seeds are the basic unit of plant life. However, these are infected by different types of microorganisms in field or during storage and result in poor yeild. the microorganisms are associated with seed surface and established inside seed coat after penetration the cotyledons and embryos. THere are two methods used for isolation of seed borne microorganisms, i.e. agar plate method and blotter method.
Standard agar plate method for isolation of seed microflora
This method has been described by the International Seed Testing Association (ISTA) (1966). In this method nutrient agar medium is used. Externally as well as internally seed borne microorganisms grow on agar surface.
- Potato dextrose agar (PDA) medium
- Seeds to be tested
- Polythene bags
- NaOCl (sodium hypochlorite) solution (1%)
- Sterile distilled water
- Illuminated growth chamber with controlled temperature
- Collect seed samples from store containers or any seed sample or just before harvesting of the food grains, and keep them in sterile polythene bags. According to ISTA (1966) 200 seeds per sample should be tested.
- Divide the seeds into two lots, the first one for externally seed borne microorganisms, and the second for internally seed borne microorganisms.
- SUrface sterilise the first lot of the seeds with 1% NaOCl solution for 2 – 5 minutes to analyse the internally seed borne microorganisms.
- Wash the surface disinfected seeds by sterile distilled water for about 5 – 6 times.
- Dry the seeds keeping inside sterile blotting paper.
- Prepare 6 plates of PDA medium.
- Inoculate 10 sterilized seeds per PDA plates in three replicates (for internal seed borne microorganisms), and 10 unsterilised seeds per PDA plate in three replicates (for surface associated i.e. externally seed borne microorganisms).
- Incubate the petri plates at 20 C with 12 hours light at NUV (near ultraviolet) and 12 hours darkness for about 6 – 8 days.
After incubation microorganisms grow on agar surface. isolate, purify and preserve (if required for further study) the microorganisms.
Dr. R. C. Dubey – Practical Microbiology