Experiment 2: Bacterial Transfer to a Stab Tube and an Agar Plate

Transferring an isolated bacterial colony from one plate to another or to a different type of growth surface is an important technique in microbiology. Frequently, a microbiologist is required to initially grow bacteria on one type of media then transfer it to a selective and/or differential plate to further characterize the bacteria. Stab tubes can be used for testing the motility of a microbe or for longer term storage at refrigerator temperature (4 °Celsius). Again, this transfer must be performed using aseptic technique to ensure the purity of the originally isolated microbe.

Procedure

Prepare the Agar Plates:

1. Prepare four Nutrient agar plates by following Steps 1 - 5 of Experiment 1.
2. Slowly pour agar into the snap-cap tubes until each snap-cap tube contains approximately 2 mL of agar. The snap-cap has a 5 mL volume capacity; therefore, your snap-cap should be approximately 40% full.
   Note: The snap-caps tubes are referred to as “stab tubes” after the agar has been poured and gelled.
3. Snap the lid on the caps and store them upright at room temperature until the agar has solidified and cooled. The tubes can be used immediately or stored at 4 °C. If stored, let warm to room temperature for approximately 1 hour before use.
   Note: Stab tubes should be vertically positioned in a 100 mL beaker to allow the agar to solidify properly.
4. Put on a pair of gloves, and find the petri plate that you reserved from Experiment 1. Locate three separate bacterial colonies. If possible, these colonies should have different morphologies from each other. Record observations of their physical characteristics in Table 2.
5. Remove about half of 1 of the selected colonies on the plate by scraping it up with an inoculating loop.
6. Remove the lid from one of the agar plates prepared in Step 1. Remember to hold the lid closely over the top of the plate to prevent airborne contamination of the plate.
7. Gently streak an inoculating loop over the surface of the agar in a zig-zag pattern, taking care to not dig into the surface of the agar with the loop. Dispose of the loop.
8. Place the lid on the plate and seal it with Parafilm™.
9. Turn the plate upside-down and label it as “Plate #1” with your permanent marker.
10. Use a new inoculating loop to remove the other half of the previously selected colony.
11. Stab/push the loop approximately half way into one of the stab tubes.
12. Pull the inoculating loop straight up and out through the same path that it went down into the media. Dispose of the loop.
13. Snap the lid on the tube, and label the tube as “Tube #1” with your permanent marker. Be sure to press the lid all the way down to create an air-tight seal.
14. Repeat Steps 6 - 9 and 10 - 13 with two other bacterial colonies from your reserved plate. Label the plates and tubes as “Plate #2” “Tube #2”, “Plate #3”, and “Tube #3”, respectively.
15. Label the fourth tube and plate as “Control”. Do not streak or stab anything onto/into them. Leave these containers covered.
16. Incubate the plates and tubes at room temperature or warmer (not to exceed 37.7 °C or 100 °F) for 3 days and observe for growth. Record your results in the Table 3.
17. Pour approximately 5 mL of the 10% bleach solution onto the surface of the remaining agar plates, allowing it to cover the entire surface. Incubate them for 20 minutes, and then pour the bleach down the sink with running water.
18. Seal the petri dishes with Parafilm™ and dispose of them in the trash.
19. Fill the stab tubes with the 10% bleach solution and incubate them for 20 minutes at room temperature.
20. Pour the bleach down a drain with running water, snap the caps onto the tubes, and dispose of them in the trash.