EXERCISE 13
TO IDENTIFY ANTIBIOTIC-RESISTANT MARKED STRAINS OF RHIZOBIA IN NODULES
Antibiotic resistant marked strains of rhizobia may be identified by their ability to grow on media containing antibiotics. The antibiotic marker technique is applied in ecological studies where strain identification is not possible by serology due to cross reactions of the strains, or because of unavailability of antisera. Antibiotic markers also provide useful confirmatory data.
Key steps/objectives
1)Set aside inoculated soybean plants (from Exercise 8)
2)Prepare antibiotic plates for nodule typing
3)Harvest soybean plants; clean, trim, and sterilize roots; type nodules
4)Read results
5)Compare results to those obtained by the serological methods (Exercise 9 and 10)
(a)Culturing plants inoculated with antibiotic resistant marked strain(s) of Rhizobium
(Key step 1)
Soybean plants which have been set up for Exercise 8 will also be used in this exercise. They have been inoculated separately with TAL 379 str, TAL 378 spc, and a mixture of TAL 379 str and TAL 378 spc.
Obtain the viable cell counts of the inocula as determined in Exercise 4.
(b)Preparing YMA containing antibiotics for nodule typing
(Key step 2)
Prepare plates containing: a) streptomycin (40 μg ml1 YMA); b) spectinomycin (250 μg ml1 YMA); and c) plain YMA as in Exercise 12.
Draw a grid pattern on the bottom of each plate. Draw approximately 20 squares, each of which can be individually identified by letter and number (Figure 13.1). Squares with identical number and letter combinations on each of the three plates are meant to correspond to the same nodule.
(c)Typing nodules using antibiotic resistant markers.
(Key step 3)
Harvest one of each inoculation treatment from Leonard jars, saved from Exercise 8.
Figure 13.1. Plate with grid pattern for nodule identification by antibiotic resistance.
Detach and surface sterilize nodules as in Exercise 1.
Pick up one nodule with a pair of sterile, blunttipped forceps. While holding the nodule between the tips of the forceps, apply just enough pressure until the milky nodule content emerges. Spread this nodule material within its allotted square of the grid pattern on each plate.
Inoculate the plain YMA plate last to check for sufficiency of nodule inoculum. Process at least 20 nodules from each replication in this way. Flame forceps thoroughly between fresh nodules.
Alternatively, sterile toothpicks or pins may be used to transfer bacteroids from the nodules to the plates. This method is especially useful for smaller nodules.
In another method, all non-nodulated excess root material is trimmed off with a pair of scissors and discarded. The trimmed nodulated part of the root is then sterilized and placed onto sterile filter paper in a sterile Petri dish. A sketch is made of the nodulated root system on a record sheet and the nodules assigned reference numbers. The nodules are then detached with sterile forceps, one at a time, and processed as described above, starting with the first nodule on the upper part of the root. In this way it is possible to identify not only which nodules on the plants were formed by the introduced, marked strain but also the specific location and distribution of those nodules in the root system.
Incubate the plates at 2530C and make daily observations. Some contaminants (bacteria & fungi) may be resistant to the levels of spc and str used. Therefore, if the nodules have not been properly surface sterilized, these contaminants may appear on the plates earlier than the rhizobia.
(d)Interpreting the growth patterns
(Key steps 4 and 5)
Five to 8 days after inoculation, inspect the plates for signs of growth. Since corresponding squares on the three different plates have been inoculated with bacteroids from the same nodule, it should now be possible to determine which strain or strains occupied the nodule by the presence and absence of growth (Figure 13.2).
Compare the results from this exercise with those obtained in the identification by the agglutination method (Exercise 8) and the gel immunodiffusion method (Exercise 9).
Figure 13.2. Interpreting growth patterns on antibiotic
Plates.
Requirements
(a)Culturing plants with antibiotic resistant marked strains of B. japonicum.
Leonard jar with 2 soybean plants inoculated with TAL 379 str
Leonard jar with 2 soybean plants inoculated with TAL 378 spc
Leonard jar with 2 soybean plants inoculated with a mixture of TAL 379 str and TAL 378 spc
(b)Preparing YMA containing antibiotics for nodule typing (Exercise 12)
Plates containing YMA + streptomycin (40 μg ml1)
Plates containing YMA + spectinomycin (250 μg ml-1)
YMA plates
Felt pen with permanent ink, ruler
(c)Typing nodules through the use of antibiotic resistant strains of B. japonicum
Incubator (2530C)
Requirements for sterilizing nodules (Appendix 10)
Soybean plants listed in (a)
Running water
Scissors, forceps (2)
Plates prepared in (b)
Optional: sterile toothpicks or pins
(d)Interpreting the growth patterns
Inoculated plates from (c)