UNKNOWN LAB REPORT
Rio Hall
Microbiology BIO:203.604
Spring Semester/2013
INTRODUCTION
The ability to determine an unknown microorganism and isolate more than one unknown microorganism from a mixed culture is beneficial to humankind for multiple reasons. Predominantly, healthcare professionals need to ascertain the specific cause of an illness in order to be able to properly treat it with the most effective antibiotic. Microorganisms can make people ill, however, most of the microorganisms on Earth are beneficial and provide symbiotic relationships that are essential to the ecosystems on the planet. A few examples of advantageous microbial use are: waste management (i.e. specific organisms, like Alcanivorax (1) that can eat away an oil spill such as the one we had in the Gulf in 2010), food supply (2), and chemical uses, such as making polymers from genetically-modified Escherichia coli (3). This study was carried out by application of procedures learned in the microbiology laboratory course for the purpose of the identification of two unknown bacteria.
MATERIALS AND METHODS
An unknown, labeled as number 108, was given out by the lab instructor. Procedures followed used previous lab exercises from the course laboratory manual by McDonald et al. (4), unless otherwise noted, to discern the metabolic characteristics of the bacteria.
The first procedure was to streak a nutrient agar plate with mixed culture # 108. After Gram stains were performed, a mixed culture still existed, so a second nutrient agar plate was streaked with the mixed culture. Upon the second attempt, a second Gram stain was done and the two bacteria were still not isolated. The mixed culture was incubated at 35 degrees Celsius on MacConkey and EMB agar in order to inhibit the growth of the Gram-positive organism. The growth on the MacConkey and EMB plates were Gram stained as a mixed culture.
Since an isolated culture had not yet been achieved, a third isolation streak was performed from the mixed culture onto a nutrient agar plate; this time carefully streaking the bacteria into four quadrants attempting to procure an isolated colony. Finally, Gram stains showed two distinct colonies: Gram-positive cocci and Gram-negative rods; the unique colonies were grown in the incubator on separate nutrient agar plates and Gram stained to verify isolation.
The Gram positive cocci were grown on a Mannitol Salt Agar plate and displayed a positive result for mannitol fermentation by turning the phenol red in the medium to yellow. This step narrowed down the unidentified Gram positive bacteria from five choices to two. A final Blood Agar test displayed a negative result by showing partial to zero hemolysis of red blood cells indicating that the Gram positive unknown was Enterococcus faecalis.
Simmons Citrate was inoculated by a single stab with the Gram negative rods and showed a positive result by the medium turning from green to blue. Two Gram negative bacteria were eliminated using this step. Next, a Methyl Red test was completed and yielded a negative result, thereby eliminating yet another organism. The final determinant was a Voges Proskauer test, which had a negative result as indicated by the tube turning yellow not red. The Gram negative bacteria is Pseudomonas aeruginosa.
All of the following tests were performed on this unknown:
- Simmons Citrate
- Methyl Red
- Voges Proskauer
- Mannitol Salt Agar
- Blood Agar
- MacConkey and EMB tests were also performed, but did not prove to be determinative.
RESULTS
TABLE 1: Physiological and Biochemical Results
|
TEST |
PURPOSE |
OBSERVATIONS |
RESULTS |
INTERPRETATIONS |
|
Gram stain |
To determine the Gram reaction of the bacterium |
Pink rods & Purple cocci |
Gram negative rods & Gram positive cocci |
Use of Crystal violet, Iodine, Alcohol, and Safranin was successful in determining the Gram stain reaction |
|
Mannitol Salt Agar |
To determine the ability of a bacterium to ferment mannitol | Color of medium changed from red to yellow | Positive | Organism is able to ferment mannitol and produce an acid that turns phenol red to yellow |
|
Blood Agar
|
To determine the ability of a bacterium to lyse red blood cells | White streak of colony growth on top of plate; no clearing in the red medium | Negative | Gamma; organism is not able to break red blood cells apart |
|
Simmon’s Citrate |
To determine if an organism can use citrate as its sole carbon source | Color of medium changed from green to blue | Positive | Organism uses citrate as its sole carbon source and produces pyruvic acid and carbon dioxide (an alkaline compound) that changes the medium from green to blue |
|
Methyl Red
|
To determine if an organism produces acids that cause the pH to drop below 4.4 during glucose fermentation | Color of medium stayed yellow; it did not turn red | Negative | Organism does not produce acid when it ferments glucose |
|
Voges Proskauer |
To determine if an organism produces acetyl methyl carbinol | When reagents A & B were added to a cultured broth, the tube turned yellow. | Negative | Organism does not produce acetyl methyl carbinol |
FLOWCHART UNKNOWN #108 – A & B
CONCLUSION
Ultimately, the test results led to correct identification of both Gram + and Gram – microbes, however, significant problems were encountered during the process. The main difficulty was isolating the bacteria from the mixed culture of two species of microbes. Although aseptic technique was used, the problem occurred during the first two attempts to grow isolated colonies on a solid medium. Specifically, the first two attempts merely smeared both bacteria across the entire plate. Upon the third attempt to inoculate the nutrient agar plate with the mixed culture, a proper inoculation streak was used and both samples were isolated. The two bacteria were Pseudomonas aeruginosa and Enterococcus faecalis.
Enterococcus faecalis (E. faecalis) are Gram +, commensal bacteria that can live in many diverse environments. The human intestinal and genital tracts house E. faecalis, proving not harmful to humans, however, can sometimes be detrimental because of their resistance to antimicrobial drugs, “including cell-wall active agents; aminoglycosides, penicillin and ampicillin, and vancomycin” (5). This can promote serious problems for people with interococcal infections (6). “Since the resistance genes are carried on a plasmid they are readily transferable, E. faecalis can transfer these plasmids by conjugation” (7). E. faecalis are associated with both community and nosocomial infections, with the latter on the rise. A common nosocomial infection of E. faecalis is a urinary tract infection. Apparently, E. faecalis prefer to colonize the kidneys over the bladder (8). Kidney affinity and resistance to antibiotics have made E. faecalis a force to be reckoned with.
REFERENCES
- Hazen, Terry, et al. “Deep-Sea Oil Plume Enriches Indigenous Oil-Degrading Bacteria.” Science. 8 October 2010. Vol. 330, No. 6001. p. 204-208.
- Yasuda K., Taga N. 1980. “A mass culture method for Artemia salina using bacteria as food.” Mer. Vol. 18:53–62.
- Biello, David. “Turning Bacteria into Plastic Factories.” Scientific American. September 16, 2008.
- McDonald, Virginia et al. Lab Manual for General Microbiology (BIO 203) STLCC at Meramec, revised April 2011.
- Paulsen, I.T., et al. “Role of Mobile DNA in the Evolution of Vancomycin-Resistant Enterococcus faecalis.” Science. March 2003. Vol. 299, No. 5615. p. 2071-2074.
- Gilmore, Michael. “The Enterococci: Pathogenesis, Molecular Biology, and Antibiotic Resistance.” Washington, DC: American Society for Microbiology Press, 2002.
- Murrary, BE. May 1998. “Diversity Among the Multidrug-Resistant Enterococci.” Emerging Infectious Diseases. Vol. 4, No. 1. p. 46-65.
- Andrew L. Kau, et al. April 2005. “Enterococcus faecalis Tropism for the Kidneys in the Urinary Tract of C57BL/6J Mice.” Infection and Immunity. Vol. 73, No. 4. p. 2461-2468.
