Link to Flowchart
 

	Isolate 1	Isolate 5	Isolate 7	Isolate 8	Isolate 10	Isolate 12	Isolate 13
gram	positive	negative	negative	negative	positive	positive	negative
voges-proskauer		negative	negative	negative	negative	negative	negative
acid-fast	negative				negative	negative
amylase		positive			negative
catalase	negative	positive	positive	positive	positive	positive	positive
citrate		positive	positive	positive	negative	negative	negative
triple sugar iron		negative	negative	negative	negative	negative
cytochromoxidase		positive	positive	negative	negative	negative	negative
dextrose		positive	positive	positive	positive	positive	positive
endospore	negative		negative	negative	negative	negative	negative
indole		negative	negative	negative	negative	negative	positive
lactose		positive	negative	negative	positive	positive	positive
lipase		negative	negative	negative			negative
methyl red		positive	negative	negative	positive	positive	positive
nitrate		negative	negative	error	positive	positive	positive
sucrose		positive	negative	negative	positive	positive	positive
urea		negative	negative	negative			negative

This was the first isolate we were able to identify completely. This isolate was obtained from the 1000 mL level of the Winogradsky column that was always under artificial lighting. First, a Gram stain was performed to determine the Gram nature of this bacteria. The result was that it was a Gram negative bacteria. This particular bacteria was also a cocci. The fact that this bacteria grew on an EMB plate provided further support that it was a Gram negative bacteria. We then proceeded to perform a battery of biochemical tests on this unknown. The tests performed on this bacteria include: nitrate reduction, Simmons citrate, MR-VP, indole, urea, catalase, oxidase, triple sugar iron, and dextrose, sucrose, and lactose carbohydrate tests. Because many of these test results are not included in the charts in Bergey's Manual, we examined the summary that was written for every Gram negative aerobic and microaerophilic rod and cocci. The summaries of these bacteria usually contained the results from a few of the tests we had performed. By examining these summaries only for test results that matched ours, we were able to eliminate the vast majority of genera contained in this section of Bergey's manual. By doing this, we were able to limit our possibilities to the following genera: Alcaligenes, Bordetella, Cupriavidus, Methylococcus, Morococcus, and Neisseria. Once we had limited our choices to these genera, we examined the summaries more closely and also studied the charts of test results found in Bergey's Manual for these genera. By either discounting genera because of their test results or because of the natural environment in which they were found, we were able to eliminate all but four of the possible genera. The isolate was determined to be from the genera Alcaligenes or Neisseria. The test results were studied more closely for these two genera and the isolate was found to be either Alcaligenes latus or Neisseria macacae. At this point, we believed we had determined as closely as possible what the isolate was, but we checked the summaries for these genera once more. When we checked this last time, we found that Alcaligenes are obligate aerobes, while Neisseria can exist under decreased oxygen conditions. Because this isolate was found at the 1000 mL level of the Winogradsky column, Alcaligenes latus can be eliminated. It is likely that an obligate aerobe would not be growing at this depth of a Winogradsky column. Because of this, we determined this isolate to be Neisseria macacae.
Evan Haas and Dustin Romine
Lactobacillus - 1

This isolate was obtained from the Winogradsky column that was always exposed to artificial lighting. It was isolated from a sample of water taken from the very top of the column. The first test performed was a Gram stain to determine its Gram nature. We found that this bacteria was Gram positive and we identified it as a rod. We then followed the flow chart found on page 113 of our laboratory manual. We next performed a catalase test and found our isolate was negative for this reaction. Next, an endospore test was completed and this test was also negative. Finally, an acid-fast was performed and this test was negative as well. According to the laboratory manual, this isolate belonged to the genus Lactobacillus. Because of the variety of species found within this genera and because these species are so similar, further tests were not completed to determine the exact specie of this unknown. This bacteria is a member of the genus Lactobacillus.

Josh Melton
Brachybacterium, Propionibacter, or Rothia - 12

This isolate was obtained from the 1400 mL mark in the Winogradsky column that was not under artificial light the entire time that growth occured in our column. The first test performed on this unknown was a Gram stain. We found that this isolate was a Gram positive bacteria and we also determined that it was rod shaped. Because it was a Gram positive bacteria, we performed both an endospore test and an acid fast test on this isolate. This bacteria did not form endospores and it did not stain acid-fast. After these results were obtained, a variety of biochemical tests were performed on this bacteria. Tests on this bacteria included: MR-VP, nitrate reduction, triple sugar iron, Simmons citrate, indole, catalase, oxidase, and lactose, sucrose, and glucose carbohydrate tests. However, because many of these test results are not contained in Bergey's Manual, we examined the summaries of all bacterial genera that were found in the non-sporing Gram-positive rod section of the manual. As usual, these summaries contained some results from tests we had performed. By examining these results and paying close attention to what environment different genera of bacteria were found, we were able to eliminate many possible choices for this bacteria's genus. There were four genera that seemed like good choices for our bacteria. They were Brachybacterium, Caryophanen, Propionibacter, and Rothia. After examining the summaries of these genera again, we found that Caryophanen are strictly aerobic bacteria that grow best between 25 and 30 degrees Celsius. However, our bacteria was isolated from a moderately low depth of our column and it grew best at 35 degrees Celsius so this genus could be eliminated. However, we were unable to determine any further what our isolate was because these three genera were so similar. Therefore, this bacteria was Brachybacterium, Propionibacter, or Rothia.

Evan Haas and Dustin Romine
Brachybacterium, Caryophanen, or Rothia - 10

This isolate was obtained from the 2000 mL mark of the Winogradsky column that was under artificial light at all times. The first test performed was a Gram test and this resulted in the identification of this isolate as a Gram positive rod. We then performed an endospore test and an acid-fast test on our isolate. Both of these tests were negative. After this, the biochemical tests we had learned in previous laboratory exercises were performed. Tests on this isolate included: MR-VP, Simmons citrate, nitrate reduction, triple sugar iron, catalase, oxidase, indole, and lactose, sucrose, and glucose carbohydrate tests. Bergey's Manual was consulted for information from the summaries of genera contained within the non-sporing gram positive rod categories. As always, we were careful to examine both the test results contained within the summaries and the natural environment where these genera were found. By examining the various summaries, we determined that our bacteria was from one of the following genera: Brachybacterium, Caryophanen, Propionibacter, or Rothia. We examined the information for these genera again and found that Propionibacter bacteria were facultative anaerobes. Because this bacteria was taken from the very top of our Winogradsky column, we determined that it was unlikely that it was a facultative anaerobe. We were unable to further identify our bacteria because of the similarities within the other three genera. Therefore, this isolate is from one of the following genera: Brachybacterium, Caryophanen, or Rothia.

Evan Haas and Dustin Romine
Azomonas, Azotobacter, Pseudomonas syringae, or Pseudomonas viridiflava - 8

This bacterial isolate was obtained from the 1000 mL mark in the Winogradsky column under artificial light at all times. The first test performed was a Gram stain. From this, we determined that this isolate was a Gram negative cocci. We then ran a variety of biochemical tests learned from laboratory exercises. Tests performed on this isolate included: triple sugar iron, nitrate reduction, urea, lipase, catalase, oxidase, Simmons citrate, indole, MR-VP, and lactose, sucrose, and dextrose carbohydrate tests. As we did with the other bacterial isolates, we consulted Bergey's Manual and examined the summaries for genera of bacteria that were classified as Gram negative rods and cocci. By comparing our test results with those contained in the summaries of these genera, we were able to eliminate a number of possibilities. From our first examination, we limited this bacteria to seven possible genera. Those considered were: Azomonas, Azotobacter, Bordetella, Brucella, Methylococcus, Moraxella, and Pseudomonas. We then examined these seven genera closer than we had earlier. We first eliminated Bordetella as a possibility because these bacteria are strictly aerobic. Our isolate was obtained from a depth in our column that made it unlikely it was a strictly aerobic microorganism. We then eliminated Brucella because bacteria in this genera are oxidase positive and our isolate was oxidase negative. The genera Methylococcus and Moraxella were then eliminated because the test results found in various tables in Bergey's Manual did not match up with the test results we found for our isolate. We were able to determine that if our bacteria was from the genus Pseudomonas, then it was either P. syringae or P. viridiflava. However, we were not able to further determine what the specie might be if our isolate belonged to either Azomonas or Azotobacter. Therefore, we determined that this isolate was either Azomonas, Azotobacter, Pseudomonas syringae, or Pseudomonas viridiflava.

Evan Haas and Dustin Romine
Azomonas agilis, Azomonas insignis, Azomonas macrocytogenes, or Neisseria gonorrhoeae - 7

This bacterial colony was isolated from the 1000 mL level in our Winogradsky column that was under artificial light at all times. As always, the first test performed was the Gram stain. We determined that this particular isolate was a Gram negative cocci that was very small in size. We decided to perform the biochemical tests that we had performed for our other isolates. These included: triple sugar iron, urea, nitrate reduction, MR-VP, Simmons citrate, catalase, oxidase, indole, and sucrose, lactose, and dextrose carbohydrate tests. We examined the summaries found for Gram negative rods and cocci contained in Bergey's Manual to see what genera we could eliminate based on general characteristics. By doing this, we were able to narrow our possibilities for this isolate's genera to the following six choices. They were as follows: Alcaligenes, Azomonas, Brucella, Methylococcus, Neisseria, or Psychrobacter. We were then able to eliminate some of these genera by examining the summaries more closely and examining some tables of results found in Bergey's Manual. We were able to eliminate Alcaligenes because it is obligately aerobic and our isolate was found from a depth that made it unlikely that it was obligately aerobic. Test results concerning dextrose fermentation eliminated both Brucella and Methylococcus. We were able to eliminate Psychrobacter as a possibility because it generally does not grow between 35 and 37 degrees Celsius, and this is the temperature at which our bacteria grew. The remaining two choices for a possible genera worked according to our data. We were able to further distinguish within these genera. If our bacteria belonged to Azomonas, then we found that it would be Azomonas agilis, Azomonas insignis, or Azomonas macrocytogenes. If it was a member of Neisseria, then it would be Neisseria gonorrhoeae. We determined that these were the four best possibilities for this particular isolate.

Evan Haas and Dustin Romine

This particular isolate was obtained from the very top portion of the mud in the Winogradsky column that was not exposed to artificial light at all times. After performing the Gram stain, we determined this unknown to be a Gram negative rod. We then proceeded with our battery of biochemical tests that we performed on all of our unknowns. For this unknown we performed the following tests: an endospore test, MR-VP, catalase, Simmons citrate, oxidase, indole, lipase, nitrate, urea, and lactose, sucrose and dextrose carbohydrate tests. Because this bacteria came from the top of the mud, we treated it as a facultative anaerobe. We then examined the section in Bergey's Manual for facultatively anaerobic gram negative rods. By comparing our test results with the information contained in these summaries, we were able to eliminate many of the genera listed in this section. We were able to eliminate many genera based on the conditions in which they grew or in which they are found in nature. We determined that this unknown was Escherichi coli. This identification made sense because this bacteria is associated with fecal matter and the contents of our column came from behind a waste-water treatment plant. To be sure we had identified our isolate correctly, we streaked a sample onto an EMB plate. The growth on this plate had a green metallic shine, which is very indicative of E. coli. Therefore, we were convinced that this particular isolate was Escherichi coli.

The Winogradsky column is a very useful method in understanding the basic principles of microbiology. This column allows the scientists to observe microbial growth and succession in a controlled environment. The column not only serves as an observable entity, but as an actual ecosystem in which microorganisms grow and flourish. The Winogradsky column serves as an ecosystem in which different groups of bacteria can grow. One can expect to find anaerobic microorganisms towards the lower levels of the column due to the lack of oxygen at this level. Using this same logic, one can expect to find aerobic bacteria towards the upper reaches of the column, where oxygen is more available. In between the areas of aerobic and anaerobic growth, groups of facultative anaerobic or microaerobic bacteria can usually be found. If dealt with in a scientific manner, this column can teach beginning microbiologists many aspects of this subject.

Through the observations and experimentations that are required when examining a Winogradsky column, a microbiologist can gain valuable practice with microbiology laboratory techniques. As our group attempted to identify our isolated bacterial colonies, we were forced to practice the same techniques time and time again. Despite the fact that we performed the same experiments on a repeated basis, this did not get redundant. On the other hand, it served as valuable laboratory practice. I believe that after dealing with our column, the members of our lab group would feel comfortable if we were ever asked to perform any of the basic laboratory techniques required for the successful study of microbiology. Our group became acquainted with two other tests that we did not learn in our regular laboratory sessions. These two tests were the endospore staining test and the acid-fast test. Because we were responsible for identifying our unknown organisms, we were forced to further explore the avenues available to us in laboratory techniques. Because we performed extra tests that were not included in our regular laboratory, we learned as any good scientist should learn. We had to discover on our own what tests worked well for identifying organisms.

Through experimentation with our columns we were also forced to learn how to use Bergey's Manual of Determinative Bacteriology. Although this book seemed daunting at first, we were able to learn how to use it to help with the identification of our bacteria. Because the book is divided into many subsections, it made the task of identification somewhat easier. One problem we found with this manual was that it seemed that there were many test results not contained within the charts found in this book. We ran a variety of biochemical tests for our unknowns and then were unable to use the information derived from these tests to determine the identity of our bacteria. This proved to be a very frustrating aspect of using Bergey's Manual. However, the summaries in this manual for each bacterial genera were very complete. They contained enough information to make it possible for our group to eliminate many of them as possibilities.

When we set up our Winogradsky columns, we hypothesized that the column under artificial light at all times would grow a greater variety of microorganisms. However, we were not able to fully explore this hypothesis. Our group was unable to obtain as many isolated colonies from the column that was not constantly exposed to the artificial light. One possible reason why we were not able to obtain equal numbers of isolates from both columns was that our column that was exposed to light only at certain times simply did not grow as large of a variety as our other column. However, this explanation is very unlikely, because with the vast amount of microorganisms that exist in nature, there should have been a plethora of growth in both columns. A more likely explanation is that somehow, we did not collect samples as well from this column as we did from our column that was exposed to the light at all times. I cannot speculate as to why this would be so, but this is a likely explanation to the fact that we did have unequal samples from our two Winogradsky columns. There was one other problem with our Winogradsky columns that our group should have addressed. The majority of our isolated microorganisms came from the upper levels of our columns. Because of this, we did not find any true anaerobes. To obtain a good sampling of both aerobic and anaerobic organisms, our group should have taken more samples from the lower depths of our columns.

Although we were not able to fully identify all of our isolates, we did identify them to the most specific level we could using the tests we performed. We were not able to use morphological and behavioral characteristics of the bacteria as much as we should have to help identify our isolates. We did not take detailed enough observations as to whether or not our bacteria were motile, and if motile, then by what means they used to move. These observations would have aided in the identification of some of our bacterial isolates. Also, we did not take detailed enough observations as to in what arrangement we found our bacteria. We identified them as either being Gram positive or Gram negative and we took general shape characteristics, but we did not identify exactly how we found them. We did not record whether they were in sheets, long chains, huge clumps or other categories like these. In order to fully identify all of our isolates, our group should have taken more detailed observations. Despite these limitations, we are confident in the identifications that we did make. Once we had identified an unknown, we checked with Bergey's Manual to make sure our choice made sense. Often, we had identified our bacteria as those that are commonly found associated with soil, water, and most interestingly, feces. Because the mud and water for our columns came from behind a waste-water treatment plant, our identifications made sense.

As stated earlier, the Winogradsky column is a very useful tool if one wishes to understand the principles of microbiological laboratory techniques. Working with these columns was a tremendous learning tool for our lab group. Because it was expected that we would have to identify isolates obtained from our column, it forced us to learn how to perform good laboratory work. The identification process associated with these columns forced us to behave as scientists. Although we did not follow the scientific method exactly, we did use the basic principles outlined in this method. We made observations of our columns, asked questions as to what was happening in our columns and what this meant, and performed experiments to identify microorganisms found within our columns. By dealing with our Winogradsky column, we learned the principles of any scientific research activities. Because the word science is derived from Latin words that are associated with knowing or knowledge, scientists should be expected to learn, or become knowledgable with new ideas and techniques in science. The laboratory should serve as a place where this learning can take place. The Winogradsky column allowed the laboratory to serve its proper function.