Bacterial Lab Report 1 - Grade: 45/50 PDF

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BACTERIAL DIVERSITY Lab Project #1

Brooklynn Freas Section A17 Pair 7 | Laxmi Annapureddy, Corey Lewis, Adrianna Meyer 22 February 2020

Introduction: During the duration of this lab, we were presented with an unknown bacterial sample and tasked to find its identity through a series of different tests and procedures. Classifying bacteria based upon its physical and chemical properties is important in understanding how bacteria grows, evolves and survives. In this lab, we will use experimental and observational procedures in order to identify and unknown bacterial species. Physical examination of bacteria prior to manipulation is a very important part of the classification process. Different types of bacteria all have different properties: circular versus filamentous colonies and flat versus raised surfaces. The elevation of a bacterial colony can distinguish whether a species is Gram-positive or Gram-negative. The microscopic examination of bacterial samples is also important. When observed under a microscope, the cells of bacteria differ greatly. These bacterial cells all differ in both shape (cocci, bacilli, and spirochete) and their colonizing nature (clusters, chains, singular) These cellular features can tell us about the nature of the bacteria. Differing chemical tests, such as growth on different medias, will also be used in order to identify the chemical properties of the unknown bacterial sample. One of the most important portions of this lab in identifying the unknown species of bacteria is the PCR amplification of the 16S ribosomal gene. By amplifying a the 16S ribosomal gene, is becomes possible to identify and detect pathogens within different species. The PCR amplification was used to run a gel electrophoresis and ultimately to sequence the unknown bacterial sample. This was the most valuable portion in the identification process because it provided the most accurate and abundant piece of information that lead to the discovery of the genus and species of the unknown bacterial sample. Using a variety of examination and testing methods is extremely important in the identification process because it allows for the conclusion to be derived from the largest amount of knowledge about the unknown

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bacterial sample. This approach also ensures that the conclusion is still accurate in the case that a portion of the testing process fails.

Materials and Methods: This began by learning and understanding the importance of aseptic techniques in order to protect ourselves, and to get the best, most accurate results through the entirety of the three weeks. Once confident in the techniques and understanding of them, the unknown bacteria identification process began. A PCR amplification of the unknown bacterial sample was created. For this process, both a bacterial and a control PCR sample were created in order to accurately compare the results and expectations of the PCR. The tubes were incubated for use during week 2. After the liquid culture and PCR amplification were completed, the unknown bacterial samples were examined under a microscope to determine their shape and nature. The unknown bacterial sample was then also examined without the use of a microscope. This bacterial sample was then subjected to both an oxidase and a catalase test to determine whether the unknown bacterial sample contained the enzyme catalase and cytochrome c oxidase. For these tests, a chemical reaction between the unknown bacterial sample and hydrogen peroxide and a reaction of the unknown bacteria sample with an oxidase slide were performed. A portion of the unknown bacteria was also transferred to an MSA plate and incubated for 7 days. The MSA plate will determine whether the unknown bacteria sample was salt tolerant (growth) or salt intolerant (no growth). During week 2 of the identification process, the PCR reaction created during week 1 was purified using a PCR purification kit. After purification, the bacterial PCR sample and the control PCR sample were run through and 2

analyzed using gel electrophoresis. The samples and a size standard were loaded into the wells of an agarose gel inside an electrophoresis chamber. The gels were then run for approximately 45 minutes on high, until the bands had moved approximately 3/4th of the length of the gel. Using the left-over purified bacterial PCR sample, a sequencing PCR of about 500 bp was created. The sequencing samples were then placed into a thermocycler for approximately 2 hours. Upon completion of the cycle sequencing, the samples were cleaned and sequenced using the ABI 3730. While the gel electrophoresis was running, two additional tests were run to aid the identification of the unknown bacterial sample. The first of the tests was a KOH string test. This test determines whether the bacterial sample is Grampositive or Gram-negative. The unknown bacterial sample was combined with 3% KOH and mixed. If a string of cells formed, then the bacteria is Gram-negative, if no string formed, then the bacterial sample was said to be Gram-positive. The second test that was run is bacterial plating. The bacterial sample was placed onto three different Petri dishes containing EMBlactose, PEA, and vancomycin-containing agar. These plates were incubated for 7 days and examined during week 3 for the presence of growth. Once the PCR samples had been sequenced and distributed accordingly, the sequences were examined using a program called 4Peaks (If a PC is used, Chromas is the program necessary to analyze the sequences). When imported into 4Peaks, the sequence was then analyzed, and the badquality bases were trimmed from both the beginning and end of the sequence. Throughout the sequence there are positions where the bases have been replaced with “N”, this is due to the program being unable to identify the base at that location. These bases can sometimes be “called” manually if one of the bases is clearly indicated over the others present at that location. All of the “N”’s must be called throughout the sequence wherever possible. Once properly trimmed and edited, the sequence is then subject to BLAST (Basic Local Alignment Search Tool). This program compares the sequenced DNA sample to millions of gene sequences that 3

have previously been collected. Once BLASTed, the program provides results that have gene sequences that strongly match the unknown sequence. Results: The unknown bacterial sample that were we given was labeled A17-7. The sample was provided in a Petri dish and had been grown on an agarose gel within the Petri dish. The unknown bacterial sample had grown in large, irregular shaped colonies across the plate. The colonies of bacteria were white with a smooth, raised or convex surface.

Test

Results Positive

Negative None

Strong Weak Catalase X Oxidase X Table 1.1: Oxidase Catalase test results. X within the box represents the observed outcome of the test for each of the oxidase and catalase tests.

The results from the oxidase and the catalase tests, which can be observed in Figure 1.1, determined that the unknown bacterial sample contains the enzyme catalase but not cytochrome c oxidases. Both tests provided very strong and evident reactions, allowing for easy identification and understanding of the results of this test. Had the tests provided little reaction to the hydrogen peroxide or the N’-tetramethylphenylenediamine dihydrochloride, the results of the test would have been much harder to distinguish. The catalase test provided an abundant formation of bubbles at a quick rate and the oxidase test strip had little to no color change when it came in contact with the unknown bacterial sample. 4

Growth Present

No Growth (May

Red/Pink

Yellow/Orange

have sparse

Growth on

Medium Around

Medium Around

layer of cells if

Mannitol Salt

Colonies

Colonies

a large number

Agar (MSA)

of cells were

Plate

spread) XX

Table 1.2: Results of the Mannitol Salt Agar Test. Boxes with “X” indicate the observed results of the MSA plate test.

The MSA plate that was contaminated with the unknown bacterial sample presented a large amount of growth after a 7-day incubation period. The bacterial growth on the MSA plate presented with a reddish-pink color around the colonies. The presence of growth on the MSA plate concludes that the bacteria is a salt-tolerant species. Because the bacteria are observed to be salt tolerant, that also means that it is more than likely a Gram-positive bacterium. The coloring around the colonies of bacteria related to the fermentation of the mannitol in the MSA plate by the unknown bacterial sample.

Growth on Selective Medium Vancomycin EMBPEA Lactose

Forms a string n

-

-

++

KOH? n/a

+, +, -) Colony

White

White

White

n/a

Color Gram-

G+

G+

G+

No String

Amount of Growth (+

Positive or Gram5

(G+)

Negative? Table 1.3: Classification of Unknown Bacteria. Results provided within the table determine whether the unknown bacterial sample is Gram Positive of Gram Negative.

Testing the growth of the unknown bacteria on the selective mediums helped to determine whether the unknown sample is Gram-positive or Gramnegative. Table 1.3 records the observations of the unknown bacteria sample and its reaction with the three designated growth mediums: vancomycin, EMB-lactose and PEA. It is known that “most Gram-positive bacteria a vancomycin sensitive and inhibited by EMB-lactose medium but are able to grow on PEA medium,” which mean that Gram-negative bacteria will grow on both vancomycin and EMB-lactose but will not grow on PEA (Holbrook et. al, pg. 30). The unknown bacteria sample was observed to have abundant growth on the PEA plate but no growth on the vancomycin or the EMBlactose plates. These results in combination with the KOH string test conclude that the unknown bacteria sample is in fact a Gram-positive bacterium.

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Figure 1.4: Agarose gel of PCR amplified 16S ribosomal RNA gene. Well contents are labeled above. B1 and C1 are the bacterial and control samples for pair 7 and B2/C2 are the bacterial and control samples for pair 8.

The results of this agarose gel were not what was expected. As seen in figure 1.4, there is a band present in the C1 lane but not in the B1 lane which is inconsistent with the expectations. There should be a band present in the B1 lane and not in the C1 lane. An unknown bacterial sample was loaded into lanes 2 and 5 and a control sample was added into lanes 3 and 6. Lan 4 contains a size standard in order to measure and compare the bacterial bands to the known standardized sizes of the DNA.

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Figure 1.5: Unedited original sequence chromatogram. The sequence is unusable and unable to be edited, it therefore requires a backup. 9

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Figure 1.6: Edited backup sequence chromatogram. The initial sequence was unusable and therefore this sequence is analyzed in place of the original one. 11

The original sequence chromatogram was deemed unfit and unusable; therefore, a backup sequence was required in order to properly analyze and BLAST the sequenced PCR sample. As shown in figures 1.5 and 1.6, the chromatogram found in figure 1.6 contains a large number of uncalled bases. The waveforms of this chromatogram also have very drastic peaks or waveforms that are hardly differentiable. This makes it nearly impossible for the 4Peaks program to distinguish between the peaks and call the base that is clearly present over the others. This is also true for manual calling of bases. The backup chromatogram provides clear and obvious peaks at each location, making for an easy sequencing. This also allowed the uncalled bases to be identified easily because they were clearly present over the others.

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Figure 1.7: BLAST results. The image above is the primary match and the corresponding alignment to the 16S ribosomal RNA sequence. There was a 99.53% match. With respects to all of the tests run and the data collected, it can be concluded that the unknown bacteria sample is a Gram-positive bacterium strain called Sporosarcina ureae. The exact strain of this bacteria is unknown and undeterminable based upon the collected data and BLAST results. The BLAST results provide a 99.53% match to Sporosarcina ureae. There is only one inconsistency between the two sequences and that occurs at location 42. At this location, the BLAST program has recorded a base that does not exist in the backup sequence.

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Discussion: The purpose of this laboratory project was to determine the species of an unknown bacterial sample. Using a series of tests and observations, conclusions are made about the properties of the unknown sample and ultimately, what the unknown sample is. During the first week of this laboratory project, it was established that our unknown bacterial sample was a Gram-positive bacterium that contained the enzyme catalase but did not contain cytochrome c oxidase. During the physical analysis of the bacterium, it was observed that the irregular shape of the bacteria was raised in a convex elevation on the growth medium. The elevated growth of the bacterium is consistent with Gram-positive bacteria. With the negative oxidase results, it is expected that the unknown bacterial samples are anaerobic. Enterobacteriaceae are common oxidase-negative bacteria. Common enterobacteria include E. coli, Serratia, Lactococcus, Sporosarcina and Kurthia. Similarly, bacteria commonly associated with a catalase positive test are Bacillus, Staphylococcus, Micrococcaceae, Enterobacteriaceae, and Pseudomonas. With the data from these two tests it can be assumed that the unknown bacterial sample is an Enterobacterium. Though consistent to one another, these results do not set in stone that the unknown is an Enterobacterium because false positives and false negatives do occur. During week two of the bacterial diversity lab, a gel electrophoresis was run with a purified bacterial PCR sample and a control PCR sample. Unfortunately, this gel electrophoresis run failed. In this test, a band appeared in the control 1 lane but there was no band in the bacteria 1 lane. This leads to the conclusion that the two samples were switched and put into the wrong loading wells. It is possible that the control sample was contaminated, but that does not explain why there was no band in the bacteria 1 lane. Therefore, it makes the most sense that the two PCR’s were switched when they were loaded into the gel. If this is true and the bacterial PCR is in the control lane, it would appear that the band aligns perfectly with the 500bp of the size standard. Unfortunately, the data from this test cannot be relied on 14

and is therefore omitted from the information used to identify the unknown bacteria sample. During week two, growth from the MSA plate prepared in week one was also observed. The growth on this MSA plate presented with a reddish pink medium around the colonies of bacteria. Not all types of bacteria can thrive on an MSA plate, which means that the bacteria would be saltintolerant. Because the unknown bacterial sample being examined in this lab grew abundantly on the MSA plate, it can be determined that the unknown bacteria is salt-tolerant. “Bacteria growing on mannitol salt agar are positive for mannitol fermentation if the culture medium under their colonies is changed from its normal red color to yellow, indicating acidic growth products. Bacteria unable to use mannitol usually change the medium to a magenta color in the vicinity of growth. This color change indicates medium ingredients other than mannitol supported growth.” (Mannitol Salt Agar, vumicro.com). This information concludes that the unknown bacteria sample being observed is in fact a salt tolerant bacterium, but it does not ferment using the mannitol present in the MSA plate, hence why there is bacteria growth but no color change. The KOH test during week two also indicated that the unknown bacteria sample is a Gram-positive bacterium. The KOH test involves mixing the unknown bacteria with 3% KOH for approximately 1 minute. During this time, if a string forms then the bacteria is said to be Gram-negative, if a string of cells does not form from this mixture, then the bacteria is Gram-positive. No string formed when the unknown bacteria was mixed with a 3% KOH solution, therefore resulting in a Gram-positive bacterium. These results are consistent with the observations of the bacteria made in week one. During the final week of this laboratory project, observations of the growth on three different mediums and the analysis of the PCR sequencing were made. The unknown bacterium was added to an EMB-lactose, PEA and vancomycin plates and incubated for 7 days. When examined, an abundant amount growth was observed on the PEA plate but there was no growth on 15

the EMB-lactose and very little to no growth on the vancomycin plate. The growth patterns across the three plates is consistent with a Gram-positive bacterium, which is also consistent with the observations and conclusions made in previous weeks. Based on the growth pattern on the EMB-lactose plate, it can also be concluded that the bacterial species does not ferment lactose. The PCR sequencing played the largest role in determining the identity of the unknown bacterial sample. Unfortunately, the initial sequence chromatogram was unusable and required a backup chromatogram. The failure of this sequencing was most likely due to the lack of DNA within the sequencing PCR. Because the gel electrophoresis failed, the sequencing PCR was diluted based upon a standard dilution, not based upon the actual bacteria. Therefore, the dilution was made, and it did not contain enough DNA to properly sequence. With the backup sequence, the chromatogram required only two N’s to be called. This indicates a very good sequence chromatogram and an even higher match when BLASTed. Once the backup chromatogram was put through the BLAST sequence, a match of 99.53% to Sporosarcina ureae occurred. Of all of the tests and observations of the bacteria sample that was made, the PCR sequencing is by far the most accurate. This process analyzes the individual genes of the bacteria and compares it to millions of other sequences. With such a high match percentage between the unknown bacteria and Sporosarcina ureae, as well as the information collected from all of the previous tests, it can be very confidently concluded that the identity of the unknown bacterial sample, that was examined throughout the entirety of this lab, is Sporosarcina ureae.

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References: “4Peaks: For Peaks, Four Peaks. The DNA Sequence Trace Viewer for OS X.” Nucleobytes, nucleobytes.com/4peaks/index.html. “Bacterial Diversity Project: Identification of and Unknown Bacterial Species.” Diversity of Form and Function Biology 1412, by Brenda G Leicht et al., 8th ed., Department of Biology at University of Iowa, 2019, pp. 9–40. Hillis, David M., et al. Principles of Life. 3rd ed., Oxford University Press, 2019. Knisely, Karin. A Student Handbook for Writing in Biology. fifth ed., Sinauer Associates, Inc., 2017. “Mannitol Salt Agar.” Mannitol Salt Agar, vumicro.com/vumie/help/VUMICRO/Mannitol_Salt_Agar.htm. “Nucleotide BLAST: Search Nucleotide Databases Using a Nucleotide Query.” National Center for Biotechnology Information, U.S. National Library of Medicine, blast.ncbi.nlm.nih.gov/Blast.cgi? PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome. Rampini, Silvana K, et al. “Broad-Range 16S RRNA Gene Polymerase Chain Reaction for Diagnosis of Culture-Negative Bacterial Infections.” Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Soci...