Culturing and Enumerating Bacteria from Soil Samples

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.

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,

Therefore,

Table 1: Dilution and plating of the samples.

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.