Culturing and Enumerating Bacteria from Soil Samples
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.
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. L
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,
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
Skip to...
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved