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.
b.
c.
d.
e.
f.
Culturing strains of rhizobia in YM broth
Germinating seeds
Preparing a Fahraeus-slide
Inoculating the seedlings
Observing the root-hairs under the microscope
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 slowgrowing 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.
b.
c.
d.
e.
Preparation of somatic antigens from cultured cells
Dilution of stock antiserum
Performing agglutinations in microtiter trays
Performing agglutinations in tubes
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.
b.
c.
d.
e.
f.
g.
Fractionating serum globulins
Purifying the serum globulins
Determining the protein content of the dialyzate
Conjugating the globulins with fluorescent dye
Purifying the fluorescent antibodies
Testing the quality of fluorescent antibody
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. . . . . . . . . . .
a.
b.
c.
d.
e.
f.
g.
191
Culturing strains of Rhizobium
Preparing seedling-agar tubes and Leonard jars
Preparing germination plates
Surface sterilizing seeds
Planting and inoculating
Observing periodically and harvesting
Evaluating the experiment
Requirements
Exercise 16. TO SCREEN RHIZOBIA FOR NITROGEN FIXATION
POTENTIAL. . . . . . . . . . . . . . . . . 201
a.
b.
c.
d.
e.
f.
Experimental design and treatments
Preparing Leonard jars
Culturing the rhizobia for testing
Surface sterilizing the seeds
Planting and inoculating of seeds
Harvesting the plants
Requirements
Exercise 17. SELECTING EFFECTIVE STRAINS OF RHIZOBIA IN
POTTED FIELD SOIL. . . . . . . . . . . . . 210
a.
b.
c.
d.
e.
f.
g.
h.
Designing the experiment and treatments
Preparing the inoculum
Choosing the site for collecting soil
Collecting, preparing, and potting field soil
Adjusting moist field soil to field capacity
Applying fertilizer
Planting and inoculating the seeds
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.
b.
c.
d.
e.
f.
g.
h.
i.
j.
Setting up the experiment
Selecting strains for the experiment
Preparing inoculants
Preparing seeds for inoculation and planting
Preparing the field
Controlling cross-contamination by modifying
irrigation methods
Applying fertilizer
Planting the experiment
Monitoring the trial and harvest
Analyzing the data
Requirements
Exercise 19. TO INVESTIGATE THE IMPORTANCE OF OPTIMAL
FERTILITY IN THE RESPONSE OF A LEGUME TO
INOCULATION WITH RHIZOBIUM. . . . . . . .
a. Setting up the experiment
b. Preparing the mixed inoculant and inoculating the
236
c.
d.
e.
f.
g.
h.
seeds
Choosing a site and preparing the field
Applying fertilizers
Planting the experiment
Monitoring the trial and harvest
Harvesting nodules for strain identification
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. . . . . . . . . . . . . . . .
a.
b.
c.
d.
262
Inoculating starter cultures
Assembling simple fermenters
Operating the glass fermenters
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. . . . . . . . . . . . . . . . . . .
a.
b.
c.
d.
Culturing rhizobia in YM broth
Making a culture dilution flask and its operation
Preparing the diluents
Preparing packaged presterilized peat and
checking for sterility
e. Preparing presterilized peat in
polypropylene trays
293
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. . . . . . . . . . . . .
a.
b.
c.
d.
311
Preparing inoculants for seed inoculation
Preparing adhesives
Inoculating and pelleting seeds
Determining the number of viable rhizobia on seeds
Requirements
References and Recommended Reading. . . . . . . . . . . 323
Appendices. . . . . . . . . . . . . . . . . . . . . . . 328
TABLE OF CONTENTS FOR FIGURES
FIGURE
TITLE
PAGE
1.1
Streaking the plate
15
1.2
Isolation procedures as used
by Date and Halliday (1979b)
17
1.3
Ceramic bead method for
storing Rhizobium
24
2.1
Petri dish with components of
Fahraeus slide
33
2.2
Placement of seedling on
Fahraeus slide
33
2.3
Roothair deformation showing
shepherd’s crook
36
2.4
Selective proliferation and
colonization of Rhizobium
trifolii on a roothair
37
2.5
Rhizobium trifolii inside infection
thread of clover roothair
37
3.1
Shapes of bacteria
44
4.1
The Petroff Hausser counting
chamber
56
4.2
Procedure for serial dilutions
62
4.3
Growth of colonies of Rhizobium
sp. From drops plated by the dropplate method
65
5.1
Soybean plants growing in growth
pouches
77
B.1
Lattice formulation in an antigenantibody reaction
93
B.2
Precipitin reactions
96
B.3
Direct immunofluorescence
98
B.4
Indirect immunofluorescence
99
7.1
Scheme for antiserum titer
determination in agglutination
tray
117
7.2
Agglutination reactions in wells
of agglutination tray
121
7.3
Agglutination reactions in
agglutination tubes
121
8.1
Identification of nodule bacteroids
by agglutination in an agglutination
tray
131
9.1
Hexagonal pattern template for Petri
dishes
136
9.2
Well pattern for immunodiffusion
137
9.3
Immunodiffusion reactions showing
precipitin bands
139
10.1
Scheme for identifying nodules
inoculated with a mixture of two
strains
147
11.1
Scheme of nodule smears for strain
identification by FA
165
13.1
Plate with grid pattern for nodule
170
identification by antibiotic resistance
13.2
Interpreting growth patterns on
antibiotic plates
172
16.1
An example of randomized complete
block design experiment
203
18.1
Field layout and dimensions
222
18.2
Diagram of field plot
223
19.1
Field layout and dimensions
239
20.1
Scheme of simple fermenter unit
265
20.2
Simple fermenter in operation
266
20.3
Modified fermenter
267
22.1
Apparatus for diluting liquid
cultures of Rhizobium
296
APPENDIX CONTENTS
Appendix
Number
1
Title
Page
Figure
Number
Number
Characteristics 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
3
Media and staining solutions
340
4
Reagents
351
5
Buffers
355
6
McFarland nephelometer barium
358
sulfate standards
7
Preparation of seedling-agar
360
slants for cultivating small
seeded legumes
Simple set up for dispensing seedling-
A.9
Agar into tubes and forming slants
8
Building a rack for growth pouches
Rack for growth pouches
363
A.10
9
Recommendations of hosts and
365
growth systems for authentication
10
Surface sterilization of seeds
369
11
Preparation of Leonard Jars
375
The Leonard Jar
12
Injecting and bleeding rabbits
A.11
378
Bleeding rack
A.12
Bellco bleeding apparatus
A.13
Collecting blood from a rabbit by
A.14
cardiac puncture
13
The indirect FA technique
386
14
Additional explanations to the
391
calculations of the most probable
number (MPN)
15
The 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
16
Methods for determining lime
410
requirements of acid soils
17
Analysis 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
18
Computing 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
19
A brief description of inoculant
427
carrier preparation
20
Seed inoculating procedure
430
21
Determining field capacity of
432
field soil
Determining field capacity of
A.19
Field soil
22
Simple transfer chamber
436
Cross section of chamber
A.20
Illustrating working principle
Simple transfer chamber
23
Freeze drying cultures of
A.21
441
Rhizobium
24
Sealing ampoules
A.23
Sealing ampoules (close up)
A.24
Source of Rhizobium strains
454