Unknown Microorganism

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Introduction

The purpose of this lab is to classify and identify unknown microorganisms, specifically to identify unknown microorganism number 9. The health of humanity has increased as the identification of microorganisms has become available. The disease can now be prevented by the understanding of inter-bacterial relationships, how microorganisms reproduce and how they feed. As Harper et al. (2011), wrote in the paper “Phenotype Sequencing: Identifying the Genes That Cause a Phenotype Directly from Pooled Sequencing of Independent Mutants,” “Random mutagenesis and phenotype screening provide a powerful method for dissecting microbial functions” (p. 1). An organism can be identified by a series of positive or negative results based on the phenotype being present or absent.

An isolated sample of the pure culture must be established before laboratory testing can be initiated. A microbiologist can quickly identify an organism based on the organisms’ metabolic profile by completing differential tests. The difference between gram-negative and gram-positive bacteria can be established with a differential stain. Gene sequencing, however, is the most accurate tool used to identify the genes of microorganisms. Gene sequencing can compare existing sequenced microorganisms with a DNA profile of interest. For this to be accomplished in the lab, the amplification of an organisms’ 16S gene is necessary. The three-step process of PCR can be used to rapidly replicate the organism for testing. By heating to approximately 95 degree Celsius, denaturation separates the double DNA strand. The primer binds to the template strand during the annealing process which occurs at about 55 degree Celsius. The elongation step adds dNTPs to extend the complementary strand. 16S gene is a gene on the ribosomal RNA that is often used for microorganism identification purposes. The genomic DNA was isolated before the PCR amplification through gene extraction, as was written in Maxwell (2016) “Lab Experiment 6.”

The hypothesis for this lab is that if the unknown microorganism shows a positive result for the Citrate Test and a positive result for the Lactose fermentation test, then a positive result for the VP Test then identifies the organism as Enterobacter.

Methods

Isolation Procedure:

An unknown organism number 9 was assigned to the group. Two TSA plates were provided to isolate the bacteria and were incubated at 30 degrees Celsius for two days. The different colonies on the plates were documented. A plate was chosen for the most different colony types. One colony was harvested and inoculated in a TS broth tube, then incubated overnight at 37 degrees Celsius. That container was labeled unknown A. Another colony type was harvested and inoculated in a TS broth tube, then incubated overnight at 37 degrees Celsius. That container was labeled unknown B.

Gram Staining:

Unknown sample number 9 was Gram stained. The shape and arrangement of cells were documented. Identification was assisted by using a dichotomous key

Biochemical Tests:

After approval, the test was carried out. A dichotomous key was used, with the gram stain results, to assist in identifying unknown organism number 9.

A tube containing simmons citrate agar was obtained and inoculated with a culture of bacteria for the Citrate Test. The inoculation process was completed by using flame sterilization with a metal loop. The tube was incubated at 37 degrees Celsius for 24 hours. The color change was then observed and recorded.

Another tube was obtained and inoculated with the culture of bacteria for the Voges-Proskauer test. The inoculation process was completed by using flame sterilization with a metal loop. The tube was incubated at 37 degree Celsius for 24 hours. The color change was then observed and recorded.

A Petri dish containing lactose was obtained for the lactose test. The loop was sterilized under the flame, extracted a culture of the bacteria from the isolation plate, and inserted onto the plate containing lactose. The plate was incubated at 37 degree Celsius for 24 hours. The color change was then observed and recorded.

DNA Extraction:

Two heating blocks were brought to 55 degree Celsius and 37 degree Celsius, respectively. The reagents were mixed thoroughly and heated to 60 degree Celsius until all the precipitate dissolved, then cooled to room temperature. Two microcentrifuge tubes were used; one was labeled as A, the other labeled as B. A pipette was used to insert 1.5 mL of bacterial culture into each tube. The cell pellets were harvested by centrifuging both tubes at 12,000-16,000 x g for approximately two minutes. The culture medium was poured out of the tube leaving the pellet. Cells were re-suspended by adding 200 microliters of Lysozyme Solution into the pellet and then incubated at 37 degree Celsius for approximately 30 minutes. The microcentrifuge tubes were then removed from the heating block. 20 microliter of Proteinase K solution and then 200 microliters of Lysis Solution C was added into the tube. The tube was then mixed throughout using a vortex for approximately 15 seconds. It was then incubated for 10 minutes at 55 degree Celsius. 500 ml of Column Preparation Solution was added to a 2mL collection tube (Maxwell 2016). Tubes then centrifuged for approximately one minute at 12,000 x g, and the tubes were removed after the eluate was discarded. The centrifuge tube was removed from the heating block. 200 microliters of ethanol (95–100%) were added to the lysate, then mixed by vortex for 10 seconds and transferred to the column. Centrifuged was carried out again at 6500 x g for one minute. Eluate was then thrown away, and the column was placed in the 2mL collection tube. 500 microliters of Wash Solution was added to the column. The centrifuge was carried out again at 6500x g for one minute; the eluate was discarded and placed into a 2ml tube. 500 uL of Wash Solution was added into the column and centrifuged for approximately 3 minutes at 14,000 x g. A pipette was used to put “200 μL of the Elution Solution directly onto the center of the column” and was centrifuged at 14,000 x g for one minute, as was written in Maxwell (2016) “Lab Experiment,” (p. 5).

PCR Procedure: 

Each tube was labeled with the unknown number. Tubes were inserted one microliter of 16s Forward Primer, one microliter of 16s Reverse Primer, three microliters of Unknown sample DNA, and 20 microliters of Sterile water. The tube was gently mixed until the PCR bead was fully dissolved and it turned clear. The tube was placed into the thermocycler. The thermocycler made multiple copies of DNA by initially heating to 95 degree Celsius for approximately five minutes, then another 30 seconds at the same temperature. It then lowered to 55 degree Celsius for 30 seconds, then rose to 72 degree Celsius for 30 seconds and continued at the same temperature for another 2 minutes until it cooled down to 4 degree Celsius, as was written in Maxwell (2016) “Lab Experiment 6.”

Gel Electrophoresis Procedure: 

The 1% gel was prepared before the start of the lab by using 75 mL of 1% agarose with 7.5 μL of Syber® Safe DNA stain (Edvotek.com. 2019). The gel ran at 125 V for approximately 20 minutes. The FotoDyne Foto was used to visualize the gel. In a clean microcentrifuge tube, 8 microliters of the product from the PCR procedure was inserted into the tube. 2ul of loaded dye was then added. 10ul of PCR product was then into the 1% agarose gel. Data was documented.

Purification of PCR Product Procedure: 

The GenElute plasmid mini spin column was inserted into the collection tube.  0.5 millimeters of Column Preparation Solution “was added to each mini spin column. It was centrifuged at 12,000 x g for approximately 45 seconds. 5 volumes of Binding Solution to 1 volume of the PCR reaction” was added and mixed (Sigma-Aldrich, 2019, p. 1). It was then transferred to the binding column and centrifuged at 12,000 g for approximately one minute.  The solution was transferred into the binding column and the column was centrifuged at maximum speed (12,000 g) for 1 minute. The eluate was discarded but the collection tube was retained; this was repeated but centrifuged for 2 minutes. The column was transferred to a clean collection tube, 50 uL of Elution Solution was added to the center of each column. It was then incubated at room temperature for 1 minute. Centrifugation was carried out for one minute at maximum speed then stored on ice, as was written in Maxwell (2016) “Lab Experiment 6.” 

Quantify DNA Procedure: 

46 microliter of deionized water was used to dilute four microliters of purified PCR product. 50 microliter of DNA mixture was then added to a cuvette. 50 microliter of deionized water in another cuvette was used as a blank. The cuvette with the DNA sample was measured using the GENESYS™ 10 Series Spectrophotometer and DNA concentration results were documented.

DNA Sequencing Procedure:

The DNA concentration was used to calculate the amount of PCR product needed. In a PCR tube, 15 ng of PCR product for every 200 bp of PCR product length, one microliter of primer, QS to 10 microliters was added and labeled with the unknown number. Sequencing analysis was then carried out for the sample, as was described in Maxwell (2016) “Lab Experiment 6.”

(Materials omitted for preview. Available via download)

(Figures 1-6 omitted for preview. Available via download)

Conclusion

Separating and isolating a pure, unknown, bacteria culture started the experiment by using the plate-streaking technique. On a plate in three quadrants, a bacterial sample was inoculated and then incubated at 37 degrees Celsius overnight. Once the pure culture was isolated, gram staining was carried out. This resulted in Gram-negative samples for both members. A dichotomous key in figure 8 follows the biochemical tests necessary to determine the unknown microorganism. The Gram-negative sample has a rod shape when viewed under the microscope, so no further test was necessary. The Gram-negative sample went through the Citrate Test as the first biochemical test. It is a test to identify the organism’s ability to utilize citrate as its source of energy. When citrate is metabolized by the organism, ammonia is produced, changing the pH which turns the bromthymol blue from green to blue. The Citrate Test results were positive since there was an observed color change of green to blue indicating a positive test, as shown in figure 1.  The next biochemical test carried out is the Lactose Fermentation Test. The color changed to a pink color in the Lactose Fermentation Test is indicating positive as the pH indicator in the medium changes color, indicating acid production, as shown in figure 2. The final biochemical test carried out is the Voges–Proskaue (VP) Test. The test identifies organisms that utilize butylene glycol pathway and produce acetoin. It determines if the organism produces acetylmethyl carbinol from the fermentation of glucose. Acetoin is oxidized by potassium hydroxide (KOH) to diacetyl which then reacts to produce a red color. The color changed to a brownish-red color indicating positive results in the VP test.

Genomic DNA was isolated through the process of gene extraction. The 16S gene was amplified using the PCR. Once amplified, the DNA was sent to the Nanospec for quantification. The value of A260/A280 has to be greater than 1.8 to indicate a purified DNA sample. The experimental value was 1.829. Gel electrophoresis is another technique used for the identification of the unknown microorganism by comparing to the ladder strand. The negatively charged DNA fragment migrates toward the positive end of the gel. The rate of migration is relative to size. Bigger fragments travel a smaller distance than smaller fragments. DNA concentration from the SYBR safe was 10,000x concentration (10 microliters of SYBR safe) Cuvette spectrophotometer 1:10 Agarose 1:100 (1g agarose). Gel: lane 3 had 16s rRNA, the smudged line was DNA with other things.  This process makes the DNA sequence test more confident than microbiological tests. The Thermo Scientific Dream Taq PCR Master Mix was used which is “a ready-to-use solution containing DreamTaq DNA Polymerase, optimized DreamTaq Green buffer, MgCl2, and dNTPs” (ThermoScientific, 2019, p. 1). Sequencing analysis was then carried out for the sample which led to the final step. The final step was to analyze the unknown microorganism by comparing it to other closely related microorganisms on a phylogenetic tree

The results conclude that the unknown microorganism is Enterobacter, which is consistent with the original hypothesis and confirmed by the gene sequenced and the generated phylogenetic tree in figure 7, B. Subtilis, S.epidermidis, P. fluorescens. Enterobacter is Gram negative is pathogenic and causes infections in immunocompromised; respiratory tracts and urinary tracts are the most common infection sites. “Enterobacter infections can necessitate prolonged hospitalization, multiple and varied imaging studies and laboratory tests, various surgical and nonsurgical procedures, and powerful and expensive antimicrobial agents” (Fraiser, 2018, p. 1). Enterbacter is part of the coliform group of bacteria.  The bacterium is responsible for many opportunistic infections for patients after a surgical procedure in the hospital. An organism can be identified by a series of positive or negative results based on the phenotype being present or absent but makes DNA sequence tests are more confident than microbiological tests.

(Appendix omitted for preview. Available via download)

( Figure 7 omitted for preview. Available via download)

References

Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic acids research, 25(17), 3389-402. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/9254694.

Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet, F., … Gascuel, O. (2008). Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Research, 36(2), W465–W469 Retrieved from https://doi.org/10.1093/nar/gkn180.

Edvotek.com. (2019). EDVOTEK Quick Guide: SYBR Safe DNA Stain. Retrieved from http://www.edvotek.com/site/pdf/SYBR_Guide.pdf.

Fraser, S., MD. (2019). Enterobacter Infections. Medscape. Retrieved from https://emedicine.medscape.com/article/216845-overview.

Harper, M., Chen, Z., Toy, T., Machado, I., Nelson, S. F., Liao, J. C., & Lee, C. L. (2109). Phenotype sequencing: Identifying the genes that cause a phenotype directly from pooled sequencing of independent mutants. PloS One, 6(2), e16517. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3041756/

Maxwell A. R. (2016). Lab experiment 6: Unknown identification. Georgia State University. Retrieved from http://gastate.view.usg.edu.

Sigma-Aldrich. (2019). GenElute PCR Clean-Up Kit Catalog Number NA1020. Retrieved from https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/na1020bul.pdf.

ThermoScientific. (2019). Thermo Scientific DreamTaq PCR Master Mix (2X). Retrieved from https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012702_DreamTaq_K1071_UG.pdf.