Bacterial isolation

Purpose

To isolate bacteria from plant tissue and obtain pure cultures

Materials

• Scalpel or shears
• Sterile Water
• 2 Nutrient agar plates or other bacterial media per sample
• Tissue homogenizer (Dounce or Potter-Elvehjem type)
• Inoculating Loop

Instructions

Typical pattern used in streaking bacteria. Streaking is done in the order of Black, Orange, Blue, Purple.

1. Select a piece of symptomatic tissue
2. cut along into small pieces- not much tissue is needed
3. Add a small amount of water
4. Grind tissue and mix
5. Use pestle to inoculate plates in initial streak in first section of plate (black streak on picture)
6. Using inoculating loop and aseptic technique, streak plate on the second quarter of the plate, crossing over the initial streak area one time (orange streak on picture)
7. Heat sterilize the loop between sections
8. Repeat on third quarter, crossing over the second quarter only once- being careful not to touch the first or second quarter (blue streak on picture)
9. If there is enough room on the plate, repeat for a fourth quarter (purple streak on picture)
10. Incubate plates at 30°C for 48 hours
11. Observe plates for growth
12. Choose individual colonies- lightly touch the top of an individual colony with a sterile loop or needle and streak on a fresh agar plate like above

Notes

• Plant pathogens are usually shiny/mucoid looking-use these for further testing
• Usually four colonies for each sample are used for further testing to get a representative sample

Contributed by

From the University of Florida Plant Disease Clinic-GNV Systems Manual: originally created by Anne Vitoreli; contributed by Anne Vitoreli.