, thus playing an extremely important ecological role (9,12,19,39).The growing interest in various aspects of phage biology must be connected to isolation and characterization of newly discovered viruses. However, contrary to well-investigated model bacteriophages, in most cases of newly isolated phages there are problems with their propagation under laboratory conditions. This is partially due to the fact that the number of indicator strains used in laboratories is rather limited and various limitations arise from using nonpermissive or suboptimal hosts. The first step in getting an uncontaminated lysate of a particular bacteriophage strain is obtaining single plaques on the host lawn. However, this first and obligatory stage of analysis may be, in fact, a limiting step as there are many examples (though mostly unpublished) of serious problems with getting plaques of newly discovered bacteriophages. This problem seems to be common in the case of lambdoid bacteriophages (27, 42). We met this problem when investigating phages coding for Shiga toxins, whose genomes are present as prophages in chromosomes of some Escherichia coli strains. For example, phage 24B (⌬stx::cat) (4) and phage ST2-8624 (⌬stx::cat gfp) from E. coli O157:H7 strain 8624...

Heterogeneity in Induction Level, Infection Ability, and Morphology of Shiga Toxin-Encoding Phages (Stx Phages) from Dairy and Human Shiga Toxin-Producing Escherichia coli O26:H11 Isolates

“…In addition, the phage titers determined by plaque enumeration were lower than the concentrations of phage genomes determined by qPCR, as reported previously (45). Several factors have been proposed to influence plaque enumeration, including the susceptibility of the host strain and the presence of noninfectious particles in the phage suspension tested (45,46). Nevertheless, despite this difference, the relative levels of Stx1 and Stx2 phages determined by the two methods among the panel of STEC O26:H11 strains were in agreement.…”

Analysis of Novel Mycobacteriophages Indicates the Existence of Different Strategies for Phage Inheritance in Mycobacteria

“…Since we tested the influence of temperature in this phenotype by using the indicator strain grown at either 30°C or 37°C with negative results, we discarded changes in the cell envelope composition as the cause for the failure in propagation at the higher temperature. Thus, based on a previous report [32], we sought to improve their detection by using sub-inhibitory amounts of INH, a mycobactericidal drug that affects mycolic acid synthesis, thus damaging the cell envelope integrity. As shown in Fig.…”

Phenethyl Isothiocyanate Inhibits Shiga Toxin Production in Enterohemorrhagic Escherichia coli by Stringent Response Induction

“…Supernatants were diluted in TM buffer (10 mM Tris, 10 mM MgSO 4 , pH 7.2), and 100 l of each dilution was mixed with 0.5 ml of a culture of the indicator strain. Then, 2 ml of a prewarmed (to 45°C) top nutrient agar was added to each mixture, which was poured onto an LB agar plate supplemented with 2.5 g/ml chloramphenicol, according to a previously published procedure (34). Results of experiments performed in triplicate are shown as mean values with standard deviations (SD).…”