Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Authors: Bradley Schmitz and Luisa Ikner
Surface soils are a heterogeneous mixture of inorganic and organic particles that combine together to form secondary aggregates. Within and between the aggregates are voids or pores that visually contain both air and water. These conditions create an ideal ecosystem for bacteria, so all soils contain vast populations of bacteria, usually over 1 million per gram of soil.
Bacteria are the simplest of microorganisms, known as prokaryotes. Within this prokaryotic group, there are the filamentous microbes known as actinomycetes. Actinomycetes are actually bacteria, but they are frequently considered to be a unique group within the classification of bacteria because of their filamentous structure, which consists of multiple cells strung together to form hyphae. This experiment uses glycerol case media that select for actinomycete colonies, during dilution and plating. Typically, actinomycetes are approximately 10% of the total bacterial population. Bacteria and actinomycetes are found in every environment on Earth, but the abundance and diversity of these microbes in soil is unparalleled. These microbes are also essential for human life and affect what people eat, drink, breathe, or touch. In addition, there are bacterial species that can infect people and cause disease, and there are bacteria that can produce natural products capable of healing people. Actinomycetes are particularly important for producing antibiotics, such as streptomycin. Bacteria are critical for nutrient cycling, plant growth, and degradation of organic contaminants.
Bacteria are highly diverse in terms of the number of species that can be found in soil, in part because they are physiologically and metabolically diverse. Bacteria can be heterotrophic, meaning they utilize organic compounds, such as glucose, for food and energy, or autotrophic, meaning they utilize inorganic compounds, such as elemental sulfur, for food and energy. They can also be aerobic, utilizing oxygen for respiration, or anaerobic, utilizing combined forms of oxygen, such as nitrate or sulfate, to respire. Some bacteria can use oxygen or combined forms of oxygen and are known as facultative anaerobes.
One way to enumerate the number of bacteria present in a soil sample is to utilize dilution and plating methodology. This methodology utilizes agar as a medium for bacterial growth, a process termed, “culturable technology.” Because of the vast numbers of bacteria found within soils, a small sample of soil is serially diluted in water, prior to being plated on agar within a Petri plate. Typically, a small amount of soil contained within 0.1 to 1 mL of the diluted soil suspension is “spread” over the surface of the agar plate. The plates contain agar, which is molten when hot, but solid when cool. In addition to the agar, nutrients, such as peptone yeast or a product commercially available as R2A, are added to the medium to allow for the growth of heterotrophic bacteria.
Dilution and plating is an inexpensive and relatively simple technology for the enumeration of soil bacteria. However, there are several drawbacks to the technique. Some common errors and assumptions associated with dilution and plating assays are as follows: it is assumed that every single soil bacterium gives rise to a colony, but in reality a colony may arise from a clump of cells, resulting in an underestimation of true culturable count. During serial dilution of the soil, soil particles can settle out (fall to the bottom), so the true aliquot of soil is not passed on into the next dilution. Many soil microbes are viable but non-culturable. Slow growing bacteria may not result in visible colonies within a reasonable time frame (1-2 weeks).
Also, anaerobic bacteria do not grow under aerobic conditions, and bacteria that do grow are selected for by the nutrients added to the medium. Thus R2A selects for heterotrophic bacteria, while elemental sulfur selects for autotrophic sulfur oxidizers. Overall, it is estimated that only 0.1 to 1% of all soil bacteria can be cultured. Therefore, dilution and plating of soil bacteria only accounts for culturable bacteria and underestimates the true viable soil population by one to two orders of magnitude. An example of heterotrophic bacterial colonies that resulted from soil dilution and plating is shown in Figure 1. Note that approximately 1 million bacterial cells are needed for a colony to be visible to the naked eye.
This experiment demonstrates the dilution and spread plating methodology used to enumerate the number of bacteria within a soil sample. Specifically, two media are used: one designed for all bacteria, and the other that selects for actinomycetes. Once the bacterial colonies have grown on the agar plates, isolate the pure cultures of selected colonies by using a streak plate technique. Such pure cultures can then be further analyzed and characterized for specific traits and functions.
Figure 1. Heterotrophic colonies on an R2A agar plate. A number of discrete colonies with diverse morphology arise after dilution and plating from soil. Permission for use granted by Academic Press.
1. Preparation of Soil Dilutions
- 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 a 9-mL deionized water blank. Vortex thoroughly, and label as “B”.
- 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
- To grow bacterial colonies, take three pre-prepared peptone-yeast 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).
- 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 (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.
- Re-flame the spreader and repeat the process with the next plate, working quickly so as not to contaminate the agar with airborne organisms
- 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
- 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 (B = 10-2, C = 10-3, D = 10-4).
- Incubate the actinomycete plates (inverted) at room temperature for 2 weeks.
4. Bacterial and Actinomycete Counts
- 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.
5. Isolation of Pure Cultures
- 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.
- 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.
- 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.
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!
A 10-g sample of soil with a moisture content of 20% on a dry weight basis is analyzed for viable culturable bacteria via dilution and plating techniques. The dilutions were made as shown in Table 1. 1 mL of solution E is pour-plated onto an appropriate medium and results in 200 bacterial colonies.
But, for 10 g of moist soil,
|10 g soil (weight/volume)||95 mL saline (solution A)||10-1|
|1 mL solution A (volume/volume)||9 mL saline (solution B)||10-2|
|1 mL solution B (volume/volume)||9 mL saline (solution C)||10-3|
|1 mL solution C (volume/volume)||9 mL saline (solution D)||10-4|
|1 mL solution D (volume/volume)||9 mL saline (solution E)||10-5|
Table 1: Dilution and plating of the samples.
Applications and Summary
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. For example, if there are 106 to 108 culturable bacteria present per gram of soil, this would be considered a healthy number. A number less than 106 per gram indicates poorer soil health, which may be due to a lack of nutrients as found in low organic matter soils; abiotic stress imposed by extreme soil pH values (pH < 5 or > 8); or toxicity imposed by organic or inorganic anthropogenic contaminants.
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 that may be useful in either medical or environmental applications. Examples include: antibiotic production; biodegradation of toxic organics; or specific rhizobia useful for nitrogen fixation by leguminous crops, such as peas or beans.