List of experiments

Soil Productivity - Plate Count Method Environmental Microbiology Experiments for middle and high school Christine L. Case Skyline College

Objectives

Estimate the number of microorganisms in soil using the plate count method. Describe the general activity of microorganisms in the soil. Distinguish between bacterial, actinomycete, and fungal colonies.

Background

The soil is one of the main reservoirs of microbial life. Typical garden soil has millions of bacteria in each gram. The most numerous microbes in soil are bacteria (Table 1). Although actinomycetes are bacteria, they are listed separately because conidiospores make their dry, powdery colonies easily recognizable. Soil bacteria include aerobes and anaerobes with a wide range of nutritional requirements, from photoautotrophs to chemoheterotrophs. As usable nutrients and suitable environmental conditions (such as light, aeration, temperature) become available, the microbial populations and their metabolic activity rapidly increase until the nutrients are depleted or physical conditions change, and they then return to lower levels. Human pathogens, with the exception of endospore-formed bacteria, are uncommon in the soil. Soil microorganisms are responsible for recycling elements so they can be used over and over again. The numbers of bacteria and fungi in soil are usually estimated by the plate count method. The actual number of organisms is probably much higher than the estimate, however, because a plate count only detects microbes that will grow under the conditions provided (such as nutrients and temperature). In a plate count, the number of colony-forming units (c.f.u.) are determined. Each colony may arise from a group of cells rather than from one individual cell. The initial soil sample is diluted through serial dilutions in order to obtain a small number of colonies on each plate. A known volume of the diluted sample is plated on sterile nutrient agar. After incubation, the number of colonies is counted. Plates with between 25 and 250 colonies are suitable for counting. A plate with fewer than 25 colonies is inaccurate because a single contaminant could influence the results. A plate with greater than 250 colonies is extremely difficult to count. The microbial population in the original soil sample can then be calculated. For example, if 232 bacterial colonies were present on the plate containing 0.01 mL of the 1:1,000,000 dilution, the calculation would be

		Distribution of microorganisms in numbers per gram of typical garden soil at various depths
Depth		Bacteria	Actinomycetes	Fungi	Algae
3-8 cm		9,750,000	2,080,000	119,000	25,000
20-25 cm		2,179,000	245,000	50,000	5,000
35-40 cm		570,000	49,000	14,000	500
65-75 cm		11,000	5,000	6,000	100
135-145 cm		1,400	-	3,000	-
350-450 m		100

Materials

Balance 99 mL sterile water in a bottle (3) Pipettes or calibrated one-piece plastic droppers Sterile nutrient agar plates (3) Spreading rod (L-shaped glass rod or place of piece of No. 16 shrinkable Teflon tubing over a large, straightened (L-shaped) paper clip or metal spreaders can be purchased from Carolina Biological Supply) Alcohol (rubbing alcohol, 70% isopropyl alcohol)

Procedure This procedure can also be used with food, milk, and water. Figure 2. Use of a spreading rod.

Each group should start with a soil sample collected from a different environment, e.g., leaf litter, sand, stream bottom, clayey soil, etc. Prepare a 1:100 (or 10^-2) dilution of the soil. Weigh out 1 g of soil and add it to 99 mL sterile water. Mix the soil and water. Be sure to keep all tubes and bottles capped to prevent addition of bacteria and fungi from the air. Prepare a 1:10,000 (or 10^-4) dilution by pipetting 1 mL of the 10^-2 dilution to another 99­mL bottle of sterile water. Mix thoroughly by pipetting up and down. Prepare a 1:1,000,000 (10^-6) dilution by pipetting 1 mL of the 10^-4 dilution to another 99­mL bottle of sterile water. Mix thoroughly by pipetting up and down. Label one nutrient agar plate "0.5." Label a second plate "0.1" and a third plate, "10^-4­0.5." Aseptically pipette 0.5 mL of the 1:1,000,000 dilution onto the surface of the nutrient agar. See Figure 1. Disinfect a spreading rod by dipping in alcohol, quickly igniting the alcohol in a Bunsen burner flame, and letting the alcohol burn off. Let the spreading rod cool. Spread the liquid over the surface of the agar. (See Figure 2.) Disinfect the spreading rod and return it. Repeat inoculating the second plate with 0.1 mL of the 1:1,000,000 and the third plate with 0.1 mL of the 1:10,000 dilution. Incubate the plates inverted for 24 to 48 hours. After incubation select a plate with a countable number of colonies (between 25 and 250). Count the number of colonies and calculate the number of bacteria in 1 g of that soil Record class data in a table:   Soil type Number of bacterial c.f.u./g.       Number of actinomycete c.f.u./g.       Number of fungal c.f.u./g.

Figure 1. Serial dilutions               Back

Questions

Questions Why do these numbers represent the minimum number of microorganisms in the soil? Which soil type has the most microorganisms? Why? Which type of microorganism (fungi, bacteria, or actinomycetes) predominates in each soil type?