Intro Soils – Lab 5 Soil Microorganisms – Enumerating Heterotrophic Soil Bacteria
Lecture Materials: Soil Organisms (Chapter 11)
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Lab 5 – Enumerating Heterotrophic Soil Bacteria
Soil microbiology laboratory exercises are designed to familiarize students with the basics of microbiology in general including the use of a compound microscope, sterile technique, the preparation of materials including growth media, and even molecular methods including DNA extraction, the polymerase chain reaction, and molecular marker screening.
These skills are then utilized to better characterize and understand soil microbial populations including but not limited to bacteria, fungi, and nematodes, protozoa, and cyanobacteria. Today, we will highlight a mainstay in soil microbiology: how to enumerate cultivable bacteria from soil. Bacteria are generally the most abundant and diverse organisms in soil on the range of 106 to 109 bacteria per gram of soil.
The soil bacterial population is dominated by species of Pseudomonas, Arthrobacter, Clostridium, Bacillus, Micrococcus, Flavobacetrium and others. These bacteria can be difficult to classify as many appear the same as seen with a microscope or on culture plates. Means for classifying bacteria are vast and include their physical characteristics like size, shape, and color of their colonies, nutritional requirements, metabolic products (gas, enzymes, etc.), serology, and more modern techniques which compare their genetic relatedness by characterizing their ribosomal RNA.
There are many methods for estimating numbers of bacteria in soils and include various staining techniques to directly count bacteria using a microscope, plating techniques employing a multitude of various culture media, a statistical technique called most probable number, and molecular approaches characterizing the bulk DNA extracted from soils or monitoring active RNA genes in soil.
Many soil scientists when looking to enumerate the aerobic, heterotrophic bacterial population from soil are content to use the dilution plating technique on a non-selective agar media. It is well known that this technique only measures a small portion of the actual bacterial population due to the inability to adequately replicate soil conditions where these bacteria reside and thrive.
Even with this knowledge, it is very useful to be able to characterize cultivable organisms and how they change over time and on various growth media with any number of research and/or management objectives. The goal for a growth medium is to provide the bacterial population with the carbon and energy sources it needs to grow. Media can either be non-selective or selective.
Non-selective media look to provide wide ranging nutrients and cultivate any and all organisms capable of growing on a solid agar plate or liquid medium. Selective media are used for the growth and cultivation of specific groups of organisms and generally include or exclude nutrients, particular metabolites, or even antibiotics to support the growth of a population of interest.
For plating techniques, media is prepared with agarose, a natural gelling agent, to provide a solid surface where the bacteria can grow and contained on a petri plate routinely 90 mm wide. As soil bacterial numbers are in the billions if not trillions, it is necessary to dilute these samples to reduce the number of colonies on the growth medium down what is called a countable range. Preparing and plating the dilution series is illustrated below.
To prepare the dilution series, first place 10 grams of soil into 95 ml of water; accounting for pore space this is 1:10 dilution. The sample is shaken to mix the soil and water. Then 1 ml is added to a 9 ml dilution tube for another 1:10 dilution; over all in this tube is a 1:100 from the original sample. This is the basis of a serial dilution, each step down the line is another ten-told dilution. For instance, if you started out with 1000 organisms, the 10 fold dilution would net 100 organisms, next dilution down would be 10 organisms, and then another would net 1 organism.
Depending on the range of bacteria in a soil sample you might need more or less dilutions to achieve colony counts on the plate that are in the countable range. If colonies are crowded on the plate as to not be able to see them individually they are said to be ‘too numerous to count’. For this procedure, between 30 and 300 individual colonies is the target on at least one dilution to calculate the colony forming units per gram of soil.
To this end, serial dilutions are made of the soil sample in water and then plated or spread evenly onto the agar media, placed in an incubator at normal growth conditions (approximately room temperature or slightly higher), and then enumerated or counted approximately 24 hours later. The goal is to be able to count individual colonies on the agar plate of at least one dilution range. Each plate is enumerated and data recorded.
The dilution series plate (routinely these series are done in triplicate to gain an average for each dilution) which meets the ’30 to 300’ criteria is used to calculate the number of ‘colony forming units per gram of soil’ (CFU/gram). It is difficult to know whether each of those individual colonies counted on the plate are from one or more than one actual bacteria, so to account for this ambiguity, the term colony forming units is used. Simply multiply the count on the plate by the reciprocal of the dilution plated (swap the sign on the exponent).
For instance, in the example below, the 10-6 plate had 81 colonies counted, so this soil had 81 x 106 CFU/gram of soil. Adjusting for proper scientific notation, you move the decimal one place over so your figure is less than ten, and add one to the exponent: 8.1 x 107 CFU/gram of soil. Lab Reference: Laboratory Exercises in Soil Microbiology, Texas A&M University, Agronomy 405 – Soil Microbiology, Dr. David Zuberer.
Images from a Bacteriology course at the University of Wisconsin (Link provided below). Items of note: (1) Diversity in color, shape, and size of bacterial colonies, (2) Reduction in number of colonies as go from least dilute (top left) to most dilute (bottom right), (3) Individual colonies are too close together to be able to count in top two plates (TNTC) and countable in lower dilutions. )
Intro Soils – Lab 5 – Assignment Questions Soil Microorganisms – Enumerating Heterotrophic Soil Bacteria
Utilize Lab, Lecture and Text Materials: Soil Organisms (Ch 11)
1.) Farmer Jim’s daughter was taking soil microbiology and decided to enumerate the heterotrophic bacteria from the alfalfa field her family limed back in Lab 4. As she collected her soil sample, she noticed a neighbor had recently also added lime to his field which was in corn last fall, but this family routinely utilizes tillage in their operation and had incorporated the lime into the soil. She decided it might be interesting to see if there was a difference in the two cultivable bacteria counts. Below are the results, using the illustrations and information provided in the lab, determine the CFU/gram of each soil. Discuss some reasons why the two soils might not have similar bacterial counts.
Alfalfa Field: Plate 10-3 – TNTC Plate 10-4 – TNTC Plate 10-5 – TNTC Plate 10-6 – 98 Plate 10-7 – 9 Corn Field: Plate 10-3 – TNTC Plate 10-4 – 65 Plate 10-5 – 6 Plate 10-6 – Zero Plate 10-7 – Zero
2.) Rank these soil organisms in order of overall abundance (number per gram) in soil: Earthworms, Bacteria, Actinomycetes, Fungi, Nematodes
3.) Fill in the following table with the appropriate metabolic group (4 in grey):
Source of Energy
Source of Carbon Reduced Inorganics / Biochemical Oxidation
Light / Solar Radiation
Organic Carbon / Combined Organic Carbon
4.) Name at least one positive contribution to soil health for each of the following soil organisms: a. Earthworms b. Bacteria c. Fungi d. Protozoa
5.) Name at least one negative contribution to soil/plant health for each of the following organisms: a. Ants/Termites b. Fungi c. Nematodes
6.) What is the rhizosphere and why is it such an important area of activity in soils?
7.) Discuss some defining characteristics of soil Actinomycetes. Why are they culturally and
8.) Why is an active soil microbial community so important to soil health and productivity? What are some managerial activities to help promote this community?
BONUS: In your own words, describe the Universal Phylogenetic Tree.