Bacteriophage host range determination (Culture-Based Approach)

Bacteriophage host range is by definition a repertoire of bacterial species and strains that are able to support the multiplication of the given virus. It is therefore impossible to determine a complete host range practically since in no experiment can all of the existing bacterial strains are tested for susceptibility to the chosen bacteriophage. In laboratory practice, the bacteriophage’s host range is normally determined against a pre-selected panel of bacterial test cultures (for example, a set of clinical isolates of a given pathogenic species obtained in some region or during some period of time or some other strain set made on purpose).

Phage host range

We shall also note that assessing the bacteriophage growth directly is difficult, so the researcher generally has to resort to a number of proxies of this parameter, based mostly on the bactericidal action of the bacteriophage;
The ability of the bacteriophage to cause clearance of growing liquid bacterial cultures.
The ability of the bacteriophage stock to form spots of reduced or inhibited growth of the bacterial lawn on solid media.

Requirements

1. Bacterial cultures and bacteriophage stock(s) with known titer (s)
2. Sterile liquid medium appropriate for the bacterial strains used.
3. Plates with solid medium containing 15 g of bacto-agar per 1 L. For E. coli and many other heterotrophic bacteria, tryptic soy broth or LB medium containing 10 g of Tryptone, 5 g of yeast extract, and 5 g of NaCl per 1 L of deionized water is suitable. For bacteria with different nutrient requirements use specialized media.
4. Sterile soft agar containing 6 g of bacto-agar per 1 L and the same nutrients as the bottom agar.
5. Sterile 13 (170 mm) glass tubes with caps. 6. Sterile Eppendorf tubes of 1.5 and 0.5 mL capacity.
6. Table-top Eppendorf centrifuge with a maximum speed of 10,000 * g or more.
7. Water bath adjusted for 48°C or thermoblock with 4 to 10 or more holes the right size for the glass tubes.

Determination of the Bacteriophage Infectivity by Top Agar Method

This technique is very similar to the drop assay method of bacteriophage titration.

1. Remove the required number of plates with bottom agar from 4°C storage. One needs to use plates poured in advance and preferably incubated overnight at 37°C, as they give out much less moisture during cultivation. At least one plate per bacterial strain is required (see Note 2). If they are too recently poured, it is possible to put them partially open in a laminar flow hood to dry the agar surface and to adjust the medium to room temperature. Label the plates.
2. Melt the top agar in a microwave oven until it comes to a gentle boil and no visible clumps are left, dispense 3 ml aliquots to sterile glass test tubes (e.g. 13 (170 mm)), and place them in a water bath or dry block at 48°C, so the medium is kept molten. Use within a few hours.
3. Prepare the suspensions of log-phase liquid cultures of bacterial strains to be tested. For aerobic bacteria, it is better to grow them with vigorous agitation in the shaker-incubator under their optimal growth temperature in small (50 ml) Erlenmeyer flasks or in test tubes containing 3 ml of the medium and covered by loose caps or aluminum foil. In many cases, cells from a fresh plate resuspended in the liquid medium or in physiological saline work as well. The density of the bacterial suspension should be 108 –109 CFU/mL (normally about OD600 ¼ 0.4–1.0).
4. Take the tube with top agar, immediately add 50 μL of bacterial suspension, mix by rotating the tube between the palms (the vortexing may produce foam), and pour to the center of the plate containing solid medium. Do it quickly enough, so the bacteria will not perish due to a thermal shock. Spread the top agar layer over the whole surface of the bottom agar by gently tilting the plate, and let solidify on a horizontal surface. Do not cover the plate, as the condensing water would cause undesired “swamping”, causing the mix-ups of bacterial cultures. The plates with the inoculated lawns prepared in such a way should be used within 3 h. Inoculate one or more (if many bacteriophages have to be tested) plates for each bacterial strain. If more than five bacteriophages are tested it is convenient to use square plates (for 10 � 10 cm square plate, 6–8 mL of the top agar should be used instead of 3 mL).
5. Centrifuge your bacteriophage stocks for 5 min at the maximum speed of a table-top centrifuge to remove possible bacterial contamination. Prepare serial dilutions of the stocks. To do this, for each stock prepare six Eppendorf tubes with 990 μL of physiological saline or bacteriophage SM buffer. Label them as “-2”, “-4”, “-6”, and “-8” and indicate also the bacteriophage name. Add 10 μL of the bacteriophage stock to the first tube, mix by brief vortexing. Transfer with a new pipette tip 10 μL aliquot of this dilution to the second tube and repeat the same operations until the last tube is used. This will yield the dilutions by 102 , 104 , 106 and 108 times respectively. The tenfold dilutions may be used instead of 100-fold to ensure the countable number of the plaques in at least some of the drops, however, it will increase the time and material consumption that may be crucial for a large-scale screening experiment.
6. Draw on each plate the grid and mark the lines according to the bacteriophages. Apply drops of the bacteriophage stocks dilutions on the surface of the top agar in the corresponding line of the grid. One may use a single pipet tip for each bacteriophage if one moves from the highest dilution (10power8 ) to the lowest. Apply also a drop of nondiluted bacteriophage (100 ). We 80 Andrey V. Letarov and Eugene E. Kulikov suggest to use small drops of 5 μL to avoid smearing of them by the water expelled from the solid medium.
7. Let the drops to be absorbed by the agar; dry the plate 10 min under the laminar hood (no UV irradiation!).
8. Incubate the plates under the optimal conditions for the growth of the bacteria. For rapidly growing cultures an overnight incubation is sufficient, for slower growing a longer incubation may be needed. In the latter case seal the plates with parafilm to avoid the drying of the agar. It is important to keep the plates inverted to avoid water condensation on the agar surface.
9. Read your plates. If the bacteriophage produces isolated plaques in any dilution, the bacterial strain used for the lawn can be considered sensitive. A separate estimate of EOP would be useful for distinguishing the fully sensitive strains and those to which the bacteriophage may be adapted by further passaging. If the inhibition of the lawn growth is observed at high bacteriophage concentrations but no plaques appear in stronger dilutions, the strain may be considered as basically resistant (or more detailed evaluation of its interactions with a particular virus should be performed).

The bacteriophage host range is one of the most practically important characteristics of each bacterial virus. Here the classical plate-culture-based approach for bacteriophage host range determination is described. The important considerations related to the interpretation of the data and limitations of the methods are discussed.