Determining an accurate count of environmental bacteria is critical to assessing the health of a soil ecosystem. This can be accomplished by culturing bacterial colonies with appropriate dilutions.

Soil bacteria are an important part of a healthy soil ecosystem, playing roles in decomposition, nitrogen fixing, and nutrient cycling. Surface soils are a substrate of organic and inorganic particles forming a complex matrix surrounding pores that fill with air or water. These conditions create an ideal ecosystem for bacteria, with surface soils typically containing upwards of a million bacteria per gram.

Because of this high concentration of bacteria, dilution is necessary before plating onto growth media. Serial dilutions can reduce the concentration of the original soil sample to levels low enough for single colonies to be grown on media plates, allowing for the calculation of the initial counts of bacteria in the soil sample.

This video will illustrate how to prepare serial dilutions of soil samples, how to plate these bacterial samples, and how to calculate soil bacterial counts from the dilution plates.

Bacteria are simple prokaryotic organisms, but highly diverse in terms of species and ecosystem. Actinomycetes are a subset of bacteria that are frequently considered a unique group due to their filamentous structure, where they grow strung together to form hyphae.

Typically, actinomycetes account for 10% of the total bacterial population in soil. Bacteria and actinomycetes are abundant in almost every environment on Earth, but are found in unparalleled diversity and abundance in soil.

Soil bacteria are enumerated, and potentially cultured and identified by dilution plating. Here, a soil sample is serially diluted in water, and then dispersed onto agar growth plates. The resulting colonies are then counted. This value, along with the dilution factor, is used to elucidate the initial concentration of bacteria in soil.

Dilution plating is a common, inexpensive, and simple method for bacterial enumeration, but it does suffer from several drawbacks. The soil should not be allowed to settle during dilutions, which could lead to biased growth. Some soil bacteria will not culture on the plates, or grow too slow to be observed. Additionally, this method assumes that colonies are formed from a single bacterium, but they may arise from clumps of multiple cells.

Certain bacterial species grow better on different growth media. A common substrate to culture a wide range of bacteria is an agar-peptone yeast plate. Actinomycetes preferentially grow on glycerol-casein based plates, which better suit the growth of these filamentous bacteria.

Now that you understand the concept behind bacterial enumeration, let’s see how this process is carried out in the laboratory.

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”. Before the soil settles, remove 1 mL of the suspension with a sterile pipette and transfer it to 9 mL of deionized water. Vortex thoroughly, and label as “B”.

Repeat this dilution step 3 times, each time with 1 mL of the previous suspension and 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.

To grow bacterial colonies, take 3 pre-prepared peptone-yeast agar plates and label them as C, D, and E. Vortex the corresponding samples and pipette 0.1 mL onto each plate. Since CFUs are reported per mL, using 0.1 mL further increases the dilution by a factor of ten.

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.

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 to cool, and then spread the drop of inoculum around the surface until traces of free liquid disappear. Replace the plate lid.

Re-flame the spreader and repeat the process with the next plate, working quickly so as not to contaminate the plate with airborne organisms. Invert the plates to prevent moisture drops falling onto the agar, and incubate the plates at room temperature for one week.

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 1/10th of the bacterial population.

Finally, invert these plates and store at room temperature for two weeks.

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.

Count and record the number of bacterial colonies, including any actinomycetes. Only count plates with 30-200 colonies per plate.

To grow cultures of specific individual bacteria, select discrete colonies from any of the plates, choosing colonies that are well separated from neighboring colonies. Sterilize a spreading loop, then open the plate and touch the loop to an empty spot to cool. Next, pick a small amount of the colony of interest onto the loop.

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.

From the bacterial and actinomycete colony counts, the Colony Forming Units per gram of soil can be determined. The number of colonies per gram of soil is equal to the number of colonies counted on the plate, multiplied by the reciprocal of the dilution plated. For example, if 46 colonies are counted on a dilution plate of 10-5 with a 0.1 mL inoculant, then the CFU per gram of soil is equal to 10-6 by 46, or 46 million CFUs per gram of soil.

Performing bacterial counts and cultures is a key first step in many scientific investigations or protocols.

Healthy soil contains between 106 and 108 bacteria per gram. Bacterial enumeration of soil can be used to determine the health of soils of interest, and counts of less than 106 and 108 bacteria per gram indicate unhealthy or poor soil. This may be caused by a lack of nutrients or organic matter, abiotic stress due to extreme soil pH, or soil contamination.

Many types of antibiotics used in medicine today were first identified from soil dwelling bacteria or fungi. Isolating pure strains from soil cultures can help in potentially help scientists identify new antibiotic compounds. Here, test bacteria or isolated compounds of interest can be added to plates grown with bacterial lawns, and their effects on the lawn growth recorded. Clear patches in bacterial lawns where growth was inhibited by the test bacteria or compound may indicate antibiotic activity.

Leguminous plants including peas and beans rely upon symbiotic relationships with nitrogen-fixing bacteria. These bacteria live freely in the soil or within nodules in the root system, and produce nitrogen compounds that are utilized by the plant. In poor soils, supplementing leguminous crops with cultured nitrogen-fixing bacteria from soils may boost the growth and health of the plants. This can result in bigger, hardier plants, which in turn give greater crop yield.

You’ve just watched JoVE’s introduction to bacterial enumeration. You should now understand how to perform dilutions of soil samples, how to plate for colony counting and colony isolation, and how to calculate the numbers of bacteria in soil samples. Thanks for watching!