The purpose of this lab report was to identify the unknown bacteria given to us by our professor.
Requirements: 500
Unknown Lab Report #2 Unknown G+ A Student November 22, 2022 Professor Shawn Mustain Microbiology 2120 Fall 2022 Section 500
Introduction The purpose of this lab report was to identify the unknown bacteria given to us by our professor. The methods and procedures learned in this class were applied in the process of identifying the unknown bacteria. Being able to identify bacteria is crucial in preventing and treating different diseases. When determining the best course of treatment for a patient, it is essential to know which bacteria you are dealing with. Knowing the identify of a bacteria infecting a patient is also helpful in preventing antibiotic resistance, giving accurate diagnoses, and tracing diseases back to the microbes that cause them. Materials: • Microscope • Slide • Tryptic Soy Agar • 3% Hydrogen Peroxide • Bunsen Burner • Inoculating loop • Test Tubes • Phenol red dye • 1% Lactose • 1% Sucrose • 0.1% Glucose • Sodium thiosulfate • Ferrous sulfate
Methods: A gram stain and TSA plate for the unknown bacteria labeled G+ was given by the professor. The gram stain showed a purple, sphere shaped bacteria in clusters. A catalase test was performed to determine if the unknown could produce the enzyme catalase, Ex which allows organisms to escape the damage done by hydrogen peroxide. (American Society for Microbiology, 2010.). With a sterilized inoculating loop, a small amount of the unknown bacteria was smeared onto a dry slide. A drop of hydrogen peroxide was placed on top of the bacteria. If bubbles appeared, the test was positive. If no bubbles, the test was negative. The result of the unknown was positive. The control organisms used in the catalase test were Staphylococcus epidermidis and Lactococcus lactis. When a catalase test was performed on Staphylococcus epidermidis, bubbles appeared and the result was positive. On the other hand, Lactococcus lactis failed to produce bubbles which resulted in a negative catalase test. The purpose of the controls was to ensure that the catalase test was performed correctly. After the result of the catalase test was determined, a Triple Sugar Iron Agar slant was performed. A TSI Iron Agar slant is used to test the ability of an organism to ferment lactose, glucose, and sucrose and determine whether or not the organism can produce hydrogen sulfide. The TSI slant was inoculated with a loop and incubated for 48 hours at 37C. The TSI slant was observed for a change in color from red to yellow. (American Society for Microbiology, 2005). The result retrieved from this test for unknown G+ was a red slant and yellow butt with cracks or bubbles. The control organisms used in this test
were Staphylococcus epidermidis and Pseudomonas aeruginosa. Staphylococcus epidermidis produced the same results as Unknown G+. Pseudomonas aeruginosa produced a red slant and an unchanged butt. Gas was also produced. Results: The Gram stain revealed a Gram-positive cocci. After reviewing the Gram stain and eliminated all Gram-negative species from the list of potential bacterial species that Unknown G+ could be, a catalase test was performed and brought back positive results. After the positive catalase test, a TSI test was performed to narrow down the possible list of species that Unknown G+ could be. This test resulted in a red slant and yellow butt with bubbles showing that the unknown bacteria was able to ferment glucose and produce gas. Because only one remaining bacterial species matched this criteria, the unknown was determined to be Staphylococcus epidermidis. The results of all tests performed can be seen in Table 1. Table 1: Biochemical Tests Test Purpose Reagents Observations Results Catalase Used to test a bacteria for the production of the enzyme Catalase Hydrogen peroxide Bubbles The unknown G+ tested positive for catalase. Triple Sugar Iron Agar To differentiate enteric based on the ability to reduce sulfur and ferment carbohydrates Lactose, sucrose, glucose (dextrose), sodium thiosulfate, ferrous sulfate, phenol red Red slant/ Yellow butt. Cracks/bubbles present. The unknown G+ was only able to ferment glucose. Gas was produced.
Flow Chart for Unknown G+ Gram stain (Positive result) Positive (Purple) Negative (Pink) 1.Staphylococcus aureus 2.Alcaligenes faecalis 3. Enterococcus faecalis 6.Klebsiella pneumonia 5. Bacillus cereus 7. Pseudomonas aeruginosa 11. Bacillus subtilis 8. Escheria coli 13. Bacillus megaterium 9. Shigella flexneri 16. Lactococcus lactis 10. Salmonella typhimurium 17. Staphylococcus epidermidis 12. Serratia marsescens 20. Corynebacterium diphtheriae 14. Proteus mirabilis 22. Micrococcus luteus 21. Providencia alcalifaciens Catalase Test Performed (Positive result) Positive (Produced bubbles) Negative (No bubbles produced) 1.Staphylococcus aureus 3.Enterococcus faecalis 11.Bacillus subtilis 5.Bacillus cereus 17. Staphylococcus epidermidis 13.Bacillus megaterium 20. Corynebacterium diphtheriae 16.Lactococcus lactis 22.Micrococcus luteus TSI Slant test performed (Red Slant/Yellow butt. Cracks/bubbles present) 1.Staphylococcus aureus (Yellow slant/Yellow butt. Cracks/bubbles present) 11.Bacillus subtilis (Yellow slant/Yellow butt. Cracks/bubbles present) 17. Staphylococcus epidermidis (Red slant/Yellow butt. Cracks/bubbles present) 20. Corynebacterium diphtheriae (Red slant/Red butt. No cracks or bubbles) 22.Micrococcus luteus (Yellow slant/Yellow butt. No bubbles present) Unknown G+ = Staphylococcus epidermidis
Discussion/Conclusion After viewing the Gram stain initially given by the professor, it was clear that the bacteria was a Gram-positive cocci. This eliminated nine of the eighteen potential bacterial species that Unknown G+ could be. A Catalase test was performed on the Unknown G+ and the control organisms Staphylococcus epidermidis and Lactococcus lactis were used to ensure accuracy and that proper procedures were being followed. Unknown G+ produced bubbles and it was concluded that the catalase test performed on the unknown was positive. The results of the Catalase test left five potential bacterial organisms that Unknown G+ could be. The TSI Iron Agar slant seemed like the perfect test to further narrow down the result because not only does it result in a change of colors based on whether or not the organism can ferment glucose, lactose, and sucrose, but it can also determine if gas and HS2 can be produced. (Microbiology Info, 2022.) The TSI Iron Agar slant were performed and the results were a red slant and yellow butt with bubbles which meant gas had also formed. Only one organism was a match for all of the results above and it was ultimately determined that the identity of Unknown G+ was Staphylococcus epidermidis.. References American Society for Microbiology. (2010, November 11). Catalase Test. Asm.org
https://asm.org/Protocols/Catalase-Test-Protocol Microbiology Info. (2022, August 10). The Triple Sugar Iron (TSI) Test – Principle, Procedure, Uses and Interpretation. MicrobiologyInfo.com https://microbiologyinfo.com/triple-sugar-iron-tsi-test/ American Society for Microbiology. (2005, September 30). Triple Sugar Iron Agar Protocols. Asm.org https://asm.org/ASM/media/Protocol-Images/Triple-Sugar-Iron-Agar-Protocols.pdf?ext=.pdf
Unknown Lab Report #2 Unknown A- A Student November 22, 2022 Professor Shawn Mustain Microbiology 2120 Fall 2022 Section 500
Introduction The purpose of this lab was to identify an unknown organism given to us by the professor. The significance of being able to identify an unknown bacterial species is of the utmost importance when providing excellent and accurate care to patients. In severe cases, being able to identify a bacteria that is causing infection or disease in a patient could mean the difference between life and death. Some antibiotics work well against specific strains of bacteria. Therefore, tracing a disease back to a specific bacteria helps medical professionals provide an accurate diagnosis and effective treatment. Materials • Test tube • MRVP broth • Peptone • Glucose • Inoculating loop • Methyl red • Refrigerator • Needle • Gelatin Methods The procedures learned in this class were used to carry out these tests to identify the unknown bacteria. When performing the Methyl Red test, the MRVP broth was inoculated with the unknown organism using an inoculating loop. The broth was then incubated for 24 hours at 37C. Then, Methyl Red dye was added to the medium and observed for a color change to red. (Microbiology Info, 2022) If the medium turned red, the result would have been positive but the unknown species remained yellow, returning a negative result. The controls used in this test were Shigella flexneri and Alcaligenes faecalis. The same procedure was used for both controls. The Gelatinase test was performed by first using a needle with the Unknown bacterial
species to inoculate the gelatin media. The gelatin was then incubated at 35C – 37C for two weeks. The gelatin medium was removed from the incubator daily and placed at 4C to see if liquid would form. (Microbiology Info, 2022) The controls used in this experiment were Pseudomonas aeruginosa and Alcaligenes faecalis. The same procedures were followed for both controls. Observation of the controlled medium at 4C was observed for any liquid. Liquid was only observed in Pseudomonas aeruginosa. Results After reviewing the Gram stain given by the professor, Unknown A- was determined to be a Gram-negative rod shaped bacteria. A Methyl Red test was performed on the unknown and two control organisms for accuracy. The MR test remained yellow instead of changing colors to red. Thus, returning a negative result. The last test performed for Unknown A- was this Gelatinase test which was done by inoculating a gelatin broth to observe for any liquid formation. Again, two unknown organisms were used for accuracy and to make sure the tests were done properly. The Gelatinase test yielded a negative result because no liquid was formed. It was concluded that the Unknown A- was incapable of producing gelatinase enzyme. Table 1 and the flow chart below show how Unknown A-‘s identity was determined.
Table 1: Biochemical Tests Test Purpose Reagents Observations Results MR test To test an organism’s ability to ferment glucose and produce acid. Peptone, Buffers, Glucose Remained yellow Negative Gelatinase To determine the organism’s ability to make an enzyme called gelatinase that can turn gelatin into liquid. Beef extract, gelatin No liquid was formed Negative
Flow Chart for A- Gram stain (Negative) Positive Negative 1.Staphylococcus aureus 2.Alcaligenes faecalis 3.Enterococcus faecalis 6.Klebsiella pneumoniae 5.Bacillus cereus 7.Pseudomonas aeruginosa 11.Bacillus subtilis 8.Escheria coli 13.Bacillus megaterium 9.Shigella flexneri 16.Lactococcus lactis 10.Salmonella typhimurium 17.Staphylococcus epidermidis 12.Serratia marsescens 20.Corynebacterium diphtheriae 14.Proteus mirabilis 22.Micrococcus luteus 21.Providencia alcalifaciens MR test performed (Negative result) Positive Negative 6.Klebsiella pneumoniae 2.Alcaligenes faecalis 8.Escheria coli 7.Pseudomonas aeruginosa 9.Shigella flexneri 10.Salmonella typhimurium 12.Serratia marsescens 14.Proteus mirabilis 21.Providencia alcalifaciens Gelatinase test performed (Negative result) Positive Negative 7.Psuedomonas aeruginosa 2.Alcaligenes faecalis
Discussion/Conclusion The Unknown organism A- was correctly identified as Alcaligenes faecalis based on the positive Gram stain, negative Methyl Red test and negative Gelatinase test. It was the only bacterial species to meet all three of these criteria. A first look at Unknown A-‘s Gram stain showed a pink rod shaped bacteria. It was concluded that the unknown organism was a Gram negative species. The Gram stain eliminated half of the potential species that the unknown could possibly be. With nine species left, a Methyl Red test was performed and produced a yellow color. The result of the MR test was negative and ruled out seven of the nine species that were left. Pseudomonas aeruginosa and Alcaligenes faecalis were the two bacterial species left. A Gelatinase test was performed to narrow it down to one species. This test is often used to differentiate Staphylococcus aureus and Staphylococcus epidermidis. (Microbugz, n.d.). In a Gelatinase test, Pseudomonas aeruginosa would liquify and have a positive result while Alcaligenes faecalis would still be able to solidify and have a negative result. When performed, the Gelatinase test had a negative result. The only bacterial species to match the above criteria was Alcaligenes faecalis which was the identity of Unknown A-. References Microbiology Info. (2022, August 10). Methyl Red (MR) Test – Principle, Procedure and Result Interpretation. MicrobiologyInfo.com https://microbiologyinfo.com/methyl-red-mr-test-principle-procedure-and-result-interpretation/ Microbiology Info. (2022, August 10). Gelatin Hydrolysis Test – Principle, Procedure, Uses and Interpretation. MicrobiologyInfo.com https://microbiologyinfo.com/gelatin-hydrolysis-test/ Microbugz. (n.d.). Gelatinase Test AustinCC.Edu
https://www.austincc.edu/microbugz/gelatinase_test.php
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