https://app.jove.com/v/10099/culturing-and-enumerating-bacteria-from-soil-samples

1. Preparation of Soil Dilutions

1. To begin the procedure, weigh out 10 g of soil sample and add to 95 mL of deionized water. Shake the suspension well, and label as “A”.
2. Before the soil settles, remove 1 mL of the suspension with a sterile pipette and transfer it to a 9-mL deionized water blank. Vortex thoroughly, and label as “B”.
3. Repeat this dilution step three times, each time with 1 mL of the previous suspension and a 9-mL deionized water blank. 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

2. Making Spread Plates for Bacterial Culture

1. To grow bacterial colonies, take three pre-prepared LB agar plates and label them as C, D, and E. Vortex samples C, D, and E, and pipette 0.1 mL onto each plate. This increases the dilution value further, by a factor of ten (C = 10^-3, D = 10^-4, E = 10^-5).
2. Next, dip a glass spreader into ethanol. Place the spreader in a flame for a few seconds to ignite and burn off the ethanol. This will sterilize the spreader.
3. Hold the spreader above the first plate until the flame is extinguished. Open the plate quickly, holding the lid close by. Touch the spreader to the agar away from the inoculum (Inoculum = cells used to begin a culture) to cool, and then spread the drop of inoculum around the surface of the agar until traces of free liquid disappear. Replace the plate lid.
4. Re-flame the spreader and repeat the process with the next plate, working quickly so as not to contaminate the agar with airborne organisms
5. 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.

3. Making Spread Plates for Actinomycetes

1. To grow actinomycetes, take three pre-prepared glycerol-casein plates and label them as B, C, and D. Using the techniques shown previously, spread plate 0.1 mL from the suspensions B, C, and D. The lower dilutions are used because actinomycetes are typically present as 1/10^th of the bacterial population (B = 10^-2, C = 10^-3, D = 10^-4).
2. Incubate the actinomycete plates (inverted) at room temperature for 2 weeks.

4. Bacterial and Actinomycete Counts

1. After incubation, examine all of the bacteria plates carefully, and note differences in colony size and shape. When grown on agar, bacteria produce slimy colonies ranging from colorless to bright orange, yellow, or pink. In contrast, actinomycete colonies are chalky, firm, leathery, and will break under pressure, where other bacterial colonies will smear. This allows colonies to be distinguished by touch with a sterile loop.
2. Count and record the number of bacterial colonies, including any actinomycetes. Only count plates with 30-200 colonies per plate.

5. Isolation of Pure Cultures

1. Select individual bacterial colonies from any of the plates. More colonies can be selected if there is particular interest in the soil. 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.
2. Sterilize the loop by dipping it in alcohol and flaming it. Quickly open the Petri dish of interest, and touch the loop to a bare spot in the agar to cool it. Then, remove a small amount of a colony of interest onto the loop.
3. Taking a fresh peptone-yeast plate, make a streak a few centimeters long on one side. Sterilize and cool again, then make a streak that crosses the initial streak only on the first pass. Repeat this process twice more in the same manner. This streaking “dilution” results in cells on the loop being separated from one another. Place the plate in a dark area to incubate at room temperature for two weeks.