Bacteriopolis

1. Place the funnel on the top of your clear container. 
2. Place the mud or dirt in a basin. 
3. Add a little water to the mud or dirt and mix with your hands (or a spoon), so that the resulting mud is loose enough to be squished through the funnel. Try not to add too much water—you can always add more, but you can’t take it out.
4. Add nutrients to your mud culture. You can add a handful or so of finely shredded paper, a raw egg, both, or neither. For faster, more colorful results, we suggest adding both.
5. Fill the clear container with mud by pouring and squishing it through the funnel with your hands. This can make a mess, so you may want to cover your work space with newspapers. 
   Optional: Another variation is to add an iron source to your culture by dropping a nail or two into the container, roughly in the middle of the mud. 
6. Put the lid on the container, but keep the lid cracked so that air can enter the container. If your container doesn’t have a lid, cover the mouth loosely with plastic wrap. 
7. Optional: To investigate the effect that light has on your culture, cut a small piece out of foil or cardboard and tape it to the outside of the container. 
8. Place your container in a sunny window (with the foil side facing the window, if you used it), and let it sit undisturbed for about 24 hours.
9. After 24 hours, the mud should have settled to the bottom of the container, with a layer of water sitting on top. If there is more than 1 or 2 cm of water above the mud, pour a bit off until there is only 1-2 cm of water. If there is less than 1-2 cm of water, add a little tap water to the top of the container.

Observe your mud periodically, watching for changes over days, weeks, months, or even years. What patterns do you notice over time? When and where do various colors appear or disappear? Does the side of the column facing the window appear different from the side facing away? If you used foil or cardboard, peek underneath: does the covered part look different than the uncovered part? 

What evidence do you have that the colors you see are living things?

You may want to draw and measure the way that different colored patches grow, shrink, and change over time—a grid printed on a transparency and colored pencils can help you to capture this information. 

To keep your culture growing, just add a little water to the top of the container if you notice it has dried out. 

The colorful patches that grow in your container of mud are actually clusters of diverse species of bacteria that live naturally in the soil. Each different color represents a group of microbial species. Allowed to grow undisturbed, these bacteria form colonies large enough to see by eye. 

Another name for your container of mud is a Winogradsky column, named for the scientist that came up with this way of growing and studying soil microorganisms. A Winogradsky column captures the processes that are continually happening in soil and mud in nature, a self-contained, dynamic ecosystem in which energy flows and matter cycles between different metabolically diverse organisms. 

The green bacteria in your culture make their food like plants—they get their energy from sunlight by photosynthesis, and they get their matter (that is, carbon) from the air in the form of carbon dioxide. If your column is in a sunny window, you might have encouraged the growth of lots of these photosynthesizing species, and the side of the column that got less sun likely had fewer.

Other bacteria in your column are more similar to animals and fungi—they get their energy and matter by metabolizing carbon compounds made by other organisms (like plants, animals, fungi, or other microbes). This is similar to the way that you get energy and matter by digesting and breaking down the molecules in the food you eat. If you added shredded paper to your column, which is made largely of cellulose, a carbon compound made by plants, you likely encouraged the growth of some of these species, which tend to appear grey.

The egg or nails you might have added to your column represent another type of energy source that can only be used by certain bacterial species. These organisms get their energy through reactions they conduct with sulfur or iron, minerals that are present in the egg and nails, respectively. Iron-oxidizing species can often be identified by the reddish brown color of their colonies—you may have noticed that iron rusts to a similar color. 

Each of these different metabolic reactions produces different waste byproducts. And each byproduct may in turn be used by another organism to survive. For example, photosynthetic cyanobacteria produce oxygen gas as a byproduct of photosynthesis. This oxygen gas is crucial for the survival of aerobic bacteria—as well as for animals like us. In fact, we have photosynthetic cyanobacteria to thank for creating the relatively oxygen-rich atmosphere of our planet several billion years ago, without which we humans could never have evolved.

Although your column might start as a uniform mass of mud, over time it develops gradients and microenvironments with different resources, such as oxygen and light. Oxygen is rich at the top of the column, but is nearly absent towards the bottom of the column, where anaerobic (non-oxygen-using or producing) species are usually found. The side of the column facing the window or other light source will tend to have more photosynthetic species than the side of the column receiving less light, which will be richer in heterotrophs, or non-light-using species. Each species occupies a particular niche or area of the column, depending on their energy and matter needs. The waste products of each species create new niches for other species to grow in. 

In this way, matter recycles within and energy flows through the ecosystem of the column, and with continual inputs of light energy, these organisms can continue using their diverse metabolic processes to live, change, and grow for many years.

Make multiple Winogradsky columns using different ingredients—with or without egg, paper, or nails—and then watch and compare the different bacterial cultures that develop as a result. Different mud or dirt sources will also produce different microbial communities. What beautiful, biochemically diverse bacteria can you discover?

If you have a UV flashlight, you can use it to identify photosynthesizing bacteria in your column.  Chlorophyll and other photosynthesizing pigments (not all of which are green) will glow or fluoresce with a reddish-magenta color when you shine UV light on them. You’ll probably find that some colonies that aren’t green fluoresce with this distinctive magenta color, meaning that they’re probably photosynthetic. 

Our understanding of the phenomenon explored in this Science Snack is built on the work of many scientists.

Dr. Rosanna "Rosie" ʻAnolani Alegado is kānaka ʻōiwi, a Native Hawaiʻian, and an associate professor of oceanography at the University of Hawaiʻi at Mānoa. Dr. Alegado studies how bacteria influence the evolution of animals and how these interactions impact their ecosystems. She completed her postdoctoral work in evolutionary biology at UC Berkeley and holds a PhD in microbiology and immunology from Stanford, in addition to a BS in biology with a minor in environmental health and toxicology from MIT. In addition to her research, Dr. Alegado works with different community groups, including tracking the influence of restoration, storms, and climate patterns on the health of a traditional Hawaiʻian fishpond. She is committed to increasing participation of underrepresented minorities in STEM and is the director of a program that provides mentoring and support to undergraduates transitioning from community colleges to her university at Mānoa. In the Science Snack Bacteriopolis, you can create an ecosystem to observe how different bacterial colonies grow and interact with one another.

This Snack makes an excellent long-term project for students to investigate and document the dynamic nature of ecosystems, the cycling of matter and flow of energy into and out of organisms, and the environmental interactions between living things and nonliving factors. 

This project can be carried out over weeks or months, although bacteria from different soil sources will tend to grow at different rates. When colonies in the columns initially begin to grow, students can gather evidence for the presence of living things in the soil. Documenting differences in bacterial populations under different conditions can be done weeks or months after initially setting up the column. 

To measure change over time in the bacterial populations, you may want to have students take data on their column once a week or so, recording and noticing changes in the size and color of the bacteria colonies through drawing and measuring. Although any clear container can be used to make a column, flat-sided tissue culture flasks make it particularly easy to measure and draw bacterial colonies by placing a grid printed on transparency over the side. 

By manipulating the nonliving factors supplied to the column, like nutrients, light, and air, students can gather evidence for the importance of these factors to the growth of different types of bacteria, and make hypotheses about the ecological niches these organisms occupy. 

Any source of soil or mud is worth investigating for signs of bacterial life. Making columns from different source soils, gathering evidence for the presence of living things in soils, and noticing the differences in the populations present is an excellent project for younger students.

See this Snack in action in this episode of Build Your Own Exploratorium.

Read more about the science behind this Snack at Microbial Life Educational Resources from SERC at Carleton College. A Field Guide to Bacteria, Betsey Dexter Dyer (Cornell University, 2003) is a thorough guide for identifying microorganisms and understanding their interactions with their environments.