ompiled By: Babu Ram Panthi Page 2
infestation by insect pests determines the seed heath status of a seed sample and by inferenceseed lots.
It is based for most diseases upon symptoms, isolation and practical identification schemesusing relatively few but important key tests. When testing for seed borne bacterial pathogen fromseeds, the following steps are usually involved:1)
Extraction of bacteria from seeds.2)
Isolation and purification.3)
Identification of the bacterial isolates.
Extraction of bacteria from seeds
Bacteria can be extracted from seeds by following methods:1.
Direct plating of seeds on various media followed by incubation at optimum conditions,especially temperatures ranging between 25 and 30°C, and darkness.2.
Soaking either whole seeds or ground-up seeds in sterile water or saline water for different durations followed by plating the water containing bacterial cells onto differentmedia including semi selective or selective ones.3.
growing seedlings from seeds on various substrates and then isolating bacteria from plantshowing symptoms followed by the streaking onto a medium, preferably general oneslike Nutrient agar (NA), Growth factor (GF), KB etc
Isolation and purification of bacteria
Bacteria thus extracted are isolated on media of special composition and purified in order to be able to identify them. The procedure is described below.1.
Surface sterilize infected plants by dipping into 95% ethanol before treating with NaOCl(0.5-1%) solutions for 2-5 minutes and rinse 2-3 times with sterile water. Leaf materialsor roots can also be washed in running tap water for 1-2 hours.2.
Cut small area of the affected tissues on a clean glass slide and mount in a drop of water.Put a cover slip and examine under compound microscope for streaming of bacterialcells. Only if bacterial ooze is observed proceed with further steps of isolation.3.
Remove the cover slip and put the infected tissue in a few more drops of water, teaseapart he tissue and leave it for 10-15 minutes to obtain more bacterial cells in the water.4.
Using a bacteriological loop streak the water containing bacteria a number of times on asuitable medium in a cross wise manner. Try to get single colony of bacterium.5.
Incubate the streaked plates in an inverted position at 25-30°C.6.
Start observing the plates after 1-2 days for pseudomonas and up to 4 days for xanthomonads. The colonies of pseudomonads on nutrient agar are white, or whitish