How Can I Isolate Bacteria From Soil?

Soil Sample

• 1

  Set aside a corked sterile test tube containg 5 ml of nutrient trypticase soy broth. Choose a sterile Popsicle stick.

• 2

  Go outside and collect a small soil sample with the tip of the Popsicle stick, using sterile technique, a method to ensure the sample is not contaminated, and the user is not infected.

• 3

  Uncap the test tube and add the soil to the nutrient broth in the test tube. Mix the soil into the broth with the Popsicle stick. Cover the test tube with the cap. Write the collection date and collection location on the side of the test tube using the wax marking pencil.

• 4

  Put the test tube in a test tube rack for about 30 minutes to allow the soil to settle to the bottom.

Inoculate Petri Dish

• 5

  Disinfect the working surface by cleaning it with 10 percent bleach solution and paper towels.

• 6

  Turn the petri dish so the bottom lid containing the agar is up. Draw a T on the bottom lid, with the crossbar of the T separating off the upper third of the lid and the stem of the T dividing the lower two-thirds in half.

• 7

  Turn on and light the Bunsen burner. Flame the inoculation loop over the Bunsen burner flame.

• 8

  Uncap the test tube and touch the loop to the upper layer of clearer liquid. Withdraw the loop and re-cover the test tube.

The T-Streak Method

• 9

  Use the crossbar of the T you drew on the back of the bottom lid as a reference point. Touch the inoculating loop at one side of the space above the T crossbar and draw the tip over the surface of the agar in a zigzag line, ending the line at the opposite side of the space.

• 10

  Remove the inoculating loop and close the petri dish. Flame the loop in the Bunsen burner, and allow it to cool briefly. Open the petri dish and touch the loop to one spot near the very end of the line already drawn by the loop. Make another zigzag line at right angles to the first line, covering the first space under the T crossbar and to one side of the stem.

• 11

  Remove the loop and close the petri dish. Flame the loop again, open the petri dish, and touch the slightly cooled loop to the second line near its end. Draw another zigzag line at right angles to the second line in the remaining open space on the petri dish.

• 12

  Close the petri dish after withdrawing the loop. Flame the loop and set it aside. Turn off the Bunsen burner.

Secondary Culture

• 13

  Put the petri dish in an incubator at 30 degrees Celsius for 48 hours. Take it out and examine the agar for growth of bacterial colonies. Choose a colony that looks uniform and discrete in overall appearance, an indication that the colony arose from just a single bacterium. Circle the location of the colony on the back of the bottom lid with the marking pencil.

• 14

  Flame the inoculating loop. Open the petri dish and touch the slightly cooled loop to the center of the colony.

• 15

  Repeat the steps for making a T-streak on a new petri plate with agar in the bottom lid. When the streak is made and the petri dish is closed, write the date and the soil sample the inoculum was taken from on the bottom side of the bottom lid. Turn off the Bunsen burner.

• 16

  Incubate the petri plate at 30 degrees Celsius for 48 hours. Examine the colonies that have grown on the agar, looking for uniformity of color, shape and texture. Repeat the secondary culture procedure if there are any colonies that deviate markedly in appearance from the others, indicating that a single bacterium was not isolated.

• 17

  Refrigerate the agar plate that contains identical-appearing colonies on it, with the uniformity being a good indication that only a single bacterium has been isolated from the soil sample.

• 18

  Perform other tests for identification of the isolated bacterium if desired, such as gram stains, microscopic examination and culture on different kinds of agar.