To Identify Nodules by Gel Immunodiffusion

EXERCISE 10

TO IDENTIFY NODULES BY GEL IMMUNODIFFUSION

In this exercise, a precise technique is described to differentiate and identify the occupant strain(s) of nodules from plants inoculated with a mixture of two serologically distinct strains. Bacteroids from nodules are used directly as antigens.

Key steps/objectives

1) Prepare Leonard jars 2) Culture strains for stock broth cultures and for antigen preparation 3) Pregerminate soybean seeds 4) Sample stock broth cultures for viable counts and prepare mixed inoculum 5) Plant and inoculate pregerminated seeds 6) Harvest nodules 7) Set up the Gelman immunodiffusion apparatus. Prepare gel in Petri dishes 8) Prepare antigen from nodules and cultured cells of inoculum strains 9) Perform gel diffusion in microscope slides and Petri dishes 10) Record precipitin bands by drawing; analyze immunodiffusion patterns

(a) Preparing mixed-strain inocula of B. japonicum (Key steps 2 and 4)

Prepare two flasks each containing 150 ml of YM-broth. Inoculate one flask with B. japonicum strain TAL 379 str and the other flask with B. japonicum strain TAL 378 spc. These two flasks will provide stock cultures of each strain. Allow 7 days for maximum growth of the strains. (The two strains used in this experiment are antibiotic resistant. TAL 379 is resistant to streptomycin (str) and TAL 378 is resistant to spectinomycin (spc)). The nodules formed by these two strains will be identified by gel immunodiffusion in this exercise and by their ability to grow in YMA plates containing antibiotics in Exercise 12.

After 7 days of growth of the stock cultures, aseptically and accurately transfer 50 ml of TAL 379 str to a 250 ml sterile flask. Do the same flask transfer 50 ml of TAL 378 spc. (Use a fresh 10 ml pipette for the transfers and pipette 5 times to remove each 50 ml. Use a fresh pipette when transferring different strains). Swirl the flask to ensure a good mixture of the two strains and label this flask M.

Use the drop- or spread-plate methods (Exercise 4) to obtain viable counts of TAL 378 spc and TAL 379 str. When the viable counts become available later, the actual ratios of the competing strains in the mixed inocula can be more accurately computed. Set aside the remaining portions of the two stock cultures and use these as inocula for single strain inoculation.

(b) Culturing of soybean plants inoculated with a single strain and a mixture of strains of B. japonicum (Key steps 1, 3, 5, and 6)

Prepare 15 Leonard jars (Appendix 11).

Surface sterilize and pregerminate 60 soybean seeds of good viability as described in Appendix 10. Allow two days for the pregermination of the seeds.

Select well-germinated seeds and plant three per jar. Inoculate each seed at sowing with 1 ml of the broth inoculum of the appropriate treatment. Plant four jars for each treatment and label adequately.

Proceed to plant another 4 jars and inoculate the seeds with 1 ml per seed of the stock culture of TAL 379 str and label. Similarly set up 4 more jars but inoculate with TAL 378 spc.

Finally, plant the remaining three jars and leave them uninoculated. These three jars serve as uninoculated controls. Seven days after planting, thin down to two uniform plants per jar. Harvest all treatment after 30-35 days. Carefully remove and wash the root-system of each plant. Count and record the number of nodules on the roots of each plant. Detach the nodules and pack them in small plastic bags as described in Exercise 8. Label the bags adequately for later identification of the treatments. (The remaining plants in the jars will be harvested at a later date for use in Exercise 13).

(c) Preparing nodule bacteroid-antigens (Key step 8)

Proceed as explained in Exercise 8. Prepare nodule bacteroid-antigens from nodules of the treatments which received the mixed broth inoculum.

(d) Preparing soluble antigen from cultured cells (Key step 8)

Inoculate one YMA flat each with TAL 379 str and TAL 378 spc. Harvest these strains after 7 days and prepare soluble antigen for immunodiffusion as described in Exercise 9.

(e) Setting up the immunodiffusion system (Key step 7, 9, and 10)

The gel (see Exercise 9) for immunodiffusion is prepared on microscope slides. Thin (1 mm) microscope slides are especially suitable for this method using the Gelman immunodiffusion apparatus (Gelman Instrument Company, Ann Arbor, Michigan U.S.A.) described in this exercise. The various components of the apparatus include the immunoframes, immunoframe holders, rinsing tanks, and the immunodiffusion punch set. Familiarize yourself with their construction and use(s). The Gelman product numbers for the various components are given in the list of requirements.

Study the immunoframe which has been especially constructed to hold microscope slides. Each immunoframe holds six standard microscope slides, three in each of its two compartments. Each compartment is divided into three windows and one slide is centered over each window. All three slides must be placed in close contact with one another. Complete the arrangement of slides in each immunoframe and place the immunoframes on a clean and level surface. (A level surface is important to obtain gel of uniform thickness.)

With a Pasteur pipette, dispense minimal amounts of the molten gel around the edges of each slide to seal off the fine gaps at all points of contact between slides and between slides and compartment walls. (Sealing is necessary to prevent leakage of the gel to the bottom when the melted gel is poured.)

Allow the gel to cool to obtain proper seal. Pipette 10 ml of the molten gel into each compartment of the immunoframe. Empty the pipette beginning at one end of the compartment and proceed to the other end moving the pipette in a zig-zag motion, to evenly spread the gel over the slides. Complete layering the gel over all the slides in the immunoframes.

Allow 1 h for the gel to cool and set, in a dust-free environment. The gel should be protected from dust particles settling on its surface during the cooling process by improvising suitable covers.

On cooling and setting of the gel, mount the immunoframes onto the immunoframe-holder. It can accommodate a maximum of three immunoframes. Place the whole assembly into a rinsing tank containing approximately 80 ml of water and replace the lid. Store the rinsing tank and its contents overnight at 4C (refrigerator) or at room temperature (26-28C) to improve the setting of the gel.

Examine the immunodiffusion gel-punch set. The gel-punch consists of a die and an attached system of cutters. The arrangement of cutters on the die allows the production of two sets of the hexagonal pattern of wells (used in this technique) on one slide at any one time. The gel punch is designed to fit the sides of the immunoframe and when the punch supports are properly mounted, the punch can be slid back and forth to desired positions.

Mount the gel punch onto the immunoframe and position it over a slide. Gently press the punch down on the gel and hold for 3-4 seconds to cut out the hexagonal patterns.

The 3 mm wells on the slides can hold 8-10 microliters of antiserum or antigen. These small volumes can be conveniently delivered with a variable volume (5-50 microliters) Finn pipette with disposable tips.

Perform the immunodiffusion with the nodule bacteroid-antigens with reference to Figure 10.1. Identify all the nodules being analyzed for strain occupancy using the scheme given in Figure 10.1.

Figure 10.1. Scheme for identifying nodules from inoculated with a mixture of two strains. Set the Finn pipette to deliver 8 microliters of the antigen or antiserum. Deliver the antigens and antisera to their respective wells in the hexagonal system according to scheme (Figure 10.1) given. (Note that each nodule formed by the mixed inoculum is identified against the antisera of the two component B. japonicum strains in the mixture.)

Assemble the immunoframes (housing the microscope slides) on the immunoframe-holders and incubate the assembly in a saturated atmosphere (provided by approximately 80 ml of water in the rinsing tank). Incubation at room temperature (26-28C) allows precipitin band development between 24 to 48 hours.

In Table 10.1 record the number of nodules giving positive precipitation bands against each antiserum. Nodules giving reactions of identity with both the antisera indicate mixed infections, i.e., the nodule contains both the strains from the mixed broth inoculum.

Use the nodule analysis data to examine whether the proportion of nodules formed by each strain was according to its representation in the mixture using Chi-square analysis.

If sufficient nodules and antisera are available, perform a parallel immunodiffusion exercise with gel prepared in Petri dishes (Chapter 11). Follow a similar scheme of nodule identification as detailed for the microscope slide method in this chapter.

Table 10.1. Identification of nodules for the mixed inoculum treatment

Ratio of No. of Chi-square TAL 378:379 nodules Nodule occupancy (%) TAL378 deviation examined TAL379 TAL378+TAL379 (1 df)

Requirements

(a) Preparing the mixed broth inoculum

Transfer chamber Agar slant cultures of B. japonicum strains TAL 379 str and TAL 378 spc YM-broth, 150 ml (two flasks) Sterile 125 ml Erlenmeyer flasks (two) Sterile 10 ml pipettes (five) Sterile 1 ml pipettes (15) Sterile calibrated Pasteur pipettes 90 ml sterile water in each of milk dilution bottles Quarter-strength YM-broth or sterile water (9 ml in 30 ml capacity screw-cap tubes) YMA plates

(b) Culturing of soybean plants inoculated with a single and mixture of strains B. japonicum

Leonard jars Soybean seeds Sterilizing solutions (see Appendix 10) Sterile water Water agar plates Pipettes, 10 ml (three-five) Spirit lamp, alcohol in spray bottle, matches Forceps Inoculant broth of TAL 379 str and TAL 378 spc

(c) Preparing nodule bacteroid antigen

See Exercise 8

(d) Preparing soluble antigens from cultured cells

Slant cultures of TAL 378 spc and TAL 379 str YMA flats (two) Other requirements as in Exercise 9

(e) Setting up immunodiffusion systems

Immunoframes (Gelman Product No. 51447) Immunoframe holders (Gelman Product No. 51448) Rinsing tanks (Gelman Product No. 51457) Immunodiffusion punch-set (Gelman Product No. 51450) Microscope slides without frosted ends (Approx. 1 mm thick) from Curtin Matheson Scientific Inc. Finn pipettes (Variable Volumetrics Inc., Woburn, MA, USA) Pasteur pipettes