EXERCISE 12
TO DEVELOP ANTIBIOTIC RESISTANT RHIZOBIA
Rhizobia bearing genetic markers are obtained through a mass
selection technique.
Bacterial strains contain small numbers of
naturally occurring mutants which are resistant to high
concentrations of certain antibiotics.
This resistance may be
used for the recognition of rhizobial strains.
Key steps/objectives
1)
Culture rhizobia in YM broth
2)
Prepare YMA plates containing antibiotics
3)
Spread selected culture(s) onto appropriate antibiotic and
non-antibiotic plates
4)
Check for natural resistance
5)
Transfer resistant colonies to YMA slants
6)
Culture resistant isolates in YM broth
7)
Spread broth culture(s) resistant to streptomycin onto
plates containing spectinomycin (and vice versa)
8)
Transfer double resistant mutants to YMA slants.
Confirm
resistance to streptomycin and spectinomycin; streak onto
plates containing both antibiotics
9)
Confirm retention of symbiotic effectiveness of resistant
strain
(a)
Culturing selected strains
(Key step 1)
Select strains for the development of antibiotic resistant
mutants.
Culture the strains in duplicate flasks containing 50
ml YM broth.
Place on a shaker for 3-7 days according to the
growth rates of the strains chosen.
(b)
Preparing YMA plates containing antibiotics
(Key step 2)
Prepare a stock solution of streptomycin (str) with a
concentration of 4 mg ml-1 (Appendix 3).
of the stock
pore size.
Filter sterilize 5 ml
through a sterile millipore filter of 0.20 micron
Add the filtrate to 500 ml of YMA (in a 1 liter
Erlenmeyer flask) kept molten in a water bath at 50C.
Mix well,
but avoid vigorous shaking to minimize the formation of air
bubbles.
Return the flasks to the water bath for 10 min to re-
equilibrate the temperature and to allow the air bubbles to
dissipate from the agar.
Pour the plates.
These plates will
have streptomycin 40 μg ml-1 agar.
Similarly prepare plates containing 250 μg ml-1 of spectinomycin
(spc) from a stock solution containing 25 mg ml-1 (Appendix 3).
Also prepare YMA plates containing a mixture of both the
antibiotics in the above concentrations for use in the selection
of rhizobia with resistance to two antibiotics.
Prepare an equal number of YMA plates without antibiotics.
All
the plates may be stored under refrigeration.
(c)
Selecting spontaneous mutants with resistance to one
antibiotic
(Key step 3,4,and 5)
Spread 0.1 ml of each broth culture on plates containing (a) no
additives; (b) streptomycin (40 μg ml-1); and (c) spectinomycin
(250 μg ml-1).
The growth rates of rhizobial strains may be retarded in the
presence of antibiotics.
Prepare to incubate up to 12 days but
check for emerging colonies everyday after day 5.
The plates of treatment (a) which contain no antibiotics should
have abundant rhizobial growth.
The plates of treatment (b) and (c) should have very little
growth compared with treatment (a).
Not more than 30 resistant
colonies are expected since the rate of mutation is 1 in 105 to 1
in 107 with most strains of rhizobia.
Pick four colonies each from treatments (b) and (c) and transfer
to separate YMA slants (containing no antibiotics) in culture
tubes.
Incubate at 25-30C for 5-9 days, then store at 4C.
These four isolates must be kept separate till the end of the
selection process.
Confirm the antibiotic resistance of str and spc isolates.
Streak the mutants on YMA containing antibiotics and on a control
plate containing plain YMA.
(d)
Incubate at 25-30C for 5-9 days.
Selecting strains of rhizobia with resistance to two
antibiotics
(Key steps 6, 7, and 8)
To develop strains resistant to both streptomycin and
spectinomycin, spread a 0.1 ml broth culture of a spc mutant on a
plate containing streptomycin (in a similar manner, a str mutant
should be spread on YMA containing spectinomycin).
Incubate at
25-30C for 5-9 days.
Check for growth of colonies on the plates containing
antibiotics.
Again, because of a similar mutation rate as with
resistance to one antibiotic, no more than 30 colonies of
spontaneous mutants with double resistance (str  spc) are
expected.
Transfer four of these colonies to YMA slants (containing no
antibiotics) in culture tubes, incubate, and store. Confirm
resistance to both antibiotics by streaking on plates containing
both streptomycin (40 μg ml-1) and spectinomycin (250 μg ml-1) and
on control plates of plain YMA.
Incubate at 25-30C and compare
growth on antibiotic and control plates.
Streptomycin, spectinomycin and streptomycin-spectinomycin
resistant strains usually retain their N2-fixing capability.
Mutant strains should be compared with their parent strain in a
symbiotic effectiveness test as described in Chapter 20 prior to
use in ecological experiments.
To be useful, mutant strains
should not show significant differences in N2-fixation from the
parent strain.
Requirements
(a)
Culturing selected strains
Transfer hood, incubator and shaker
Bunsen burner
Inoculation loop
Two flasks, 150 ml, containing 30 ml YM broth each
Culture of rhizobia
(b)
Preparing YMA plates containing antibiotics
Filled water bath adjusted to 50C
Suction pump or aspirator with moisture trap
Two sterile filter sterilizing units with sterile millipore
filter (0.20 micron)
Three 10 ml pipettes
Wash bottle with distilled water
Stock solution of streptomycin (4 mg ml-1)
Stock solution of spectinomycin (250 mg ml-1)
Sterile molten YMA, 3 l, in three 2 l or six 10 l
Erlenmeyer flasks
Petri dishes, sterile
(c)
Selecting spontaneous mutants with resistance to one
antibiotic
Incubator, bunsen burner, transfer loop, small beaker of
alcohol
YMA plates containing streptomycin (40 μg ml-1)
YMA plates containing spectinomycin (250 μg ml-1)
YMA plates
Spreading stick
Broth culture
Six YMA slants in culture tubes
Graduated pipette, 1 ml
(d)
Selecting strains of rhizobia with multiple antibiotic
resistance
Transfer or laminar flow hood, tools and incubator as in (c)
Antibiotic stock solutions and YMA plates as in (c)
Six YMA slants
Mutant broth inoculum resistant to streptomycin
Mutant broth inoculum resistant to spectinomycin
Alcohol, spreading stick