Growth of Bacteriophages

Bacteriophages are grown in either gelled or liquid solutions of nutrients. Gels are made of the polysac-charide, agar. Gel-embedded infected cells are used to count viable bacteriophage particles. The procedure is to embed bacteriophages in the gel, together with host cells. The number of host cells is much greater than the number of bacteriophages. This gelled mixture is incubated to replicate the cells and infect them. A bacteriophage particle infects a growing host cell while in the gel. The cell bursts at the end of the infection. Light-scattering (turbidity) decreases when the infected cell bursts. All progeny of a single bac-teriophage particle remain in a restricted region of the gel. This region is comparatively clear (nonturbid). The clear region is called a plaque. A viable bacterio-phage particle is assayed by the formation of a single plaque on a glass or plastic Petri dish or plate which holds the gel with plaques. Plaques are counted after incubating viable bacteriophage particles with host cells. The number of viable bacteriophage particles per plate is small enough (usually 100-2000) so that overlapping plaques do not cause difficulty in counting. The number of plaques is multiplied by a dilution factor, if the bacteriophage particles had been diluted before assay. Bacteriophage plaques are illustrated in Figure 1.

Incubation of a host-bacteriophage mixture in a gel can also be used to prepare large numbers of bacteriophage particles before purification. The bac-teriophages produced are sometimes called a plate stock. Preparing of bacteriophage particles via large volume ( > 100 mL) plate stocks is not efficient (in time and cost), in comparison to preparing bacteri-ophage particles via infection in liquid culture. However, some bacteriophages which do not grow well in liquid cultures grow comparatively well when prepared as a plate stock. Thus, plate stocks are sometimes used for the growth of bacteriophage particles before purification. To remove bacteriophage particles from the gel of a plate stock the gel is minced

Figure 1 Plaques of bacteriophage T7. A Petri plate is filled with a 1 % agar gel in an enriched growth medium. Subsequently, 0.7% molten agar in the same enriched medium is mixed with both host bacteria and bacteriophage particles. This mixture is spread on the 1.0% gel. The plate is incubated at 37°C. A plaque forms at the position of a single bacteriophage particle.

Figure 1 Plaques of bacteriophage T7. A Petri plate is filled with a 1 % agar gel in an enriched growth medium. Subsequently, 0.7% molten agar in the same enriched medium is mixed with both host bacteria and bacteriophage particles. This mixture is spread on the 1.0% gel. The plate is incubated at 37°C. A plaque forms at the position of a single bacteriophage particle.

with either a glass rod or a spatula and the minced gel is incubated with buffer. The bacteriophage particles diffuse out of the gel, into the buffer during this process. Centrifugal pelleting is then used to remove the pieces of gel, together with pieces of the host bacterium. Some bacteriophages are pelleted with the gel. Thus, the pelleted pieces of gel are resuspended in buffer and then pelleted a second time. This process is called washing. Washing is typically performed two or three times. The supernatant solutions are pooled to produce a clarified plate stock.

Details of plate stock preparation vary slightly among the various bacteriophages. A bacteriophage best prepared by plate stock is bacteriophage G. Bac-teriophage G is the largest bacteriophage (known to the author) that can be grown in culture. Bacterio-phage G has a double-stranded DNA genome 670 kilobase pairs long. Bacteriophage G can be grown in liquid cultures, but results are erratic and greater success has been achieved with plate stocks. Typically, plate stocks of bacteriophage G are clarified by centrifugation at 5000 rpm in a 250 mL bottle (or the equivalent; centrifugal force at the bottom of the bottle is 3800 g). This speed is doubled (centrifugal force is quadrupled) in the case of smaller bacterio-phages that don't sediment as quickly as bacterio-phage G. Pelleting the bacteriophage particles (or aggregates of them) works against the objective of clarifying a plate stock.

Maintaining the stability of the bacteriophage particles is an objective at all stages of purification. Known bacteriophages are stabilized by the presence of divalent cations. Magnesium is usually used. Thus, magnesium should be present in the buffer. The presence of 0.001 mol MgCl2 is usually sufficient for stability. Some salt (usually NaCl) should also be present. Some bacteriophages increasingly adsorb to fragments of host cell, as the salt concentration is lowered. Thus, salt concentrations are sometimes raised from the typical 0.1 mol L 1 to 0.5 mol L "1 or more. Adsorption to host cell debris can either inactivate a bacteriophage particle or cause it to pellet with the fragments of gel.

Storage

Plate stocks are also sometimes preferred when a new bacteriophage is isolated. The new bacteriophage might have been isolated from the wild. It might also have been produced by genetic modification of a previously isolated bacteriophage. A plate stock made with a single plate is a rapid and simple way of preserving this new strain. Preservation is completed by freezing a clarified plate stock. Freezing (typically at — 70°C) is used to prevent inactivation during storage for periods of years to decades. A cryoprotec-

tant is added before freezing. Glycerol (10%) can be used as a cryoprotectant. Alternatively, a high molecular weight cryoprotectant can be used. A high molecular cryoprotectant sometimes used is 10% dextran, average molecular weight = 10000. High molecular weight cryoprotectants are less likely to enter a bacteriophage particle. Therefore, high molecular weight cryoprotectants are less likely to cause bacteriophage particles to burst from osmotic shock during thawing. Osmotic shock during thawing occurs because freezing causes nonuniformity in the concentration of cryoprotectant. If the bacteriophage particle is in a region of comparatively low cryo-protectant concentration, an outward osmotic pressure gradient will develop. This is a demonstrated cause of inactivation of bacteriophage T4 by freeze-thawing.

Growth in Liquid Culture

A bacteriophage is usually grown in liquid culture, when the purpose is either chemical or physical characterization of either the bacteriophage or its nucleic acid. In this case, amounts in the 1-50 mg range may be needed. Either simple, well-defined media or enriched media are used. A simple, well-defined medium might have glucose as the primary (sometimes only) source of both carbon and energy. The medium is typically buffered with phosphate. The medium is supplemented with ammonium chloride to provide nitrogen for proteins. Other salts are also added. Both magnesium sulfate and calcium chloride are added after sterilization by autoclaving. Apparently, some requirements, such as iron, are present in sufficient quantity as contaminants. Alternatively, an enriched, but less well-defined, medium can be used. The major components of an enriched medium are often both tryptone and an extract of yeast cells.

Some bacteriophages are more easily (and more inexpensively) grown in minimal medium. This is true for some lytic double-stranded DNA bacteriophages, for example. A lytic bacteriophage always produces progeny when it infects a cell. The counterpart of a lytic bacteriophage is a lysogenic bacteriophage. A lysogenic bacteriophage may or may not produce progeny. The lysogenic bacteriophage genome both remains in and replicates with the host, if progeny are not produced. This state is called lysogeny. Lysogeny can simplify growth of some bacteriophages, as described below. Growth of a lytic bacteriophage, but not a lysogenic bacteriophage, encounters the following problem: cells are infected at a low ratio of bac-teriophage particles to host cells, typically 0.01-0.1. Multiple cycles of infection occur. Therefore, the concentration of cells during the final (and most critical) cycle of infection is hard to control. Overgrowth of cells can result in a suboptimal yield, because of inadequate aeration. Undergrowth of cells can result in suboptimal yield, because of the low concentration of cells. The more rapidly cells grow, the more difficult controlling their concentration during the last cycle is. Cells grow more slowly in minimal medium (typical doubling time = 1.5-2.0 h for E. coli at 30°C) than they do in enriched medium (typical doubling time = 30-40 min for E. coli at 30°C). Thus, the infection is more easily controlled in minimal medium. None the less, other factors are also involved. Some experimentation with growth medium is required to optimize conditions of growth, when these conditions have not been previously optimized.

Growing lysogenic bacteriophages is usually easier than growing lytic bacteriophages. Lysogenic bacterio-phages can be made to leave a state of lysogeny and enter a lytic cycle. Some mutants will do this when the temperature is raised. Thus, growth of lysogenic bac-teriophages (bacteriophages X and P22, for example) can be simplified. Host cells in a state of lysogeny are grown to an optimal concentration. The temperature is raised to induce the lytic cycle. The temperature is then lowered to an optimal temperature for growth. This process is useful for producing bacteriophage particles in large amount. It is also useful for producing other components of bacteriophage-infected cells. For example, some components of bacterio-phage-infected cells retain their metabolic activity when infected cells burst (lyse is a frequently used synonym for burst). These activities include packaging of double-stranded DNA in bacteriophage capsids. Thus, a fragment of foreign DNA can be cloned by, first, incorporating the fragment in a bacteriophage genome, and, then, packaging the DNA in vitro by incubating the DNA in an extract of lysed, infected cells. Extracts of lysed, bacteriophage-infected cells can sometimes be used for incorporating the foreign DNA, as well as packaging it. An extract can be made by use of a lytic bacteriophage, as well as a lysogenic bacteriophage. The process is, however, less time-and resource-consuming with a lysogenic bacterio-phage.

Equipment for Large-Scale Growth of Bacteriophages in Liquid Culture

Small scale (1-1000 mL) liquid cultures are grown in typical laboratory glassware. A flask is sometimes used, with aeration by shaking. Alternatively, a bottle is used with aeration via a bubbler and forced air. The latter alternative is simpler, less expensive and less space-consuming. A reliable shaker is needed in the former case; a source of forced air is needed in the latter. A reliable and reliably clean source of forced air is an aerator designed for use with tropical fish. The use of forced air is scalable to at least a 15 L culture. Shaking is scalable to roughly a 1 L culture.

The control of temperature can be achieved via several routes. A temperature-regulated fermentor with forced air can be used. The cost and inconvenience can, however, be dramatically lowered by using a bottle in a temperature-controlled water bath. Even a bottle in a temperature-controlled, water-filled sink can be used. In the latter two cases, a bottle with sterile medium and bubblers is aerated via forced air.

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