plant surface microbiology.pdf

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Thomas F.C. Chin-A-Woeng and Ben J. J. Lugtenberg
50 % (v/v) sodium hypochlorite can be prepared from commercial stocks. The
effectiveness of a certain procedure is dependent upon the species and source
of the seeds. To ensure sterility, checks should be performed by placing the
disinfected seeds on rich agar medium. Care should be taken to remove traces
of the disinfectant since this may influence germination efficiency as well as
the survival of the bacteria after coating or inoculation of the seed. Sterilised
tomato (Lycopersicon esculentum) seeds are obtained by rinsing tomato seeds
with household bleach (adjusted to approximately 5 % sodium hypochlorite)
and stirring in a sterile flask for 3 min. Not all seeds sink to the bottom of the
flask despite stirring. After 3 min, sterilised demineralised water is added and
most, if not all, seeds will then sink to the bottom of the flask. Seeds that
remain floating are discarded. The hypochlorite is removed by washing the
seeds five times extensively with 20 ml sterile water, followed by 2-h washing
in sterile water during which the water is replaced at least three times. Contamination
checks, carried out by placing the disinfected seeds on King’s
medium B agar (KB), show whether the seeds are free of contaminating
microorganisms. For colonisation assays, this method is a reliable disinfection
method. For disinfection of grass and wheat seeds, NaOCl/0.1 % SDS
solutions can be used.
If seedlings are used instead of seeds, the surface disinfected seeds are
placed on PNS solidified with 1.8 % Bacto Agar and placed in the dark to allow
germination. Prior to transfer to a suitable temperature for germination (e.g.
28 °C for tomato), the seeds are incubated overnight at 4 °C, which often
improves the germination efficiency and enhances synchronous germination
of the seeds. For seeds such as tomato, wheat, or radish, it subsequently takes
1–2 days before 3–5-mm root tips appear. Seeds are inspected for proper germination
and seedlings with the same length of root tips are selected.
3.3 Growth and Preparation of Bacteria
Liquid cultures of bacterial strains are grown overnight on a rotary shaker.
For colonisation experiments with a mixture of strains (e.g. wild type versus
mutant) a suspension of washed bacteria is prepared in a 1:1 ratio. A volume
of 1.0 ml of an overnight culture is sedimented by centrifugation and the
supernatant is discarded. The cells are washed with 1 ml phosphate buffered
saline (PBS: 20 mM sodium phosphate, 150 mM NaCl, pH 7.4) and resuspended
in PBS. The concentration of bacteria in this suspension is determined
by measuring the optical density (OD 600 nm ). The strains are diluted to
a concentration of 1◊10 8 CFU/ml. If a mixture of strains is to be used for inoculation,
the cells are mixed prior to inoculation of the seeds or seedlings, e.g.
in a 1:1 ratio. The suspension is vortexed vigorously to yield a homogenous
suspension of two strains.