Creating a Winogradsky Column: A Method to Enrich the Microbial Species in a Sediment Sample

1. Set-up

1. To set up a Winogradsky column, you will need some basic supplies:
   • A shovel, bucket, and bottle to collect the samples in the field
   • A vertical, transparent vessel, such as a graduated cylinder or plastic water bottle of about 1L
   • Plastic wrap and rubber bands
   • large mixing bowls and large spoon to stir
   • A sulfur source (egg yolk or calcium sulfate)
   • A source of organic carbon (cellulose, in the form of shredded newspaper)
   • A source of light (sunny window or desk lamp)
   • Soil or mud collected from a marsh, wetland, pond, or stream
   • Water from the same habitat
   • OPTIONAL The following will be necessary for some of the optional experiments described in this protocol:
     • Table salt
     • Differently colored cellophane
     • A source of iron (such as a nail or steel wool)
     • A refrigerator with a light source
     • A radiator near a light source
2. If using a plastic water bottle, cut off the neck area so that the column is cylindrically shaped. Remove any wrappers so that light can penetrate through the plastic.
3. Raw eggs may contain Salmonella and should be handled with care. Proper hand-washing techniques should be followed. Alternatively, boiled egg can be used. Additionally, there is no way to know for sure if mud or sediment is contaminated with sewage or other harmful substance. Gloves should be used when mixing the mud and setting up the column.

2. Assembling a Winogradsky Column

1. Using the shovel, dig up and collect mud into the bucket. The sediments should be near the water's edge and completely saturated with water. You will need enough mud to fill each Winogradsky column. Collect some water from the same source into the sample bottle (approximately 3000 mL per column is needed).
2. In the lab, transfer enough mud to the first mixing bowl to fill ~75% of your 1-liter volume column. Next, sift through to remove large rocks, twigs, or leaves while using the spoon to break apart clumps.
3. Add some of the water that you collected to the mixing bowl while stirring. Add until the consistency of the water-mud mixture is like a milkshake. Continue to make sure there are no clumps.
4. Transfer about ⅓ of the water-mud milkshake to the second bowl. Add the egg yolk and a handful of shredded newspaper and mix.
5. Add the mixture of mud, egg yolk, and newspaper to the column until the column is about ¼ full.
6. Add the regular water-mud mixture into the column until the column is about ¾ full.
7. Add the additional water to the column, leaving only a small space (~½ inch) of air on top.
8. Cover the column with plastic wrap and secure with a rubber band.
9. Incubate the column in the light at room temperature.
10. For the next 4 to 8 weeks, monitor changes in the Winogradsky column for the development of different colored layers and the formation of bubbles, as described in Table 1. Additionally, you should record the time it takes for different layers to develop.

3. Optional Modifications to the Classical Winogradsky Column

1. Add 25-50g of salt per 1L Winogradsky column to the collected mud before adding water and stirring (step 2.3). The addition of salt selects for halophilic (salt-loving) bacteria.
2. Alternate substrates, such as iron, in the form of a nail or steel wool, can be added to the column along with the egg yolk and the shredded newspaper (step 2.4). This will enrich iron-oxidizing bacteria, such as Gallionella, and will appear as a rust-colored layer.
3. Instead of room temperature (step 2.9), the column can be incubated near a radiator to select for thermophilic (heat-loving) bacteria or in a refrigerator with a light source to select for psychrophilic (cold-loving) bacteria.
4. The amount of light a column receives as it incubates (step 2.9) can also be varied by placing different columns in high light, low light, or darkness.
5. The wavelength of incoming light can be limited by covering the column with differently shaded cellophanes as it incubates (step 2.9) to determine which colors select for different bacterial groups.

4. Data Analysis

1. After 1-3 weeks, some green coloring on the top of the mud layer of the classical Winogradsky column should be visible (Fig. 2A). These are the first signs of growth of the cyanobacterial layer.
2. Over time, continue to monitor the appearance and evolution of the different layers, each indicative of the different bacterial types. HINT: Refer to the Concepts and Table 1 to understand which bacteria contribute to the different layers.

Figure 2A: A photo of a classical Winogradsky column that has incubated at room temperature for 21 days. Note the green sediment, indicative of cyanobacteria, in the upper portion of the column.

3. If modifications to the classical Winogradsky column were also prepared, compare the results of each column.
   1. Observe the layers in each of the modified Winogradsky columns. Take note of the following:
      • Do the columns exhibit the same number of layers?
      • Are the layers the same color and thickness?
      • Do the layers occur at the same depths?
      • How long did each column take to develop?
      • Did one column develop more slowly than the others?