cgualtieri's blog

This experiment was successful in showing the wide variety of organisms found in biofilms that form on a toothbrush. Using selective and differential agar plates, a total of 9 different colony morphologies were observed from the same source of inoculum. This clearly shows that a variety of organisms can be present in a biofilm simultaneously. If the toothbrush had been put in the saline immediately after being used by my lab partner, I would expect the number of colonies to increase significantly. I think that due to the toothbrush drying out between the time it was used and the time it was put in the saline, the number of microorganisms available to be captured from it decreased. Despite this, this experiment clearly shows the bacterial diversity present in our mouths and on biofilms on our toothbrushes.

This experiment showed that biofilms from two different bacterial sources could be grown in the lab and visualized using microscopy. P. aeruginosa was shown to be an excellent biofilm former, producing a thick, gooey, green slime. The EPS and rod shaped bacteria were clearly visible under the microscope. The environmental sample taken from the soil also produced a biofilm. It was yellow in color, smelled like rotting organic material, and was not as thick and gooey as the biofilm produced by P. aeruginosa. Under the microscope EPS and bacteria were visible, showing that there were some bacteria in the soil and root sample that had the ability to produce biofilms. Keeping the biofilms wet using the flow through gram stain was essential to maintaining the structure of the biofilm and allowed for visualization under the microscope. This experiment showed that two different sources of bacteria both produced biofilms, which were visualized under a microscope using a technique that kept the biofilms wet.

Once isolated colonies had been grown on the CDC agar plate, the next step of the experiment was to test them for their ability to eliminate two reactive oxygen intermediates (ROIs), hydrogen peroxide and singlet oxygen. To test for the bacteria’s ability to degrade hydrogen peroxide, a catalase test was done. It was expected that the anaerobic bacteria would contain the enzyme catalase and be able to degrade hydrogen peroxide. It was expected that when these bacteria were exposed to hydrogen peroxide, a chemical reaction would take place and small bubbles would form indicating that catalase was present and able to degrade hydrogen peroxide into water and gaseous oxygen. Staphylococcus epidermidis was used as a positive control for the catalase test, as this organism is known to have the catalase enzyme. It was expected that bubbles would form when S. epidermidis was exposed to hydrogen peroxide. Streptococcus agalactiae was used as a negative control for the catalase test, and was expected to show no bubbling when exposed to hydrogen peroxide due to the absence of catalase.

Wild type C. violaceum will produces a purple colored antibiotic called violacein. Violacein production is regulated by quorum sensing. C. violaceum’s AHL synthase gene, CviI, synthesizes the signal molecule C6-HSL which then binds to the regulator protein CviR. The CviR/C6-HSL complex regulates an operon responsible for violacein production. On the Petri plate streaked with wild type C. violaceum, it was expected that the heavy band of inoculum at the top quarter of the plate would turn completely purple. The wild type strain was expected to produce violacein because the AHL synthase and regulator protein were not mutated. Thus, this strain could produce its own signal molecule and receptor to activate the operon responsible for violacein production.

The aim of this experiment was to use wild type and mutant strains of Chromobacterium violaceum to explore N-acyl-homoserine lactone (AHL) based quorum sensing in Gram negative bacteria. AHLs are signal molecules produced by Gram negative rods. They regulate antibiotic synthesis, expression of virulence genes, biofilm formation, and several other cellular activities. Two genes are responsible for AHL mediated gene regulation. One encodes a transcriptional regulatory protein (R gene), and the other encodes the enzyme AHL synthase (I gene). The presence and proper functioning of these two genes is essential for the target genes to be transcribed. AHL synthase produces AHL molecules, which are classified by their side chain length and molecular structure. AHL synthases differ between each genus of bacteria, and produce AHL molecules that are slightly different from each other. Most regulator proteins that bind AHL molecules are specific for a certain AHL structure, but some can bind more than one type of AHL. This can create the phenomenon of cross-communication between different species of bacteria. This experiment was done to explore quorum sensing in Gram negative bacteria and determine if different species of bacteria could communicate with C. violaceum.  

The aim of this experiment was to explore the wide variety of organisms found in biofilms that form on toothbrushes. My lab partner’s used toothbrush was the source of these bacteria, and was sacrificed for the purposes of this experiment. The toothbrush head was cut off and placed into sterile saline to create a fluid suspension. An inoculum from the toothbrush-saline suspension was streaked onto TSA, SBA, MacConkey agar (MAC), Mitis-Salivarius agar, and CDC anaerobe blood agar plates. After incubating the plates, it was expected that each one would contain several different colonies that would allow the bacteria growing on the toothbrush to be directly observed.

The results of this experiment were in accordance with my expected results. On the slide culture of P. aeruginosa, a thick, slimy, green goo had grown and covered the entire slide and surrounding glass dish. When I attempted to remove the glass slide from the dish, long thick strands of biofilm formed between the slide and the glass dish. It was like when you take a bite of cheese pizza and the cheese forms a long, gooey strand between your mouth and the slice. When I put the P. aeruginosa slide under the microscope at 400x, I could see long, thin, greenish grey strands of biofilm going in all different directions. It was clear from this slide that P. aeruginosa formed a biofilm that contained lots of EPS. At 1000x the strands were not as defined, but rod shaped bacteria with thin EPS and biofilm filaments in the extracellular space were clearly visible. This showed that P. aeruginosa is an excellent biofilm former, and can form biofilms in an artificial environment in the lab.

The aim of this experiment was to observe biofilms microscopically by growing them on a slide using a process called slide culture. Using a technique called flow through Gram stain, the biofilms on the slides were kept wet to maintain their complex arrangement. Keeping biofilms wet is essential to preserving their structure, and allows them to be seen under a microscope. The inoculum for these slide cultures was obtained from two different sources. The first slide culture sample was taken from a pure culture of Pseudomonas aeruginosa. This Gram negative, rod shaped bacteria is an excellent at forming biofilms, which contribute to its virulence in humans and animals. It was expected that P. aeruginosa would produce a thick, slimy, dense biofilm on the slide culture. It was also expected that extracellular polymer substance (EPS) would be able to be seen under the microscope.

When a water soluble substance, like Vitamin C, is present in the body at higher than threshold

plasma levels, it will be eliminated from the body through urination. The level of the substance

in the body must reach the renal threshold for this to begin to happen. In the case given in this

example, we are dealing with vitamin C which is a water soluble protein that can’t be stored in

the body. If you were to intake too much vitamin C into your body at a given time, then it will

remain in your blood stream until it is eliminated via the kidneys and urinated out of the body.

Taking the vitamins in smaller quantities in timed out intervals will allow all of the vitamins to

be absorbed into the body because the renal threshold will not be reached.

Overall objective: to observe the phototactic behavior of the common cellar spider through the effects of LED light on web formation

Specific Aim 1. Observe how LED lights hanging at three different distances from the top of three separate Ziploc container effect cellar spider web formation. The first container’s LED will be touching the top of the container, the second container’s LED in the middle of the container, the third located one inch from the bottom of the container.