To Identify Rhizobium Using Phages

EXERCISE 14

TO IDENTIFY RHIZOBIUM USING PHAGES

Bacteriophages of rhizobia (rhizobiophages) are isolated from the soil and used for identifying rhizobia. Strains of rhizobia vary in their resistance to rhizobiophages. The different patterns of susceptibility which result from exposure to a range of phages are used for strain identification.

Key steps/objectives

1) Collect soil samples from various soybean fields

2) Inoculate YM broth with TAL 379

3) Inoculate broth cultures of TAL 379 with soil from soybean fields

4) Filter broth cultures

5) Enrich phage suspensions by filtration

6) Assay filtrates for phage concentrations

7) Inoculate YM broth with rhizobial strains (A to F) to be typed

8) Spreadplate rhizobial suspensions and spot phages

9) Inspect plates for phage forming units (PFUs) and tabulate results

(a) Isolating bacteriophages

(Key steps 1, 2, 3, 4, and 5)

Collect soil samples from sites where soybeans are growing or have been grown. Obtain the soil from the rhizosphere of individual plants. Include some root material and, if possible, nodules. Collect samples from eight locations. Mix each soil thoroughly and store the samples at 4°C until use.

Sixteen 250 ml flasks, each containing 100 ml sterile YM broth, are required for each soil sample. Inoculate the flasks with B. japonicum strain of choice (eg. TAL 379) in batches of four, with a lag period of 1 day between each batch. This will provide cultures in the exponential growth phase when needed for subsequent phage inoculation. Incubate cultures on a rotary shaker at 25o-30oC.

When the first batch of cultures has reached its exponential phase of growth (24 days after inoculation), add 1 gram of soil to each flask of batch one. Make sure that each flask is inoculated with soil from a different location. Incubate for 1820 h at 2530°C.

Remove cells and soil by centrifugation (10,000 x g for 15 min) and filter the supernatant through a sterilized membrane filter (0.20 μm). This filtrate contains the rhizobiophages which are small enough to pass through the filter. Add 10 ml of each filtrate to a fresh culture (second batch) of the same strain of rhizobia, incubate on a shaker for 1820 h and again centrifuge and filter. Repeat this procedure two more times, making certain that the filtrate is matched to the corresponding culture.

The turbidity in the bacterial cultures should diminish noticeably 810 h after the addition of the phage filtrate. The last filtrate is the phage suspension and should contain 106109 phage particles. Dispense the filtrate into 20 ml tubes, add four drops of chloroform and store in the refrigerator at 4°C.

(b) Assaying for phage by the overlay method

(Key step 6)

Make tenfold serial dilutions of the phage filtrates in phosphate buffered saline (PBS at pH 7.1, Appendix 5). Add 0.1 ml of each dilution to tubes containing 2.5 ml melted YMA kept at 50°C in a water bath. Stir in 0.5 ml of a fresh culture of TAL 379 and immediately pour the YMA mixture over plates of MNA and distribute evenly. Prepare controls with only PBS and TAL 379 (no phage) added to agar and poured over YMA plates. Allow plates to stand for 1015 min. Incubate inverted plates for 2472 h and look for plaques (small clear zones). Count the plaques per plate. To calculate the number of Plaque Forming Units (PFU) in 1 ml of the original filtrate, multiply the number of plaques per plate by 10 and by the dilution factor. If 20 plaques were counted on a plate containing a 105 dilution, the number of PFUs is 20 x 10 x 105 = 2 x 107 PFU/ml.

(Key steps 7, 8, and 9)

Because of the specificity of bacteriophages for their bacterial hosts, each strain of bacterium exhibits a unique pattern of susceptibility against a large number of different bacteriophages. This unique pattern can be used to identify (phagetype) the organisms of interest.

Select several strains of B. japonicum and distinguish between them by their susceptibility to a range of phages. Incubate each strain in duplicate flasks of 50 ml MN broth at 2530°C. Include strains of B. japonicum TAL 379 and TAL 378.

After 59 days incubation, spread 0.1 ml of each of the cultures to be typed over a separate MNA plate with a sterile glass spreader. Spot the surface of the bacterial lawn with a small loopful of each of the collected phage suspensions. The location of the spots should be marked on the back of the plates. Allow the plates to stand for 1015 min, invert and incubate for 2448 h.

Inspect the plates for a clear zone where each of the phages was spotted. Record presence (+) or absence () of plaques in a table similar to the example in Table 14.1.

The susceptibility of a rhizobial strain to a range of phages can be regarded as its "fingerprint," enabling it to be recognized in ecological investigations.

Table 14.1. An example of results of Rhizobium identification by phage typing.

Strains of Rhizobium
Phage
Isolate / A / B / C / D / E / F / TAL379
1 / + / ─ / ─ / + / + / ─ / +
2 / ─ / + / + / + / ─ / ─ / +
3 / ─ / ─ / + / + / ─ / ─ / +
4 / + / ─ / ─ / ─ / + / ─ / +
5 / + / ─ / + / ─ / + / ─ / +
6 / + / + / + / ─ / ─ / + / +
7 / ─ / ─ / + / ─ / + / + / +
8 / ─ / ─ / ─ / + / + / + / ─

*A, B, C, D, E and F are B. japonicum isolates from nodules of soybean

Requirements

(a) Isolating bacteriophages

Refrigerator, rotary shaker, centrifuge balance

Desiccator, centrifuge tubes (50 ml), rack, pipettes (10 ml)

Membrane filter units, sterile with Millipore filters of 0.20 μm pore size

Soil samples (from four locations where inoculated soybeans are/were grown)

Digging tools, plastic bags

Erlenmeyer flasks, 150 ml, containing 50 ml MN broth each

Transfer loop, flame

Chloroform solution (1%)

Slant culture of B. japonicum (TAL 379)

(b) Assaying for phage by the overlay method

Incubator, water bath, rotary shaker

PBS (pH 7.1)

Pipettes, 1 ml

Tubes containing 2.5 ml liquid YMA (50°C)

Plates of MNA

Phage filtrates from (a)

Broth cultures of TAL 379

Inoculation loop, flame

Pipettes (1 ml)

Spreaders

Erlenmeyer flasks of 125 ml containing 50 ml MNA

MNA plates

Slant cultures of a range of B. japonicum strains including strain B. japonicum strain TAL 379)

Phage isolates from (a) as well as others if available.