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.

Day 5 of columns since adding mud

I'm posting these photos of Day 5 since I added the more correct portion of mud to water. Already I seem to be getting quite a bit of activity if the water color itself has anything to do with it. I am also seeing small blotches of dark and light color on the side of the columns that get more light. I should point out that my kitchen window does not get a lot of light, so it will be interesting to see how well my columns do.

Shown here are the two columns side by side. The column on the left is the chemosynthetic bacteria experiment (CA) and on the right is the traditional Winogradsky column with grass as the carbon source (WA).



The WA experiment still has fairly clear water, though some algea seem to be growing in it. A layer of brick red growth seems to be forming on the surface of the mud. Color changes that appear to be due to growth of bacteria may also be occuring within the mud column where grass is available.


The second column, the CA experiment has a darker red coloring throughout the water column, the coloring should be. Is this growth due to cyanobacteria or other algea? In the "aerobic zone" at the surface of the mud, a thin film has emerged. The color is greenish. In this column, the indication of bacterial growth on the filter paper is not as immediately apparent, though there are some color blotches.

My Winogradsky Protocol

For my Winogradsky expiriment, I am trying out two different protocol. I have used four 1 litter water bottles - two using a protocal from the NASA site - Chain or web? grow chemosynthetic bacteria - which can be found at: www.bigelow.org/foodweb/chian4.html. This protocol is primarily concerned with whether or not photosynthesis is the only way to make food. This experiment is meant to show how bacteria will oxidize hydrogen sulfide to produce carbohydrates without any sunlight involved. I like this version because it is set up as a lesson plan for teachers. There are learning objectives, procedures, and extension activities. I was not thrilled about the procedure section though because I found it a little difficult to follow having never done one of these before. I also substituted the call for Plaster of Paris (CaSO4) with magnesium sulfate, which I am assuming will work out ok.

For both protocols I collected mud and creek water from Bear Creek in downtown Merced. I was relieved to find that there was enough water still in the creek to collect this late in the year (we haven't had rain for a long time and probably won't until the beginning of November).


For the first protocol, I combined about 4 grams of magnesium sulfate with enough mud from the creek to fill the first 1/3 of my water bottles. I stirred it in a container and then mixed it with filter paper strips. The procedure calls for lab filter paper, which I substituted with coffee filter paper - again, I'm not too sure how well this substitution will work out. I transferred the mud mixture to the water bottles and added the creek water. Then added .2 grams of baking soda and one crushed multivitamin pill. I attempted to remove air bubbles, but I did not feel highly successful in doing so. I let the bottles sit for about 30 minutes and then poured off a little bit of water and covered with plastic wrap and a rubber band. I marked one to sit in sunlight and one to be closed in a box.


For the second protocol I used the instructions from one of the links provided: http://www.personal.psu.edu/faculty/j/e/jel5/biofilms/winogradsky.html, which was apparently created by a Penn State faculty member. I think this protocol is a more traditional source of directions on the Winogradsky Column. For this protocol I used the same mud and creek water and also mixed the mud with magnesium sulfide. However, for the carbon source, I used one of the other recommendations - cut grass. These instructions do not include the use of baking soda or vitamins. I marked both of these bottles as well for one in sunlight and one in the closed box.

Here are the bottles right after mixing, prior to settling as well as a top down image:







The bottles looked like this last night in my window, I think it's clear that I missed the boat somewhere and didn't use enough mud. I'm not sure how that will affect the experiment overall or if I will have to start over:

The chemosynthetic bacteria site was very specific about putting the bottles right away in their dark box, so I didn't take pictures of those bottles (they looked the same - as you see above, the one on the right that is more "yellow" has the baking soda and vitamins added. I packaged my dark bottles in a box and covered them with a dark plastic bag - I'm leaving the box on my kitchen counter because it asks for the conditions to be the same. It gives a good recommendation about viewing them with a red light on a weekly basis, so I'm going to try that:

A couple of other great sites are:
http://people.clemson.edu/~skipper/In%20progress/winogradsky/winogradsky.htm
http://www.sumanasinc.com/webcontent/animations/content/winogradsky.html