Culturing Bacteria from Soil Samples
Bacterial Culturing
There are two fundamental applications of dilution and plating of soil bacteria. The first application is the enumeration of culturable bacteria within a particular soil. The quantification of the number of soil bacteria gives an indication of soil health.
The second major application is the visualization and isolation of pure cultures of bacteria. The pure cultures can subsequently be characterized and evaluated for specific characteristics, or can be used for identification using sequesncing techniques.
| Step | Add to... | Dilution |
|---|---|---|
| 5 g soil (weight/volµme) | 50 mL sterile water (solution A) | 10-1 |
| 1 mL solution A (volµme/volµme) | 9 mL sterile water (solution B) | 10-2 |
| 1 mL solution B (volµme/volµme) | 9 mL sterile water (solution C) | 10-3 |
| 1 mL solution C (volµme/volµme) | 9 mL sterile water (solution D) | 10-4 |
| 1 mL solution D (volµme/volµme) | 9 mL sterile water (solution E) | 10-5 |
Table 1: Dilution and plating of the samples.
Prerequisites
Execution Order
Culturing Bacteria from Soil Samples
Prerequisite Protocols
Making LB Plates
Primary
Procedure
Step 1.
- Prepare three LB agar plates and label them as C, D, and E.
- Add 9 ml of distilled H₂O to each of six 15 ml falcon tubes.
Step 2.
Weigh out 5 g of soil sample, put it in a 50 ml falcon tube, and bring to 50 mL with deionized water. Shake the suspension well, and label as 'A'.
Step 3.
Before the soil settles, remove 1 mL of the suspension from tube 'A' with a sterile pipette and transfer it to one of the 15 ml falcon tubes. Vortex thoroughly, and label as 'B'.
Step 4.
Repeat this dilution step three more times, each time adding 1 mL of the previous suspension into a 15 ml falcon tube containing 9 ml of deionized water. Label these sequentially as tubes C, D, and E. This results in serial dilutions of 10-1 through 10-5 grams of soil per mL.
Step 5.
Vortex samples C, D, and E, and pipette 100 μl onto each corresponding plate, covering the plates as you go.
Step 6.
You will use a sterile microcetrifuge tube to spread the media evenly across the platre. Holding the tube with the hinge up, use the conical end to spread the drop of inoculµm around the surface of the agar until traces of free liquid disappear. Replace the plate lid.
Step 7.
Incubate the bacteria plates at room temperature for 1 week. Make sure the plates are inverted during the incubation to prevent drops of moisture from condensation from falling onto the agar surface.
7 days
Step 8.
After incubation, examine all of the bacteria plates carefully, and note differences in colony size and shape. When grown on agar, bacteria typically form slimy colonies ranging from colorless to bright orange, yellow, or pink, or colonies that are chalky, firm, and leathery.
Step 9.
Count and record the nµmber of bacterial colonies, including any actinomycetes. Only count plates with 30-200 colonies per plate.
Step 10.
Select individual bacterial colonies from any of the plates. Use a high dilution plate, as it tends to have pure colonies that are separated well. Choose only colonies that are well-separated from neighboring colonies and look morphologically distinct from each other.
Step 11.
Using a sterile loop, remove a small amount of a colony of interest onto the loop.
Step 12.
Using the loop, make several streaks a few centimeters long on one side. Using a fresh loop, make a streak that crosses the initial streaks only on the first pass. Repeat this process twice more in the same manner. This streaking “dilution” results in cells on the plate being separated from one another. Place the plate in a dark area to incubate at room temperature for two weeks.