Bacterial endophytes were isolated from plants grown in the foothills of the Appalachian Mountains in Central Virginia, USA (geographic location: 37.125372, −79.298415). The soil was not fertilized. Healthy plants were selected randomly from the natural environment. Plants were taken from the field and brought to the lab for isolation or kept in a refrigerator temporarily. The plants were first washed with tap water to remove the soil, and then surface was sterilized with 5% Clorox® bleach (The Clorox Company, Oakland, CA, USA) containing 8.5% NaOCl with a drop of Tween 20 for 5 min. Finally, the plants were rinsed with sterile water five times.
To confirm the sterilization efficacy, 50 µL of the last rinsate were plated on Luria agar (LA) medium. The surface-sterilized plant was divided into the root, leaf and stem, which were ground individually with sterile water. The ground tissues were centrifuged at 2000 rpm for 3 min to remove the debris. The supernatant was diluted to 10 ×, 100 × and 1000 × with sterile water, and then 50 µL were spread on LA plates. The plates were placed at 28 °C for 2–5 days. Different individual colonies were streaked onto a fresh LA plate for purification and for producing a glycerol stock for long-term storage.
To confirm the sterilization efficacy, 50 µL of the last rinsate were plated on Luria agar (LA) medium. The surface-sterilized plant was divided into the root, leaf and stem, which were ground individually with sterile water. The ground tissues were centrifuged at 2000 rpm for 3 min to remove the debris. The supernatant was diluted to 10 ×, 100 × and 1000 × with sterile water, and then 50 µL were spread on LA plates. The plates were placed at 28 °C for 2–5 days. Different individual colonies were streaked onto a fresh LA plate for purification and for producing a glycerol stock for long-term storage.
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