Laboratory Investigations in Microbiology

Course Home | Lab Manual Home

Chapter 22: Environmental sampling of microbes

Now that we have observed known microorganisms under fairly "controlled" conditions in the laboratory, we will turn our attention to the diversity of microbes found in our environment. Our task will be to collect samples (soil, water, etc) from outside and to try to determine the number of bacteria and fungi in these samples. In this process, we will also get a look at some of the diverse organisms we can find in these environments.

Soil is home to literally billions of microorganisms per gram. It is one of the richest microbial habitats (aside from, perhaps, the animal digestive tract), including aerobes and anaerobes, endosporing bacteria, molds, nitrogen fixers, denitrifiers, lithotrophs, decomposers, gliding bacteria, and many more. In soil these microbes participate in essential nutrient cycles, such as those for nitrogen, sulfur, and carbon. Autotophic bacteria (phototrophs and lithotrophs) fix carbon dioxide; decomposers return it to the environment. Of particular importance are bacteria that can break down complex plant carbohydrates such as starch (Bacillus) and cellulose (Cytophaga). Nitrogen fixers (Azotobacter, Rhizobium) add nitrogen to the soil; lithotrophs oxidize it to nitrate (Nitrobacter, Ntrosomonas), and denitrifiers (Pseudomonas) may remove it again. Other very abundant soil bacteria include the actinomycetes, bacteria that form fungus-like filaments and spores. Typical soils may contain from 1 million to 10 billion microbes per gram. To determine these numbers, a soil sample will be diluted up to 1 billion-fold and cultured using the pour-plate method. The resulting colonies will be counted (= viable plate count method).

However, the culturing of environmental microbes in the lab is not always successful. many microbes cannot be cultured in the laboratory even though they are alive when collected. Such microbes are called "VBNC" (viable but not culturable), and make up as much as 99% of all microbes in the environment! Some of the reasons that they cannot be cultured include special nutrient requirements (trace elements, vitamins/growth factors), unique growth conditions (pH, temperature, oxygen), the need for a cooperative community structure of other organisms or the requirement for a precise combination of all these factors, such as nutrient gradients found in soil.

Aquatic samples are home to many different microbes, including algae, cyanobacteria, enterobacteria, soil bacteria, and several species found only in water. Microbes such as Caulobacter, Beggiatoa, and Hyphomicrobium often make water their home. There are comparatively fewer culturable microbes in water than in soil - up to several thousand per 100 ml - and there are far fewer fungi. However, because of its importance as a source of drinking water, aquatic samples are usually monitored closely for fecal contamination. Although usually the Most Probable Number (MPN) test is used to estimate microbial (heterotrophic) numbers in water, today we will use the pour-plate (viable plate count) method to get a more accurate number.

Materials per group
Procedure
Day 1

Collect a sample of soil  from outside with your sterile screw cap tube. Use the tube to dig into the soil. Suggestions are: flower beds, lawn, wooded lots, trees...

Soil samples
  1. Label 3 sterile Petri plates 10-3, 10-5, 10-7. These represent 1,000-fold diluted, 100,000-fold diluted, 10,000,000-fold diluted samples.
  2. Label 4 saline test tubes as 10-1 10-3, 10-5, 10-7
  3. Weigh out 1 gram of your soil sample and add it to a 10 ml sterile saline test tube. Label this test tube "10-1". Mix well by vortexing.
  4. Pipette 0.1 ml of the 10-1 tube into a second 10 ml saline tube labeled "10-3". Vortex.
  5. Pipette 0.1 ml of the 10-3 tube into a third 10 ml saline tube labeled "10-5". Vortex.
  6. Pipette 0.1 ml of the 10-5 tube into a fourth 10 ml saline tube labeled "10-7". Vortex.
  7. Pipette 1 ml from the 10-3 tube into a 20ml test tube of melted TSA (from heated water bath). Vortex, then pour into the Petri dish labeled 10-3
  8. Pipette 1 ml from the 10-5 tube into a 20ml test tube of melted TSA (from heated water bath). Vortex, then pour into the Petri dish labeled 10-5
  9. Pipette 1 ml from the 10-7 tube into a 20ml test tube of melted TSA (from heated water bath). Vortex, then pour into the Petri dish labeled 10-7
  10. Let the plates sit until hardened. Incubate 5 days at room temperature.

Next lab

  1. Count all the colonies on each of your agar plates, if possible. If colony numbers exceed 1,000, write down TNTC" (too numerous to count) and count the next plate.
  2. If colonies overlap, look very carefully so you don't miss any. Keep in mind that many colonies will be embedded in the agar and very small. Most colonies will look like small ellipses within the agar.
  3. For each agar plate, determine the estimated bacterial number in the original sample by dividing # colonies/dilution factor. Since each plate represents a different dilution of the SAME sample, these calculated values should be close to each other.
  4. Calculate the average number of colonies per gram of soil. Discard any plate count value that you deem to be unreliable (eg TNTC, fewer than 5 colonies, contaminated plates, or values far out of range of the other plates)

Printable Data Sheet

 

© 2003 - 2019 Josť de Ondarza, Ph.D.