It Doesn't Happen Only in Transylvania: Bacterial Hemolysis and Our Unknown Microbe

In order to further characterize our unknown microbe, this week's lab focused on determining whether or not it is able to lyse red blood cells. 

There are two ways that bacteria are able to lyse red blood cells (RBCs). Some bacteria contain a special compound composed of lipids and proteins known as hemolysin that allows them to form pores in the phospholipid bilayer of the cell membrane of a red blood cell. Other bacteria possess a different type of hemolysin that allows them to enzymatically cleave the red blood cell's phospholipid membrane. 

Bacteria fall into one of three categories: alpha, beta, or gamma. These designations describe their ability to lyse RBCs. Alpha bacteria do not lyse RBCs fully. Because the RBC cell membrane remains intact, they are said to partially break down the cells, and reduce hemoglobin to another chemical that is found in blood agar medium (5% sheep blood). These typically appear as a bruise-like color when plated. Beta bacteria are able to completely lyse RBCs, resulting in a clear "halo" around a plated colony. Gamma bacteria do not interact with red blood cells, and form colonies with no surrounding discoloration on blood agar plates.

Because virulence refers to the pathogenicity of a microbe, we believe that hemolytic bacteria would be more virulent than non-hemolytic bacteria since they would more easily affect/harm an organism that possesses RBCs. Additionally, we would not expect a "typical" soil microbe to be capable of hemolysis because plants and other soil organisms do not contain red blood cells or hemoglobin. Therefore, it would be an unnecessary attribute for them to possess. 

As seen in the photos below, we compared our unknown microbe to other bacteria. We believe our microbe would be categorized as alpha because of the greenish brown discoloration observed on the plate. 

We are still in the process of trying to narrow down the possible identities of our unknown soil microbe.  From all of our lab analyses, this is what we have determined about our microbe:

• gram-positive
• acid-fast
• ferments lactose and/or sucrose
• catalase positive
• endospore forming
• reduces nitrate to nitrite
• motile
• alpha hemolysis

Using the dichotomous key provided by Dr. Hanson, we have concluded that our microbe could be any of the following: Mycobacterium, Nocardia, Clostridium.

Check back next week for more details on our microbe.

-Anne and Austin

http://www.microbelibrary.org/component/resource/laboratory-test/2885-blood-agar-plates-and-hemolysis-protocols
http://en.wikipedia.org/wiki/Hemolysin
http://www.encyclopedia.com/doc/1G2-3409800092.html
https://www.wisc-online.com/learn/career-clusters/health-science/mby4307/blood-agar-and-hemolysis

Need a Fix? Nitrogen Fixation and Reduction in Our Unknown Soil Microbe

      In order to further characterize our unknown soil microbe, this week's lab focused on the process of nitrate reduction. This process is an integral part of the larger nitrogen cycle that alters atmospheric nitrogen into a usable form by plants and back into its atmospheric form. This process can be seen as a balancing act between the nitrogen that is "fixed" by other species of bacteria the nitrate that is removed from the soil by "denitrifying" bacteria and accounts for the majority of the nitrogen that enters the atmosphere. They are essential for the prevention of excessive levels of soil nitrate, which is important for public health and prevent eutrification (an overabundance of nutrients) of water sources.

      Only certain species of bacterial microbes have evolved the ability to reduce nitrate. This process typically occurs in locations that do not provide enough oxygen for aerobic respiration. Though nitrate reduction provides a microbe with a source of energy, it does not produce nearly as much energy as oxygen does in aerobic respiration. Thus, it would make sense that only bacterial species that are commonly found in oxygen-defficient locations would evolve the ability to reduce nitrate.

     We were able to use a simple test to determine whether our unknown microbe was capable of reducing nitrate. Three samples were inoculated with E. coli, P. aergoginosa, and our unknown sample, respectively, along with a control that did not contain a microbe. Samples were incubated for 48 hours. A smaller tube, known as a Durham tube, was located within the inoculated sample tube. If a bubble formed in the top of the Durham tube, it would indicate that our microbe was able to reduce nitrate (NO3) to its gaseous atmospheric state (N2). As seen in the photo below, this was not the case for our microbe. If the microbe were able to reduce nitrate to another form, such as nitrite (NO2), we would see the solution turn red after adding a solution of sulfanilic acid and alpha-naphthylamine to the inoculated sample. After adding the solution, our inoculated sample turned red (bottom photo). Thus, we concluded that our microbe was able to reduce nitrate to nitrite.

Results after 48 hours. Bubble formed in Durham tube in P. aeroginosa only.

Result following addition of sulfanilic acid and alpha-naphthylamine.

Up to this point, the dichotomous key has indicated that our unknown bacterium is Clostridium. According to the internet sources listed below, this identification still holds true with the results of this week's experiment. Come back next week for more details!!

http://en.wikipedia.org/wiki/Nitrogen_fixation
http://en.wikipedia.org/wiki/Denitrifying_bacteria

- Austin

Check Out Our Microbe's Moves: An Analysis of Our Soil Microbe's Motility

This past week in lab, Austin and I discovered another feature about our little microbe. We used the soft agar deep test to determine if our microbe is motile or not. For the experiment, we used a motile control (E. coli) and an immotile control (S. aureus). However, the E. coli sample did not behave properly because the results indicated that the sample was immotile, which is not true. As seen in the picture below, our microbe was very motile and moved all throughout the agar, which was determined by the cloudiness of the sample. A thick layer of microbes also developed on top of the agar.

Soft agar deep test results

So what makes bacteria motile or immotile? The main structural feature that allows microbes to be motile is the presence of flagella, which can vary in number and arrangement. As we studied in cell and molecular biology, flagella are anchored to cells by a hook that attaches to the basal body. A rotary motor unit does the actual whipping of the tale and can vary in speeds from 200 to 1000 revolutions per second, which is crazy fast!

There are advantages for microbes to evolve to be motile. The presence of flagella allows bacteria to swim towards nutrients and dodge harmful substances. They can also swim to new areas and colonize. Many bacteria are motile, however, some bacteria are immotile and have evolved to stay that way. A reason for remaining immotile is dependent on the types of fluid the bacteria travel through. Flagella are not useful when moving through viscous media and microbes that live in these environments do not want to be motile.

Each week in lab, we are continuously getting closer to identifying our microbe. To ensure the accuracy of our endospore verification test from last week's lab, we reran the experiment. The results ended up being the same but this time the controls produced the expected results. Below is a new picture of our endospore results.

Results from endospore verification redo

Hopefully next week, we will be able to narrow down the possible identifications of our microbe.

Check back next week for an update from Mills!

-Anne

http://cronodon.com/BioTech/Bacteria_motility.html

http://www.microbiologybytes.com/video/motility.html