Modified from Characterization of Soil Microbes, by Virginia Bahlke, PhD, Delaware Technical Community College
Isolation of Soil Bacteria
One gram of soil has been estimated to contain as many as 1010 prokaryotic organisms. The number of bacterial species ranges from several hundred to almost nine thousand. These numbers have been used to estimate that there are 10 billion species of bacteria on Earth. Although the vast majority of these microbes have yet to be identified, the critical roles they play in many environmental processes that sustain life on Earth are being recognized.
Recently, scientists have begun studying the biogeography of microbes, determining their distribution on local, continental, and global scales. Soil composition, oxygen levels, temperature, pH, types of nutrients, and water availability affect the abundance of bacteria in soil. Other factors that affect the biodiversity of bacteria are land-use type, plant species present, temperature, salinity, and contamination with pollutants. Ecologists are concerned about the effects of land use on soil microbial communities and the effects on ecosystem functioning.
The first step in characterizing bacterial communities in soil is to estimate the viable numbers of microbes present in a sample. This will be accomplished by plating a sample of the soil that has been serially diluted in sterile saline and using the number of visibly growing colonies to calculate the original colony forming units per milliliter of diluted sample plated, or CFU/mL.
Identifying the species of cultured bacteria begins with an examination of their macromorphology, or visible appearance of their growth colonies without the aid of a microscope. Microbiologists use a standard set of terms when describing macromorphologies; we will use an abridged version of this. You will then perform quadrant streak for isolation on one of these colonies. Once you have a pure culture (only one type of bacteria) you will perform a Gram stain, biochemical assays and a genetic analysis of the isolates.
The purpose of this lab is to calculate the CFU/mL and describe each of the macromorphologies observed from soil samples taken at four different locations. The general hypothesis is that bacterial communities at each of these chosen locations will differ—either in the numbers of viable colonies supported, the types of macromorphologies isolated, or both. Prior to this lab, your instructor may have had you research and rationalize why you would want to study different locations, and why the bacterial communities at each may differ.
Serial Dilutions and Plating of Soil Bacteria
Bacteria are often counted in the laboratory by the viable plate method, where a dilution of bacterial culture is plated onto an agar medium. Following incubation, plates containing 30–300 colonies are counted. This range was chosen to include enough colonies for statistical accuracy, but not too many that colonies compete for nutrients, or that you can accurately count. Counts are then used to calculate the number of colony forming units per mL of diluted culture plated, or CFU/mL.
- Samples of collected soil
- Balance, weigh boats, scoopula
- Conical tube of sterile 0.9% NaCl
- Micropipettes and sterile tips
- Tryptic Soy Agar (TSA) plates
- Sterile microcentrifuge tubes
- Using a balance, weigh out 1g of soil onto a weigh boat.
- Add the soil to the provided conical tube containing 10mL of sterile 0.9% NaCl.
- Shake the tube vigorously to separate the bacteria from the soil particles.
- With a micropipette, transfer 500µL to a sterile microcentrifuge tube. This is your undiluted (100) sample (also called neat). If the solution is too concentrated and soil is getting stuck in the tip, add more sterile saline in 10mL increments until the problem ceases. Record in your notebook the final volume of saline in which you re-suspended the 1g of soil.
- Close the cap of the microfuge tube and vortex for at least 30 seconds to thoroughly mix the bacterial cells into the 500µl of sterile saline.
- Label 3 sterile microcentrifuge tubes containing 900µL of 0.9% sterile saline with the dilution factors: “10-1, 10-2, 10-3”.
- Using a micropipette and a sterile tip, transfer 100μL of cells from your undiluted (100) tube containing 500µL of re-suspended soil to the tube labeled 10-1 dilution. Vortex for at least 30 seconds to thoroughly mix the cells into the saline.
- Continue with 1:10 serial dilutions (as shown below).
- Label 4 TSA plates with your name, your soil sample, your table number & letter, and the date around the edge of the bottom of each of the plates.
- Plate 100µL (0.1 mL) of each of the 100, 10-1, 10-2, 10-3 dilutions on the appropriate TSA plates. Incubate the plates in O2 at 30°C for 24-48 hours.
Day 2 – Results and Calculation of CFU/mL
- Transfer the headings shown in Table 1 below to your notebook.
Table 1. Colony Counts from Nutrient Agar Plates
|Date Plated||Soil Location||Dilution Factor||Total # Colonies||CFU/mL|
- Pick the plate with 30–300 colonies and write down the total number of colonies of that plate in your lab notebook.
- Calculate the original colony forming units per mL (CFU/mL) using the following formula:
Example: You count 112 colonies on the plate that came from the 103 dilution. Your dilution factor is the inverse of the 103 dilution, or 103.
|x||103||=||1.12 x 106 CFU/mL|
Write your calculation in scientific notation to 2 decimal places (as shown above).
Day 2 - Quadrant Streaking for Bacteria Isolation (Sub Culturing)
- Subculture a colony with macromorphology that interests you from your plate by performing a quadrant streak for isolation onto a TSA plate.
- For a refresher on quadrant streaking, refer to the Aseptic Techniques protocol, or your lab notebook and Figure 3.
- Incubate the plates in O2 at 30°C for 24-48 hours.
Figure 3: Streaking for isolation using the quadrant streak method.
Day 2 - Macromorphology of Bacterial Colonies
Record in your lab notebook different descriptions and total number of colonies that you cultured. Include: colony ID, soil location, date plated, size, shape, color, texture, and total number of similar colonies you cultured. Use only the standard descriptions (shown below). Be meticulous in your observations and documentation. Each colony on the plate should be evaluated.
Day 3 – Simple Stain, Negative Stain, Subculture
- Record observations of your quadrant streak for isolation in your notebook. Note whether you successfully got isolated colonies, and whether the isolated colonies all have the same macromorphology. Subculture again to ensure culture purity by performing another quadrant streak for isolation on your microbe of interest.
- Perform a simple stain on a single colony from your plate. Sketch and record the results in your notebook.
- Perform a negative stain on a single colony from your plate. Sketch and record the results in your notebook.
Day 4 – Results and Gram Stain to Confirm Culture Purity
- Record observations of your quadrant streak for isolation in your notebook. Note whether you successfully got isolated colonies, and whether the isolated colonies all have the same macromorphology.
- Perform a Gram stain on a single colony from your plate. Sketch and record its Gram reaction in your notebook.