Bacteria are very diverse organisms, and their diversity is the reason multiple different tests and differences need to be taken account of in order to identify specific species. Some identification is based on morphological differences between bacteria, like: cell size, shape, color, colony shape, surface appearance, elevation, and colony edge shape. Other than by their morphology, bacteria can be differentiated by their source of energy, thermotolerance, antibiotic resistance, style of movement, salt tolerance, and if they are anaerobic vs. aerobic. The ways to determine these differences is through biochemical tests, like growth on mannitol salt agar (MSA), vancomycin, EMB-lactose, and PEA plates, response to hydrogen peroxide in the catalase test, the color change in the oxidase test, and “string” formation in the KOH test.
MSA plate growth would indicate if the strain of bacteria is salt tolerant. In addition to growth indicating tolerance, the bacteria also causes a color change to the agar. If the plate stays the same reddish pink color that means the bacteria cannot ferment mannitol; however, if the plate changes color then it means that it can ferment mannitol. Growth on the vancomycin plate shows that the bacteria is not sensitive to vancomycin, while lack of growth indicates that the strain is sensitive to vancomycin. Sensitivity primarily occurs in Gram-positive bacteria, while growth is due to Gram-negative bacteria being resistant to the antibiotic (Saleh 2018). The EMB-lactose growth is related to whether or not a bacterium is Gram-negative or Gram-positive. No growth indicates a Gram-positive species, while the opposite points to a Gram-negative species. The color of bacteria colony growth also indicates if it can actively ferment lactose (Lal 2007). The PEA plate test is also related to whether or not tested bacteria are Gram-positive or negative. The phenylethyl alcohol prevents or reduces the growth of Gram-negative bacteria by preventing proper DNA synthesis.
The catalase test shows if a bacteria strain contains the enzyme catalase which breaks down H2O2 into water and oxygen. A concentrated hydrogen peroxide solution is added to bacteria to see if bubbling occurs. A visible reaction indicates that the bacteria has the enzyme, while little or no reaction at all means that there is no enzyme present and the cellular toxin kills or damages the cells. The oxidase test is to see if bacterial species has cytochrome c oxidases, which “functions as the last enzyme of the respiratory chain that transfers electrons to oxygen” in aerobic respiration (Holbrook 2018). The test itself uses oxidase slides that cause color change in the bacteria added correlates to the presence of the enzyme, if the bacteria sample changes to dark blue or purple it means an electron was received indicating that the enzyme is present. No change means the bacteria does not have cytochrome c oxidase.
KOH “string” test shows if a bacterium is Gram-negative or Gram-positive based on if a string is formed after mixing a small sample of the bacteria into a 3% KOH concentration. The formation of a string indicates that the bacteria is Gram-negative, while no formation means it is Gram-positive.
In addition to biochemical tests and morphological differences bacteria can be identified through Sanger sequencing, which sequences genomes from purified 16S RNA PCR. The sequencer can create about 800 base pair sequences, for longer segments to be built requires smaller fragments to be primed and pull DNA together in thousands of base pairs long (Holbrook 2018). These DNA sequences can then be run through nucleotide BLAST to identify what any unknow bacteria could be in its relation to similar or the same species of bacteria.
The purpose of this bacteria project is to determine what kind of bacteria was given through the use of morphological differences, biochemical test results, and finally through Sanger Sequencing.
Materials and Methods
To identify our unknown bacteria, we needed to separate and amplify the bacteria’s RNA, to do so we used PCR amplification of the 16S RNA gene since they are universal primers. By doing DNA sequencing of this region the end result nucleotide sequence would show which species the bacterium came from.
To start, we obtained two PCR tubes with 25l of 2X PCR Master Mix and a tube with 300l of sterile water. The two PCR tubes were then designated as either “B” or “C”. Then 5l of sterile water was added to the C tube containing 25l of 2X PCR Master Mix, along with 20l of 16S RNA Primer Mix. Since this tube had no bacterial DNA it served as our negative control. For tube B we vortexed a cluster from our bacteria colony with the remaining sterile water of 295l to create a liquid bacteria culture. We added 5l of the bacterial culture to tube B containing 25l of 2X PCR Master Mix and 20l of the 16S RNA Primer Mix. Each tube was then put in the microcentrifuge and then added in the thermocycler for DNA amplification. The thermocycler ran on a cycle of 1X: 94C, 3 minutes; 30X: 94C, 30 seconds; 50C, 30 seconds; 72C, 45 seconds; 1X: 72C, 5 minutes.
During the PCR reactions we looked at our bacteria through microscopes to get its cell shape and morphology. We identified our bacteria cell shape through moving 20l of our liquid culture onto a microscope slide and observing it through 400X magnification. We also identified the colonies’ morphology through its color, specific colony arrangement, the edges of the colonies, elevation, and surface appearance.
After getting our DNA amplified, the PCR products needed to be purified so they could be sequenced. First our PCR tubes were centrifuged, and the contents of tube B were transferred to a clean 1.5 ml microcentrifuge tube and vortexed with 250l of Buffer BB. We obtained a spin column in a collection tube and added the sample B and Buffer BB mix to the spin column. The column was centrifuged for 30 seconds, and any flow through was discarded. Then 200l of Buffer WB was added to the spin column which was then centrifuged again, discarding any flow through. We repeated the same step again. The spin column was then transferred to a 1.5ml centrifuge tube and mixed with 25l of Buffer EB. The tube was allowed to sit for 1 minute and was then centrifuged for 30 seconds, after which the spin column was discarded leaving the remains for sequencing and gel electrophoresis.
To prepare our gel electrophoresis we made 40 ml of a 1.5% agarose solution with 0.6 g agarose. We put the agarose in a 125 ml flask and added 40 ml of 1X TBE Buffer, the flask was microwaved until the solution was clear. We put the flask to the side to cool, and after it cooled ethidium bromide stain was added. Then the solution was poured into a gel caster and left aside until cooled to a solid gel. After setting, the gel was put on the center platform of the electrophoresis chamber and covered with 1X TBE Buffer.
We prepped two tubes, Tube B and Tube C, with 4l of loading dye. Tube B had 6l of the purified 16S RNA PCR product added, and Tube C had 12l of the control PCR added. Both were spun in the small centrifuge and each sample was then loaded into the gel wells. Well 1 was left empty, well 2 contained 10l of our unknown bacteria, well 3 had 10l of our control, well 4 contained 10l of the DNA Size Standard (SS), well 5 had 10l of pair 12’s unknown bacteria, and well 6 had 10l of their control, wells 7 and 8 were empty. We put the electrophoresis on the high voltage setting and let the gel run for 40 minutes.
During the gel run, we did a DNA sequencing reaction. We combined 6l of Big Dye Mix with 2l of purified PCR product and 2l of ddH2O. This mix was then added to the thermocycler and run at 1X: 90C, 1 minute; 96C, 10 seconds; 50C, 5 seconds; 60C, 2 minutes; and held at 4C.
While the PCR reactions were run, we used the bacteria sample and the liquid bacteria culture to do the biochemical tests on the MSA plate, catalase test, and the oxidase test. For the MSA test we used an MSA plate and put 60l of our liquid bacteria culture to the plate and poured 10 sterile glass beads onto the plate. Then, we gently shook the petri dish to get the beads across all of the agar to thoroughly distribute the cells. Afterwards, the beads were discarded and the dish was incubated at 37C for 2 days to allow for growth, then held at 4C for a week to allow for us to observe any growth and color change in the next week.
For the catalase test we put a clean microscope slide in an empty petri dish and, using a toothpick, collected a cluster of our unknown bacteria and placed it in the middle of the slide. We put a drop of 3% H2O2 on the cell smear and waited observed the bubble reaction. If no reaction occurred, we would repeat the catalase test using 15% H2O2.
For the oxidase test we obtained a clean dry oxidase slide and petri dishes with colonies of both positive and negative controls. The oxidase slide had 4 test areas that the cytochrome c oxidase enzyme could use as artificial election acceptor instead of oxygen; N, N, N’, and N’-tetramethylphenylenediamine. We used sterile toothpicks to collect colonies from our bacteria, the positive control bacteria, and the negative control, and placed them into the corresponding test areas on the oxidase slide. The slide was incubated for 1 minute and we observed the color change reaction that would indicate if the unknown bacteria has cytochrome c oxidases. The remaining liquid bacteria culture was used to prepare a fresh culture plate that would provide bacteria cells for the next week of biochemical tests. The procedure used for preparing the MSA plate was used for this culture plate creation. The new petri dish was then incubated into the next week.
Using the second culture plate from the week before, we created a second liquid bacteria culture using the same procedure from the creation of the first liquid bacteria culture. We used this second petri dish of bacteria culture for the KOH “string” test. We obtained a clean microscope slide and placed 50l of 3% KOH on it. Then using a sterile toothpick, we collected a small cluster of cells and mixed it in the KOH for 60 seconds while observing for string formation indicating if the bacteria is Gram-negative or Gram-positive
Using the second liquid bacteria culture we plated cells onto the EMB-lactose, PEA, and vancomycin petri dishes. We added 50l of liquid bacterial cultures to each dish and used 10 sterile glass beads, for each, to evenly distribute the cells. These three agar mediums were then incubated at 37C for 2 days and stored for the next week’s colony growth and colony color observation.
Using the DNA sequenced in the second PCR reactions, we analyzed it using 4Peaks nucleotide software to get a full DNA sequence, with the completed sequence we then analyzed it using BLAST to find the most likely strain of bacteria our unknown could be.
Half of our biochemical tests on the unknown bacteria consisted of plate tests, the MSA plate growth, vancomycin growth, EMB-lactose growth, and PEA growth. The bacteria did grow a lot on the MSA plate, in a thick layer on top of the agar. The agar also changed color around our colonies, instead of staying a reddish shade it changed to a medium orange color (Figure 1). The combination of strong growth and color change means that our unknown bacteria is both salt tolerant and can ferment mannitol. There was also growth on the vancomycin and EMB-lactose plates. Neither had excessive growth, but enough that provided visible morphology and colony color. The vancomycin colony growths were rhizoid shaped with lobate edges, rough and glistening surface and raised elevation, with a slightly yellow tinge to the colonies. For the EMB-lactose the colonies were circular in shape with entire edges, smooth and glistening surface texture, with pulvinate elevation, and the colonies were light pink in color compared to the darker red of the medium. There was no growth on the PEA plate (Figure 2).
Growth on mannitol salt agar (MSA) plate Growth Present No Growth (may have sparse layer of cells if a large amount of cells were spread)
Red/Pink medium around colonies Yellow/Orange medium around colonies
Figure 1: Table stating if there was bacterial growth on the MSA plate, and if there was any color change that occurred around the colonies
Figure 2: Unknown bacteria on biochemical growth plates. From left to right the agar mediums are: MSA, EMB-lactose, vancomycin, and PEA.
The results of the biochemical plate tests alone provides conclusions that our unknown bacteria can tolerate salt environments, ferment mannitol, is not vancomycin-sensitive, can grow on and ferment lactose. The next test that also gives more context to the growth results is the KOH string test. When we tested our bacterial sample, we did observe string formation which indicates that our unknown bacterium is Gram-negative. Pairing the results of KOH with the plates solidifies the conclusion that our unknown is Gram-negative because only Gram-negative bacteria can grow on EMB-lactose and vancomycin, and they cause the color change in the MSA plate. Also PEA inhibits proper DNA synthesis in Gram-negative bacteria, either limiting or preventing growth (Figure 3).
Growth on Selective Medium Forms a string in KOH?
Vancomycin EMB-lactose PEA
Amount of Growth (++, +, -) + + – n/a
Colony Color Colorless/ slightly yellow Light Pink No Growth n/a
Gram-positive or Gram-negative?
Figure 3: Table stating growth, colony color, Gram stain, and results of KOH string test for the agar mediums of vancomycin, EMB-lactose, and PEA.
The other set of biochemical tests we did on our unknown bacteria were the oxidase and catalase tests. When we performed the oxidase test we noticed that after the 1 minute incubation period that the oxidase test slide had no visible color change with our sample, indicating that the bacterium is oxidase negative and does not have cytochrome c oxidases. Meaning that our bacteria is not able to use oxygen as an electron acceptor or uses another cytochrome to move electrons to oxygen (Holbrook 2018). For the catalase test we observed no reaction with the 3% H2O2, so we repeated the test using 15% H2O2. In the second try there was a weak bubbling reaction, our bacteria slowly broke down the H2O2into water and oxygen creating the visible reaction. This reaction indicates that our unknown has the enzyme catalase, and since it only reacted to the 15% H2O2, we can conclude that the bacteria is weakly positive (Figure 4).
Strong Weak None
Figure 4: Table stating results of catalase and oxidase tests. This table also has labels to specify the strength of the response to the tests.
Aside from the biochemical tests, we also analyzed the morphology and cell shape of our bacteria, we found that the cells of our bacteria were bacilli and clustered together. Also, the bacteria colonies were rhizoid in shape, the edges were a mix of entire and undulate, and the elevation was raised and convex at different locations along the colonies.
Through analysis of our gel electrophoresis we compared the length and band size of our unknown bacteria to that of the DNA Size Standards. Our pair’s unknown bacteria was in well 2 labeled as B1 with our negative control located in well 3 labeled as C1, while the other members in our group had their bacteria in well 5 labeled B2 and their negative control in well 6 labeled C2. We noticed that our bacteria had a band size of about 500 bp (ng) and our negative control was not visible, indicating that our controls were accurate lending to our electrophoresis to be done correctly (Figure5).
Figure 5: Gel electrophoresis of unknown bacteria DNA compared to size standard markers and controls, with band sizes measured in bp mass (ng).
The end of our lab project was to use Sanger sequencing, to create a long bacterial nucleotide sequence that could be used to closely identify our unknown bacteria. We used the 4Peaks software to visualize the nucleotide sequence down to each individual base. Once provided with our sequence we combed through looking for any bases that were unknown since the program cannot always figure out which nucleotide is associated to the bases, these unknowns are marked as red Ns. With bases that were easy to see as dominant we made edits into the base marker, we also deleted small sections of bases at the front and end of the sequence that were too small and complicated to clearly distinguish (Figure 6). In the case of our bacteria sequence we only had about 390 bp, which is not necessarily very small but does lend itself to the fact that not all of our bacteria was able to be sequenced.
Figure 6: Edited unknown bacterial nucleotide sequence. Each base pair has a different letter and color combination, cytosine is the blue C, adenine is the green A, thymine is the red T, and guanine is the black G.
In addition to looking through the nucleotide sequence, we also used BLAST to find our highest match to identify our unknown bacteria. By looking through these matches the bases that are the same to those in the query and in the subject are connected by a line. Mismatches found between them have no line, these are formally known as single nucleotide polymorphism. Sections where insertion and/or deletion are present are indicated by a dash (Figure 7). Unfortunately, our experiment showed that we had over 50 gaps between our query and the subject.
Figure 7: Alignment of a query sequence to Cronobacter strain JZ38 produced by NCBI BLASTn.
After sequencing and analyzing our bacteria, we were able to copy our sequence into the BLAST nucleotide sequencer to get close or identical matches to our bacteria. We got plenty of possible matches to our bacteria, even though our sequence was not as long as is preferable for this lab, we were able to sequence enough DNA to get close matches (Aalsmeer 2019). Our BLAST sequence provided us with 4 possible matches with the max score of 315 and over 30 with a max score of 309. All of these matches are strains of the bacteria cronobacter, with most of our matches being unidentified smaller strains of cronobacter bacteria. Our top two matches aside from the general bacteria were Cronobacter Dublinensis and Cronobacter Muytjensii (BLAST 2019).
The top match had a max score of 315, a total score of 315, Query cover of 99%, an E value of 1e-81, and an Indent of 81.77%. Since the E value is very low it means that our match to cronobacter bacteria has a large probability of being the identity of our unknown bacterium. Cronobacter is a group of Gram-negative bacteria that inhabits dry environments, and is commonly found in dry foods and waste water (Centers for Disease Control 2018).
Since it is known to be a group of Gram-negative bacteria our results that correlate with Gram-negative bacteria help us to conclude that Cronobacter bacteria is likely to have been our unknown bacterium.
Aalsmeer, R. (2019). 4Peaks. The Netherlands: Nucleobyte. https://nucleobytes.com/4peaks/index.html
BLAST: Basic Local Alignment Search Tool. (n.d.). Retrieved February 17, 2019, from https://blast.ncbi.nlm.nih.gov/Blast.cgi
Centers for Disease Control. (2018, October 17). Expanded Information | Cronobacter | CDC. Retrieved from https://www.cdc.gov/cronobacter/technical.html
Hollbrook, M. A., Leicht, B. G., Toll, C. D. 2018. Diversity of Form and Function: Biology 1412. Seventh Edition. The University of Iowa.
Lal, A., & Cheeptham, N. (2007, September 29). Eosin-Methylene Blue Agar Plates Protocol. Retrieved from https://web.archive.org/web/20111130203711/http://www.microbelibrary.org/component/resource/laboratory-test/2869-eosin-methylene-blue-agar-plates-protocol
Saleh, N. (2018, February 14). How Vancomycin Fights Drug-Resistant Infections (S. Olender, Ed.). Retrieved from https://www.verywellhealth.com/whats-vancomycin-1124179
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