Bacterial Diversity Project

Russell Valentin
Will Tollefson, Lindsay McFarland, Joe Eppard Section 5 9/24/2013

Introduction Bacteria are everywhere, often found in multitudes in places we would not always expect. In the Diversity of Form Function lab, we the students located an area within the Biology Building East where we presumed bacteria would be living to collect of sample of the bacteria and culture it on growth medium. In the following weeks, we performed many different tests and identification methods to aid in our determination of which bacterial species we found. We began by learning how to physically identify bacteria by eye, and then later examined our samples beneath a microscope for a more intimate visual experience. We also prepared our bacteria for PCR amplification of the universal 16S rRNA gene. Doing this arranged for us to later perform gel electrophoresis to confirm the genes amplification. Our PCR amplified bacterial DNA also served as the template for cycle-sequencing reactions, which would later allow for us to view the base pair sequences of our bacterias DNA and match them up to the BLAST database to further identify the species of bacteria we found. Other tests were done to determine whether the bacteria was Gram-positive or Gram-negative, such as the KOH string test and plating the bacteria on various media to analyze their growth patterns. Additionally, depending on whether the bacteria were presumed to be either Gram-positive or Gram-negative, the Catalase Test, the Mannitol Salt Agar (MSA) Test, and Oxidase Test were performed to reveal characteristics about the bacteria beyond Grampositive or Gram-negative and help affirm their previous classifications. The purpose of the multi-week project was to discover bacterial diversity, particularly in BBE. Pursuing this purpose allowed us to practice many bacteria-handling

laboratory techniques, and learn methods of distinguishing between microbial species. This, in turn, exposed us to the extensive variance in the forms and physiologies of even the smallest of organisms.

Materials and Methods During the initial handling of bacteria was when we learned and practiced good laboratory technique in handling bacteria and other microbes to prevent contamination- otherwise known as aseptic techniques. To demonstrate the prevalence of bacteria in even our laboratory setting, we practiced poor laboratory technique and touched growth medium with our fingertips, and left other medium exposed to the open air. These media were left to incubate and harvest bacteria. During the same time, we turned our focus to identifying and classifying bacteria, especially since there are more than just bacteria present in a given environment that could grow on our media. Some of these other species include fungi and mold spores. This made our ability to distinguish between the different microbial species very important. We identified bacteria by their size, color, and morphology. These physical characteristics brought our attention to groups that colonize, retain certain hues, and whether these colonies have certain types of surfaces, elevations or edge formations. In addition, microscopic examination of the unknown bacteria was done. This was to more closely observe the shape of the cells and their organization. After visually observing the bacteria we cultured, the time came to utilize other methods of analysis to determine which bacteria we had discovered. It was

during this time that preparation for and later execution of PCR amplification of the 16S rRNA gene took place. The 16S rRNA primers are universal primers- that is, the complementary sequences to the primer are preserved in all bacterial species. We prepared for amplification of the gene by setting up bacterial cells to be lysed and thus release DNA into PCR tubes, and purifying the PCR products with a buffer and filter technique. Purifying the PCR product gets rid of the extra contents used to create the product in an effort to avoid contamination and offsetting results. The nucleotide sequence of the DNA subject to amplification was expected to reveal which bacterial species it came from. Gel electrophoresis and cyclesequencing of the PCR-amplified ribosomal DNA were the two methods that we performed requiring the PCR product we prepared. Gel-electrophoresis of the purified PCR products included making an agar gel block, and inserting the PCR product and running it, thus verifying the amplification of the 16S rRNA gene from our bacteria. DNA sequence analysis of the 16S rRNA gene was done through cyclesequencing reactions, which included mixing the clean PCR product with Big Dye, a mix of DNA polymerase, normal nucleotides and fluorescently labeled ddNTPs. Following this step was a series of heating and cooling the mix to further prepare it for automated sequencing, which created DNA sequence data that could be viewed and analyzed on a computer. This precisely was done using FinchTV to produce and copy the actual sequence data for entry into the BLAST program, which compared the particular sequence data to many others within its database to locate similarities between bacterial species.

Biochemical testing was done on the bacteria as well. Two tests were done during this installment of the lab: the KOH string test, and plating on various test media to determine growth patterns. Though very different in procedure, both tests were intended to aid in determining whether the bacteria was Gram-positive or Gram-negative. Considering that bacteria can be classified into these two major types depending on layers of peptidoglycan within their cell walls, it was useful in identifying which type our unknown bacteria was. The KOH string test was simple in procedure- it simply called for looking for a string of bacterial DNA to form after being doused with potassium hydroxide (KOH) to determine the bacteria as Gramnegative. A string of DNA would form because the lack of peptidoglycan in the cell walls of Gram-negative bacteria allowed for those cells to lyse upon contact with KOH, along with getting their DNA adhered into a string once outside the cell. The plating on different test media required a different method of analysis- identifying whether the bacteria was able to grow on the various media (including an antibiotic, EMB-lactose and PEA), since Gram-positive and Gram-negative bacteria has different growth patterns on each medium. Specifically, Gram-positive bacteria are vancomycin-sensitive, do not grow on EMB-lactose medium, but do grow on PEA medium. Gram-negative bacteria follow generally opposite trends. Even further chemical classification was done, depending on whether the bacteria were presumed to be either Gram-positive or Gram-negative. Grampositive bacteria were put through the Catalase Test and the Mannitol Salt Agar (MSA) Test. The Catalase Test reveals the presence of the catalase enzyme in the Gram-positive bacteria through whether or not bubbles form in response to

hydrogen peroxide coming in contact with the bacteria. The MSA Test selects for salt-tolerant Gram-positive bacteria by revealing whether the medium changes color in response to a change in its pH by fermentation of the mannitol by the bacteria. Bacteria that are sensitive to the MSA mediums high-salt environment would not survive to grow in the mediums conditions, thus allowing us to further determine which species are present on the plate. The presumed Gram-negative bacteria were also run through the Catalase test, but were instead put through the Oxidase Test over the MSA test. The Oxidase Test determines whether bacteria contain cytochrome c oxidases. The oxidases are components of a complex that serves as the last enzyme of the respiratory chain- the biological course that transfers electrons to oxygen in aerobic respiration (Sadava et al, 2011). This test aided in determining whether the Gram-negative bacteria were aerobic or anaerobic, or used a different cytochrome to transfer electrons to oxygen.

Results Description/Morphology of Bacteria Three petri dishes of bacteria sample were initially collected. Dish 1 (BBE sample), which was collected from a microscope eyepiece, showed no visible microorganism growth. Dish 2 (exposed to room air) contained gold specks on the plate, all of which appeared to glisten. Dish 3 (my own finger sample) was similar to Dish 2- the plate contained white and yellow specs that were glisten-y.

Classification of unknown bacteria as Gram-positive or Gram-negative Our bacteria showed miniscule growth on vancomycin, some growth on EMB-lactose, and much growth on PEA. The little growth on the vancomycin and large growth on the PEA was white in color, white the small growth on the EMBlactose was purple in color. String was not formed when the bacteria were run through the KOH string test (Figure 1). Results of additional tests of Gram-Positive bacteria During the Catalase Test, our bacteria showed strong fizzing during treatment with hydrogen peroxide. When allowed to grow on the MSA medium, a scattered layer of cells was observed across it, with no apparent growth anywhere in the dish (Figure 2). Agarose gel of PCR amplified 16S ribosomal gene After gel electrophoresis, our agarose gel showed strong DNA fluorescence in our PCR well, dim fluorescence in our control well, and normal layers of fluorescence in our DNA size standards well. Edited sequencing chromatogram The sequencing diagram was messy and appeared to be showing multiple sequences. (Figure 4). BLAST Results See Figure 5, Figure 6.

Discussion Initially cultured bacteria Of the three dishes of bacteria collected (from microscope eyepiece, air exposure, and fingertips), bacteria grew on the latter two plates. No bacteria grew from the swab taken of the microscope eyepiece, and this is most likely due to good cleaning techniques done on the lab equipment, and lack of contact near the eyepiece as to avoid leaving marks that would inhibit viewing from it. The microscopes are also stored in closed cabinets, which may block off bacteria physically from entering, since most of the time the cabinet is closed. Bacteria grew on the air exposed and fingertip sample plates most likely because the air and human skin are common places that microorganisms are found.

Gram-positive or Gram-negative chemical tests There were several indications throughout our lab that led us to believe that our bacteria was Gram-positive. Firstly was the result of the KOH string test- no string was formed. This is an indication that the bacteria is Gram-positive, since the layers of their cell walls have much peptidoglycan in them and thus are not lysed by the KOH to form a string of DNA. We must be wary of our conclusion, however, since other factors such as having a sufficient density of cells to react, reacting enough KOH, and checking for a string underneath a microscope could all have affected our determination of the bacteria to be Gram-positive. Secondly, we evaluated bacterial growth on test media, which included vancomycin, EMB-lactose, and PEA. As mentioned earlier, Gram-positive bacteria are vancomycin-sensitive, inhibited by

EMB-lactose, and can grow on PEA medium. Gram-negative bacteria follow the opposite trends. In our test plates, we saw very small growth on the vancomycin, which was not expected for our presumed Gram-positive bacteria, but can be explained by slight contamination of the dish. There was slight growth along the edges of the EMB-lactose, which was also unexpected. This was probably due to handling error as well, and contamination along the edges took place and grew inside the EMB-lactose plate. There was much growth on the PEA plate, again, as expected for our bacteria. We found, generally, our chemical test results to be conclusive of culturing Gram-positive bacteria.

Additional tests of Gram-positive bacteria In the Catalase Test, catalase-positive bacteria are identified. This is also a test to help further identify bacteria as Gram-positive, with the exceptions of E. coli and Pseudomonas fluorescens, which are catalase-positive Gram-negative bacteria. Our bacteria showed much fizzing during the Catalase Test, indicating the presence of the catalase enzyme, thus revealing the bacteria to be catalase-positive. The MSA test revealed which bacteria were salt-tolerant or not by changing medium color from a pH change due to fermentation of surviving bacteria on the medium. Our bacteria did not visibly grow at all on the medium, and no color change was observed, indicating a salt-intolerant Gram-positive species of bacteria.

Agarose gel of PCR amplified 16S ribosomal gene In our completed agarose gel (Figure 3), it can be seen that our DNA in the second well traveled far and glowed very brightly, affirming our amplification of the 16S rRNA gene from our bacteria. However, there also appears to be mild glowing in our control well, which was not supposed to contain any DNA. Possible reasons for this could be that contamination of the control well took place either during preparation of the holding tubes or during injection into the wells. There could also be a chemical factor other than DNA that is causing glowing in the wells. Though the DNA well glows much brighter than the control, we cannot be completely sure that the amplification was of DNA, or that the electrophoresis was done procedurally correctly.

Edited sequencing chromatogram The edited sequencing chromatogram (Figure 4) showed to be a messy, complicated series of what appears to be multiple sequences. Some Ns were changed to the obviously correct base pair, but some were indistinguishable and left as unknown as to avoid making a false positive conclusion. In general, the sequences match up relatively well, but the most distracting factor in reading the chromatogram is the multiple lines that appear. Multiple sequences showing up in our chromatogram could be indicative of more than one bacterial species present that were sequenced. If this is the case, it may be difficult to put confidence in the sequence data as a whole, since some of the indicated base pairs may belong to a foreign contaminating species.

BLAST Results Up until sequencing the amplified DNA base pair by base pair, we believed our bacteria to be Gram-positive. This presumption was made based off of the general results of the preceding tests and techniques. After making minor edits of obvious errors to the sequence in FinchTV, the sequence was entered into the BLAST program. The top results of the BLAST database search hovered around a 94% identification match with Bacillus megaterium, a Gram-positive, mainly aerobic bacterium. Bacillus megaterium is ubiquitous in the environment around us, commonly found in soil, food, and a variety of surfaces such as leather, paper, and stone (De Vos, P. et al., 2009). This makes it no surprise that we isolated it in an environment like the laboratory. These BLAST results further validate our presumption that our bacteria is Gram-positive, since its DNA sequence has a close match to a Gram-positive bacterium. This is what makes the BLAST results different from the aforementioned tests- it profiles the bacteria on the level of the amplified nucleotide sequence, which provides a much more specific analysis than basic chemical tests would, especially since the basic chemical tests we used merely identified the general type of the bacteria. Should our BLAST results have conflicted with our presumption of the bacteria being Gram-positive, more stock should be put in the BLAST results. Since the uniquely identifiable sequence of the bacterial DNA can be identified and compared to other sequenced bacteria, the sequence comparisons made in BLAST would have been more accurate in determining the

species of bacteria. The basic lab tests cannot do this, and only determine basic qualities of the bacteria.

References De Vos, P. et al. Bergey's Manual of Systematic Bacteriology: Volume 3: The Firmicutes. Springer (2009)

Leicht B G, Holbrook M A. 2013. Diversity of Form and Function Biology 1412. USA: Fountainhead Press. 221p.

Sadava et al. (2011). Life: The Science of Biology, 9th ed. Gordonsville, VA: W.H. Freeman & Co. 1266p.

Zheng Zhang, Scott Schwartz, Lukas Wagner, and Webb Miller (2000), "A greedy algorithm for aligning DNA sequences", J Comput Biol 2000; 7(1-2):203-14.