Gram Would Be Proud

     In our project this week, we began the process of determining the identity and makeup of our chosen soil microbe. In order to have the basic skills required to do this we used the Gram-staining technique to determine whether our microbe was Gram (-) or Gram (+) in three control groups of bacteria. The reason this information is useful to is because it gave us a better idea of what broader category of bacteria our microbe is identified in. But, it also plays a significant role in the medical field when determining how to treat an infection or sickness caused by bacteria. 

     Gram (+) bacteria have thicker cell walls made of peptidoglycan than absorb crystal violet stain very well and make the bacteria appear purple in color. Gram (-) bacteria have a thinner cell wall of peptidoglycan that is sandwiched between two layers of cell membrane. This structure allows Gram (-) bacteria to absorb the crystal violet initially, then after washing and counterstaining, the bacteria appear to be a pink color. The reason this is important in the treatment of bacterial infections is because Gram (-) bacteria are much harder to combat and develop a much higher resistance to antibiotics because of the cellular structure. The outer membrane of Gram (-) bacteria can prove useful when infecting hosts because it provides more protection from autoimmune bodies or antibiotics. This makes it more deadly and harder to treat in humans or animals. 

     In order to use this technique to identify our microbe, we first stained three other control microbes that resembled different Gram identities. Each one had already been previously determined so it could give us an idea of what each type of bacteria would look like before we tested our unknown soil microbe. 

                                             Gram-Stain Micrograph of control B. megaterium

     Then, we Gram-stained our unknown microbe and determined its shape and Gram-identity. Our group's microbe appeared to be of a diplococcous shape and stained purple meaning it was Gram (+). We recorded this result, but then did further testing to confirm our results from the Gram Stain. We used McConkey agar which is selective for Gram (-) bacteria because it contains a high concentration of salt which deters Gram (+) bacteria from growing. After allowing this agar to grow at 30 degrees Celsius for five days, we found that there was no growth of our unknown bacteria on the agar plate. This result was similar to the responsiveness of B. megaterium which is also a Gram (+) bacteria. So, the result of our McConkey agar confirmed our initial hypothesis that our unknown microbe is Gram (+). 

Determining Our Soil Microbes
2/16/15

 
     Our adventure began on the small, yet highly potential campus of Birmingham-Southern College. The goal of this extensive experiment was to have a better understanding of all the microbial organisms that lie right beneath our feet. We first chose a prospective area of where we would gather our soil samples from. Our particular area of study lays in front of the Stockholm Women's Building in a small opening between shrubs in the surrounding landscape. We noticed here that plant growth within a ten yard radius of our specific sample of dirt consisted of only shrubs. The building lays about fifteen yards from our location. Our soil sample consisted of dark brown dirt that contained tiny increments of shrub roots and pebbles. It was rather soft and mildly compact with an average amount of moisture.
    In order to investigate our site's microbial activity we gathered a small sample of dirt using proper aseptic technique to avoid contamination. To prepare our sample we weighed out a small portion of dirt and mixed it with sterile water thoroughly.

    Next, we allowed the particulate matter in the soil to sink to the bottom and pipetted 1ml of this solution into a microfuge tube. From this 1ml sample we prepared five different dilutions. Each dilution contained smaller concentrations of soil extract. Furthermore, we placed 100 ml of soil extract from each dilution into five labeled TSA plates and spread them appropriately. In addition, we placed 100 ml of the 10^-4 dilution onto a Rose-Bengal Plate which allows for selective growing of different fungi from or soil sample. After six days we observed our TSA plates for microbial growth and found that nearly all of them contained growth except for our 10^-7 dilution plate. With each smaller dilution, we found that the number and size of colonies decreased. We also found that our Rose Bengal plate contained a large amount of fungal growth.