Day 52 Observations

Winogradsky Summary for Fall Semmester:
I created 4 Winogradsky columns to see if I could show some of the various ways that microbes can survive, including in both aerobic (with oxygen) and anaerobic (without oxygen) conditions.

Column CA: In the mud in this column I placed shredded filter paper (carbon or cellulose source), magnesium sulfate (sulfur source), baking soda (sodium bicarbonate) and one crushed multivitamin pill. This column was placed in my kitchen window.

Column CB: This column was created the same as column CA, however it was enclosed in a box and kept away from light. The intent of this mixture was to provide an environment where chemosynthetic organisms could thrive without light.

Column WA: This column I mixed mud with cut grass (carbon/cellulose source) and the magnesium sulfate. Column WA was placed in the window next to column CA.

Column WB: This column was mixed the same as column WA, however it was placed in the box and kept out of the light with column CB.

After 52 days, I have seen column CA go through several stages of apparent growth. The water column at the surface went completely black for about 1 week and now has become clear again. The mud has remained almost totally black throughout, though for a few weeks there was some white areas within the upper level of the mud, closer to the water column. A thin film has remained on the surface of the mud throughout. The film was originally a whitish/greenish color and now is a thicker film that is red in color. There are red particles floating in the water column. The column has an rotten-egg like odor.

Column WA has gone through more changes. At one point the water column had taken on a foggy redish hue, but now the water is clear again. There is now a thick red film at the surface and thick red layers in the mud on the side facing the window. This column does not have a strong odor.

There has been very little change in either columns’ WB or CB. Neither column has a distinguishable smell. Both columns did go through some color change in the water column, however this was not as dark black as column WA went through. Both columns have maintained a thin film on the surface of the mud. Column WB has a thicker, redder film.

I found it very exciting to look at samples in a microscope and would recommend this part to any classroom experiment. It would be even better to have an opportunity to try to identify the microbes through isolation and gram staining.

Altogether, I think that the columns will take several more months before they can show the type of nutrient cycling and photosynthetic vs. chemosynthetic growth that the columns are known for.

Here is a photo of the surface of column WA this week:

Here are two photos of the side that has been exposed to light:

Here is a photo of the side that has been opposite the window:

In retrospect, I would have liked to use a different protocol, a contorol, and different carbon sources. I also notice that neither protocol called for a nitrogen source, so you can not follow the nitrogen cycle and it may have limited the types of bacteria that thrived in my columns. I have since found two sites that I would probably use with a class & that have a much better description of the protocol to use with students: 1. http://www.kabt.org/wp-content/uploads/2008/01/winogradsky-for-kabt-web-site.pdf
2. http://www.sciencebuddies.org/science-fair-projects/project_ideas/Geo_p038.shtml

For perspective, I also liked this site: http://steel.ced.berkeley.edu/research/hidden_ecologies/?p=31 from the Exploratorium’s Invisible Dynamics Project. This will be something to follow over time though because they’ve just got started.

Two weeks ago our lab manager helped me to use a light microscope with the computer link to take these pictures of some of my little "bugs". I only took samples from my #2 Column that is exposed to daylight. We had to use the phase contrast at 100x and add oil to the slide in order to really get a sense of what we were looking at. I also found it challenging to get a small enough sample out of the mud to be able to put it on the slide & actually see anything. Our lab manager helped me with everything & mentioned that in the first year microbiology class they are doing Winogradsky columns, but they also go through the process of isolating cell colonies on agar plates so that they have an easier time with identification. He said that for the class they use the streak-plate technique. Microbes obtained from sample aproximately 1/2 inch in mud column. Unfortunately I did not want to take up my hosts time with a staining activity (nor did I want to reveal my ignorance to the process), but that would have been even more effective in identifying what I've got here. I think if I were to do this with a class I would want to isolate some colonies and then perform gram stains to look at them.

1 micrometer =aproximately "-" on photo.
Here, I beleive you can see several living microbes taken in the picture. While looking through the microscope you could see quite a bit of movement. Most of my microbes appear to be rod shape and motile. Though there are several cocci or spherical cells.


In the lower center of the screen is a cocci shape that was pretty common with a center "nucleoid" area. It is clear that I need to purchase a microbe field guide, but even then I doubt I have the skill to satisfactorily identify these little bugs. A type of spherical cell that might be found in the conditions of my Winogradsky column could be a Micrococcus species. I pick this as a possible species because according to "A Short Guide to Some Bacteria Genera", by Harold Eddleman, Ph.D., Micrococcus species are very common in soil and dust. The colonies are also pale yellow or orange - which would match what is starting to appear in my 2nd column. They are non-motile, no spores, and aerobic which could all fit. Again, this is a sample species that might be considered, I am not going to try to identify it for real!

This is my favorite picture. The long cell shows up quite nicely and you can see the cell wall and several cellular structures inside the cell. There were several of these, but this was the best shot we got & I was unable to find it on my own (lack of patience)? For this microbe, I am going to refer to the website Microbe Wiki, http://microbewiki.kenyon.edu/index.php/Beggiatoa, for my sample species. Here I am going to highlight Beggiatoa species because of the look of the cells found on this and other sites and because the Wiki definition has this to say, "Beggiatoa is a genus of colorless, filamentous proteobacteria. With cells up to 200 microns in diameter, species of Beggiatoa are among the largest prokaryotes. They are one of the few members of the chemosynthesizers, meaning that they can synthesize carbohydrates from carbon dioxide and water using energy from inorganic compounds. Beggiatoa are found in polluted marine environments, and can be seen by the naked eye as a white filamentous mat on top of the water as a sign of environmental deterioration." I liked this description, but I don't believe I have such a mat anywhere within my column - this might rule out Beggiatoa. Also good to note that the photos on the Wiki site can be found at the Cyanobacterial Image Gallery: http://www-cyanosite.bio.purdue.edu/images/images.html.

Last, but not least, I think that the image in the middle center is another rod shaped cell with a polar flagella, there were also a number of these to be found in our samples, but this was the best shot we were able to get. I like the sample microbe that I found to highlight in this category: Rhodospirillum species. These are large spiral cells with very rapid motility and a spinning movement. According to the "Short Guide", they will grow on yeast media in the dark microaerophilically with pale white color. In the presence of light, they grow a purple or maroon color in medium anaerobically via photosynthesis. They develop huge populations in algae covered sewage ponds and can also be found in pond muds.

Currently, Column #1 has not changed much, though the water has returned to clear color, but column #2 has begun to show a variety of colors and the water has maintained it's redish hue. There is no strong smell coming from any of the columns. The columns in the dark have not developed a distinctive color change. More photos on these to be posted later.

Day 25: These photos show some of the changes that have been going on for the last few weeks. The algae in the water column seems to have multiplied in both bottles, but more noticeably in the chemosynthetic protocol column where the water appears black. The 2nd column seems to have maintained its orange tint. By following the simple diagram listed at: http://www.biology.ed.ac.uk/research/groups/jdeacon/microbes/winograd.htm I might be safe in assuming that both columns have a healthy growth of green and purple sulfur bacteria at the surface of the mud and that the 1st column has a bloom of the purple non sulfur bacteria in the water column. Possibly I have been able to promote the growth of the sulfur reducing bacteria in the first column and that might also explain the color.



Column 2: Typical Winogradsky column with cut grass & magnesium sulfate

Column 1: Chemosynthetic protocol with filter paper, magnesium sulfate, baking soda, and crushed vitamin

The two columns in the enclosed box where they are not exposed to light have shown little change, however the water column is tinted orange in both.



The 2nd column or column with grass has a thicker layer of growth at the surface of the mud and more appearance of growth just below the surface. This was not as I would have expected, as it is the chemosynthetic column that has baking soda and vitamins which were both intended to assist in microbial growth without sunlight.

I have not had a chance to check the temperature or look at any of it under a microscope yet, but I hope to be able to do so this week. I am still looking to answer why I might have so much "white" growth around the edges of the grass in the 2nd column, for which my first guess has been mold or a type of fungus.