CONTENTS
Introduction...... 1
Exercise 1. TO COLLECT NODULES AND ISOLATE RHIZOBIUM. . 7
a. Recognizing legumes and identifying them in the
field
b. Recovering nodules in the field
c. Preserving nodules
d. Examining nodules and bacteroids
e. Isolating Rhizobium from nodule
f. Performing the presumptive test
g. Authenticating the isolates as Rhizobium
h. Preserving Rhizobium cultures
Requirements
Exercise 2. TO OBSERVE THE INFECTION PROCESS...... 30
a. Culturing strains of rhizobia in YM broth
b. Germinating seeds
c. Preparing a Fahraeus-slide
d. Inoculating the seedlings
e. Observing the root-hairs under the microscope
f. Comparing root hair deformations
Requirements
Exercise 3. TO STUDY CULTURAL PROPERTIES, CELL
MORPHOLOGICAL CHARACTERISTICS AND SOME
NUTRITIONAL REQUIREMENTS OF RHIZOBIUM. . . . 40
a. Preliminary subculturing of different bacterial
cultures
b. Comparing cell morphology and gram stain reactions
of Rhizobium with those of other microorganisms
c. Determining gram stain reactions of various
bacteria
d. Characterizing growth of rhizobia using a range
of media
e. Observing growth reactions on modified media
Requirements
Exercise 4. TO QUANTIFY THE GROWTH OF RHIZOBIUM. . . . . 53
a. Preliminary culturing of fast and slow-
growing rhizobia
b. Determining the total count with a Petroff-
Hausser chamber
c. Using the Petroff-Hausser counting chamber
d. Estimating cell concentration by optical density
e. Determining the number of viable cells in a
culture by plating methods
f. Determining the mean-generation (doubling)
time of rhizobia
Requirements
Exercise 5. TO COUNT RHIZOBIA BY A PLANT INFECTION
METHOD...... 73
a. Preparing inoculants
b. Setting up the plant dilution count in plastic
growth pouches
c. Planting seeds in growth pouches
d. Inoculating for MPN count
e. Determining the most probable number
Requirements
References and Recommended Reading...... 84
SECTION B. STRAIN IDENTIFICATION
Introduction...... 91
Exercise 6. TO DEVELOP ANTISERA...... 101
a. Culturing Rhizobium for antigen
b. Preparing antigens for immunodiffusion
c. Preparing somatic antigens for agglutination and
fluorescent antibody techniques
d. Immunizing the rabbit
e. Trial bleeding for titer determination
f. Collecting blood and giving booster injections
Requirements
Exercise 7. TO PERFORM AGGLUTINATION REACTIONS WITH PURE
CULTURES OF RHIZOBIUM...... 111
a. Preparation of somatic antigens from cultured cells
b. Dilution of stock antiserum
c. Performing agglutinations in microtiter trays
d. Performing agglutinations in tubes
e. Performing agglutinations on microscope slides
Requirements
Exercise 8. TO AGGLUTINATE ANTIGENS FROM ROOT
NODULES...... 125
a. Developing antisera
b. Culturing soybean plants nodulated with a
serologically marked strain of Rhizobium
c. Separating bacteroid-antigens from nodules for
agglutination
d. Agglutinating the antigens with homologous antiserum
Requirements
Exercise 9. RHIZOBIAL ANTIGEN-ANTIBODY REACTIONS IN GEL
BY IMMUNODIFFUSION...... 134
a. Preparing gel for diffusion
b. Preparing antigens
c. Setting up immunodiffusion reactions
Requirements
Exercise 10. TO IDENTIFY NODULES BY GEL
IMMUNODIFFUSION...... 141
a. Preparing the mixed broth-inoculum
b. Culturing of soybean plants inoculated with a
single strain and a mixture of strains of
Rhizobium
c. Preparing nodule bacteroid-antigens
d. Preparing soluble antigen from cultured cells
e. Setting up the immunodiffusion system
Requirements
Exercise 11. TO DEVELOP AND USE FLUORESCENT
ANTIBODIES (FA)...... 152
a. Fractionating serum globulins
b. Purifying the serum globulins
c. Determining the protein content of the dialyzate
d. Conjugating the globulins with fluorescent dye
e. Purifying the fluorescent antibodies
f. Testing the quality of fluorescent antibody
g. Typing nodules using the fluorescent antibody
technique
Requirements
Exercise 12. TO DEVELOP ANTIBIOTIC RESISTANT
RHIZOBIA...... 161
a. Culturing selected strains
b. Preparing YMA plates containing antibiotics
c. Selecting spontaneous mutants with resistance to
one antibiotic
d. Selecting strains of Rhizobium having resistance
to two antibodies
Requirements
Exercise 13. TO IDENTIFY ANTIBIOTIC-RESISTANT MARKED
STRAINS OF RHIZOBIA IN NODULES...... 168
a. Culturing plants inoculated with antibiotic
resistant marked strain(s) of Rhizobium
b. Preparing YMA containing antibiotics for nodule
typing
c. Typing nodules using antibiotic resistant strains
of Rhizobium
d. Interpreting the growth patterns
Requirements
Exercise 14. TO IDENTIFY RHIZOBIUM USING PHAGES. . . . 175
a. Isolating bacteriophages
b. Assaying for phage by the overlay method
c. Typing rhizobia using phages
Requirements
References and Recommended Reading...... 182
SECTION C. RHIZOBIUM STRAIN SELECTION
Introduction...... 187
Exercise 15. TO TEST FOR GENETIC COMPATIBILITY BETWEEN
RHIZOBIA AND LEGUMES...... 191
a. Culturing strains of Rhizobium
b. Preparing seedling-agar tubes and Leonard jars
c. Preparing germination plates
d. Surface sterilizing seeds
e. Planting and inoculating
f. Observing periodically and harvesting
g. Evaluating the experiment
Requirements
Exercise 16. TO SCREEN RHIZOBIA FOR NITROGEN FIXATION
POTENTIAL...... 201
a. Experimental design and treatments
b. Preparing Leonard jars
c. Culturing the rhizobia for testing
d. Surface sterilizing the seeds
e. Planting and inoculating of seeds
f. Harvesting the plants
Requirements
Exercise 17. SELECTING EFFECTIVE STRAINS OF RHIZOBIA IN
POTTED FIELD SOIL...... 210
a. Designing the experiment and treatments
b. Preparing the inoculum
c. Choosing the site for collecting soil
d. Collecting, preparing, and potting field soil
e. Adjusting moist field soil to field capacity
f. Applying fertilizer
g. Planting and inoculating the seeds
h. Inspecting non-inoculatd control plants for
nodulation by native rhizobia
i. Watering the pots and making periodic observation
j. Harvesting the experiment
Requirements
Exercise 18. TO VERIFY THE NITROGEN-FIXING POTENTIAL OF
GLASSHOUSE SELECTED STRAINS OF SOYBEAN
RHIZOBIA IN THE FIELD ENVIRONMENT. . . . . 221
a. Setting up the experiment
b. Selecting strains for the experiment
c. Preparing inoculants
d. Preparing seeds for inoculation and planting
e. Preparing the field
f. Controlling cross-contamination by modifying
irrigation methods
g. Applying fertilizer
h. Planting the experiment
i. Monitoring the trial and harvest
j. Analyzing the data
Requirements
Exercise 19. TO INVESTIGATE THE IMPORTANCE OF OPTIMAL
FERTILITY IN THE RESPONSE OF A LEGUME TO
INOCULATION WITH RHIZOBIUM...... 236
a. Setting up the experiment
b. Preparing the mixed inoculant and inoculating the
seeds
c. Choosing a site and preparing the field
d. Applying fertilizers
e. Planting the experiment
f. Monitoring the trial and harvest
g. Harvesting nodules for strain identification
h. Analyzing the yield data
Requirements
References and Recommended Reading...... 251
SECTION D.
INOCULATION TECHNOLOGY
Introduction...... 257
Exercise 20. TO PRODUCE BROTH CULTURES IN SIMPLE GLASS
FERMENTORS...... 262
a. Inoculating starter cultures
b. Assembling simple fermenters
c. Operating the glass fermenters
d. Producing broth inoculum
Requirements
Exercise 21. TO PREPARE A RANGE OF CARRIER MATERIALS
AND PRODUCE INOCULANTS...... 275
a. Milling carrier materials
b. Characterizing and preparing carriers
c. Preparing inoculants by impregnating dry carriers
with broth culture
d. Testing the quality of inoculants
e. Collecting, recording and analyzing the data
Exercise 22. TO PREPPARE INOCULANTS USING DILUTED
CULTURES OF RHIZOBIUM AND PRESTERILIZED
PEAT...... 293
a. Culturing rhizobia in YM broth
b. Making a culture dilution flask and its operation
c. Preparing the diluents
d. Preparing packaged presterilized peat and
checking for sterility
e. Preparing presterilized peat in
polypropylene trays
f. Preparing diluted cultures of Rhizobium
g. Preparing inoculants with diluted cultures and
presterilized peat in packages
h. Preparing inoculants with presterilized peat in
polypropylene trays
i. Determining multiplication of the rhizobia in
peat inoculants prepared aseptically
j. Determining the multiplication of the rhizobia
in the peat inoculants prepared by hand-mixing
in trays
k. Collecting, recording and analyzing data
Requirements
Exercise 23. TO TEST THE SURVIVAL OF RHIZOBIA ON
INOCULATED SEEDS...... 311
a. Preparing inoculants for seed inoculation
b. Preparing adhesives
c. Inoculating and pelleting seeds
d. Determining the number of viable rhizobia on seeds
Requirements
References and Recommended Reading...... 323
Appendices...... 328
TABLE OF CONTENTS FOR FIGURES
FIGURETITLE PAGE
1.1Streaking the plate15
1.2Isolation procedures as used17
by Date and Halliday (1979b)
1.3Ceramic bead method for24
storing Rhizobium
2.1Petri dish with components of33
Fahraeus slide
2.2Placement of seedling on33
Fahraeus slide
2.3Roothair deformation showing36
shepherd’s crook
2.4Selective proliferation and37
colonization of Rhizobium
trifolii on a roothair
2.5Rhizobium trifolii inside infection37
thread of clover roothair
3.1Shapes of bacteria44
4.1The Petroff Hausser counting56
chamber
4.2Procedure for serial dilutions62
4.3Growth of colonies of Rhizobium65
sp. From drops plated by the drop-
plate method
5.1Soybean plants growing in growth77
pouches
B.1Lattice formulation in an antigen-93
antibody reaction
B.2Precipitin reactions96
B.3Direct immunofluorescence98
B.4Indirect immunofluorescence99
7.1Scheme for antiserum titer117
determination in agglutination
tray
7.2Agglutination reactions in wells121
of agglutination tray
7.3Agglutination reactions in121
agglutination tubes
8.1Identification of nodule bacteroids131
by agglutination in an agglutination
tray
9.1Hexagonal pattern template for Petri136
dishes
9.2Well pattern for immunodiffusion137
9.3Immunodiffusion reactions showing139
precipitin bands
10.1Scheme for identifying nodules147
inoculated with a mixture of two
strains
11.1Scheme of nodule smears for strain165
identification by FA
13.1Plate with grid pattern for nodule170
identification by antibiotic resistance
13.2Interpreting growth patterns on172
antibiotic plates
16.1An example of randomized complete203
block design experiment
18.1Field layout and dimensions222
18.2Diagram of field plot223
19.1Field layout and dimensions239
20.1Scheme of simple fermenter unit265
20.2Simple fermenter in operation266
20.3Modified fermenter267
22.1Apparatus for diluting liquid296
cultures of Rhizobium
APPENDIX CONTENTS
AppendixPageFigure
NumberTitle NumberNumber
1Characteristics of the subfamilies 328
of legumes
Subfamily Papilionoideae A.1
Subfamily Caesalpinoideae A.2
Subfamily Mimoosoideae A.3
Legume pods A.4
Leaves of legumes and associated A.5
structures
Some representative shapes of A.6
leguminous nodules
Some examples of nodule distribution A.7
on roots
2 Nodule preservation vial 338
Nodule preservation vial A.8
3Media and staining solutions 340
4Reagents 351
5Buffers 355
6McFarland nephelometer barium 358
sulfate standards
7Preparation of seedling-agar 360
slants for cultivating small
seeded legumes
Simple set up for dispensing seedling- A.9
Agar into tubes and forming slants
8Building a rack for growth pouches 363
Rack for growth pouches A.10
9Recommendations of hosts and 365
growth systems for authentication
10Surface sterilization of seeds 369
11Preparation of Leonard Jars 375
The Leonard Jar A.11
12Injecting and bleeding rabbits 378
Bleeding rack A.12
Bellco bleeding apparatus A.13
Collecting blood from a rabbit by A.14
cardiac puncture
13The indirect FA technique 386
14Additional explanations to the 391
calculations of the most probable
number (MPN)
15The acetylene reduction method 399
for measuring nitrogenase activity
Simple apparatus for generating A.15
small amounts of acetylene in the
laboratory
Trace pattern from an injection of A.16
a gas mixture containing CH4,
C2H2, and C2H4 showing the sequence
of emergence of the different peaks
16Methods for determining lime 410
requirements of acid soils
17Analysis of variance for a 414
Rhizobium strain selection
experiment
Effect of various strains of R. A.17
japonicum on the dry weight of
shoots of soybean
18Computing the coefficient of 421
correlation r to show the
relationship between shoot
weight and nodule weight in a
Rhizobium strain selection
experiment
Relationship between dry weights of A.18
plant tops and nodules in cowpea
19A brief description of inoculant 427
carrier preparation
20Seed inoculating procedure 430
21Determining field capacity of 432
field soil
Determining field capacity of A.19
Field soil
22Simple transfer chamber 436
Cross section of chamber A.20
Illustrating working principle
Simple transfer chamber A.21
23Freeze drying cultures of 441
Rhizobium
Sealing ampoules A.23
Sealing ampoules (close up) A.24
24Source of Rhizobium strains 454