A dynamic approach was used order to study the ever changing ecosystems presented by both Winogradsky columns. Physical changes in the column, such as color and pattern changes, were noted every two to three days. Semester long observations on visible column changes allowed us to study microbial succession and evolution in action!
The column in the presence of continuous 75W artificial lighting was much more appealing to the eye than the untreated control column. The bacteria present responded to direct light, making apparent a wide array of colorful life in the column. Because of this, more tests were performed on the lighted column.
We began probing into our column after approximately five weeks of observations. Glass slides were electric taped to strands of fishing line and lowered to varying levels in the winogradsky columns. When at the desired height, the fishing line was secured to the column with the use of more electric tape. In the artificially lighted column, slides were hung at the water surface, 2000 mL, 1400 mL, and 1000 mL. A slide was also dropped into the column and allowed to rest on top of the mud. In the control column, slides were hung at 1400 mL, 1000 mL, and allowed to rest on the surface of the mud. Leaving the slides in the Winogradsky columns for a period of a week allowed microbes in certain vacinities attachment to the slides, as if they were an extention of the column. The slides were labeled and placed as follows:
1 - Lighted Column, Top of Water
2 - Lighted Column, 2000 mL
3 - Unlighted , 1400 mL
4 - Lighted Column, Mid Mud
5 - Lighted Column, 1000 mL
6 - Lighted Column, Top of Mud
7 - Lighted Column, 1000 mL
8 - Lighted Column, 1000 mL
9 - Lighted Column, 2000 mL
10 - Lighted Column, 2000 mL
11 - Lighted Column, 2000 mL
12 - Unlighted , 1400 mL
13 - Unlighted , Top of Mud
After giving the slides a week to collect bacteria, they were removed. That brings us to our first novel innovation for the Winogradsky experiment. The device, simply known as the lasso, can be formed by passing a long strand of fishing line down a pipet, through the narrow tip, and then doubling back through. This yields a loop at the terminal end of the pipet which can be contracted and expanded by pulling or pushing on the other end. This allows the microbiologist the option of simply dropping slides at various places within the column and then easily retreiving them. Slides can be pressed into the mud surface, and as long as a portion of the slide is protruding from the mud, it can be retrieved with the lasso. The lasso is definately a handy little invention that aided in retreival. The slides were removed and bacterial growth was streaked on aerobic TSA plates. We were not interested in anaerobic bacteria for the purposes of this experiment. We figured that most bacteria in the water were of the type aerobic or facultatively aerobic. Slides were dropped directly onto TSA plates 10 and 12. This is a technique we have labled the direct drop method. By dropping a slide directly onto a plate, an exact replica of the bacterial life on that slide is transfered to a maintainable growth medium. After approximately 48 hours, growth could be seen on plates 1,2,5,7,8,10,11,12,and 13. Bacteria from these plates were then restreaked to aid in isolation. After restreaking, growth was seen on each plate mentioned above. Using aseptic technique, bacteria from these TSA plates were transfered to TSA slants and grown up as isolated cultures. After allowing the isolated bacteria a weeks time to devolop and mature, gram stain testing was performed on all isolated bacteria. To perform a gram stain, a slide was sterilized and two circles were drawn in it using a wax pencil. A drop of water was placed into both circles. Using aseptic technique, a small amount of bacteria was transfered into the first circle using an inocculating needle. Then using an inocculating loop, a loop of bacteria from the first circle was transfered to the second circle. This was done to dilute the bacteria so it was not so concentrated when viewing through a light microscope. After approximately five minutes of allowing the slide to air dry, it was passed over a bunsen burner once or twice to 'heat fix' the sample to the slide. Then crystal violet was placed on the slide for one minute, rinsed with water, and iodine was added for one minute. After the application of iodine, the slide was rinsed with water and then rinsed for 1 second with alcohol and then 5 seconds with alcohol. The slide was then covered in safranin for one minute before it was rinsed with water, blotted dry, and viewed under oil immersion on the light microscope. If the bacteria was stained purple, we noted them as gram (+). If the bacteria was pink, we noted them as gram (-). If both purple and negative bacteria were present, the test was inconclusive due to an unisolated slant. Also noted was the general shape of the bacteria, the relative size of the bacteria, and if groups of bacteria formed certain patterns (clumps, chains, etc). Then metabolic tests were administered to aid in the identification of unknown bacteria. If the bacteria was gram (+), endospore, catalase, and oxidase testing were performed prior to other metabolic tests. Then all other metabolic tests were performed. If the bacteria was gram (-), all metabolic tests were ran immediately. The metabolic tests performed are as follows:
1 - Acid-Fast
2 - Amylase
3 - Catalase
4 - Citrate
6 - Cytochromoxidase
7 - Endospore
8 - Indole
9 - Lipase
10 - Methyl Red & Voges-Proskauer
11 - Nitrate
--> Dextrose, Lactose, and Sucrose
13 - Urea
After all metabolic tests had been run, we turned to Bergey's manual with our acquired physical and biochemical information in hopes of identifying our bacterial isolates.