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Unknown Lab Report
There are many reasons for knowing and identifying microorganisms. Medical reasons range from knowing the causative agent of a disease in a patient, so as to know how it can be treated, to applying practical applications in the laboratory so as to correctly identify microorganisms to be used in making certain foods or antibiotics. This study was done so as to be given the opportunity to apply some or most of the methods that have been learned so far in the microbiology laboratory class for the identification of an unknown bacterium.
Material and Methods
Two unknown bacteria in a nutrient broth tube, labeled number 109, were given to me by my instructor. The instructor said one of the bacteria would be gram-positive and one would be gram-negative. Some things learned in lab class were used to identify these unknown bacteria. The first procedure that was done was a streak plate. A nutrient agar plate was inoculated with the unknown bacteria. This was done to try to isolate colonies from each one of the bacteria. The nutrient agar was then incubated for about 48 hours. After 48 hours an isolated colony was found. A new streak plate was inoculated for that isolated colony and incubated for 48 hours. After the second 48 hours of the original nutrient agar plate being incubated, another isolated colony grew. A new streak plate was started for this second isolated colony and incubated for 48 hours. The next test that was done on the first isolated colony that was found was a gram stain. It was gram-positive and the shape was cocci. Both bacteria from the original nutrient broth were inoculated in a Simmons citrate agar tube. This test was done because it would only grow the gram-negative bacterium that was trying to be identified. The second isolated colony that was found was a contamination, a gram stain was done on it and it was positive. After the Simmons Citrate test was incubated for 48 hours, it had a positive result. A gram stain was then done on this bacterium and it was gram negative and its shape was rod. A Mannitol Salt agar plate was inoculated with the gram positive bacteria and then incubated for 48 hours. The Mannitol salt test came out positive. A methyl red test was then done on the gram negative bacteria and a nitrate test was done on the gram positive bacteria. After the tubes were inoculated with the bacteria they were incubated for 48 hours. Then methyl red was added to the methyl red test tube and reagents A and B were added to the nitrate test tube. The Nitrate test that was done on the gram-positive bacteria was negative. The methyl red test that was done on the gram-negative bacterium was negative. A Mannitol salt test was then done on the gram-negative bacteria. It was incubated for 48 hours and the results came back as positive. The gram-negative bacterium was thought to be Enterobacter aerogenes. The Methyl red and Mannitol tests for the gram-negative bacteria were ignored. A Urea test tube was then inoculated by the gram-negative bacteria and incubated. The result of the urea test was positive.
All of the following tests were used on these unknown bacteria:
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Enterococcus faecalis Klebsiella pneuminiae
1. Gram Stain 1. Gram stain
2. Mannitol salt agar 2. Simmons Citrate
3. Urea 3. Nitrate
Mannitol Salt Agar Test (Positive)
Staphylococcus aureus Bacillus cereus
Enterococcus faecalis Bacillus subtilis
↓ Staphylococcus epidermidis
Nitrate Test (Negative)
Stapyhlococcus aureus Enterococcus faecalis
Unknown – Enterococcus faecalis
Gram Negative rod
Simmons Citrate (Positive)
Klebsiella pneumoniae Escherichia coli
Enterobacter aerogenes Proteus vulgaris
Urea Test (Positive)
Klebsiella pneuminiae Enterobacter aerogenes
↓ Pseudomonas aeruginosa
Unknown – Klebsiella pneuminiae
The test results led to the identification of both bacteria by eliminating them from a list of bacteria that were given by the instructor. The identification of the gram-positive bacteria is Enterococcus faecalis. The identification of the gram-negative bacteria is Klebsiella pneumoniae.
Enterococcus faecalis is a nonmotile, gram-positive, circular-shaped bacterium. It can be observed singly, in pairs, or in short chains. It is a facultative anaerobe with a fermentative metabolism. Enterococcus faecalis along with all of the enterococci are closely related to streptococci and is found in the bowel of humans and animals alike. It can also be found in the water, soil and in nature due to contamination by animal excrement and untreated sewage. Enterococci tolerate many ranges of temperatures and conditions. They can survive in temperatures ranging from 10˚C to 45˚C and in hypotonic, hypertonic, acidic, or alkaline environments. Sodium azide and concentrated bile salts are tolerated and used as selective agents in agar-based media. Enterococci are usually considered strict fermenters because they lack a Kreb’s cycle and respiratory chain. (Emerging Infectious Diseases, Vol. 4) Enterococcus faecalis is one of the leading causes of nosocomial infections with most of the infections being urinary tract infections. However, there have been many cases following surgery, usually abdominal surgery, and it also has been linked to endocarditis. (Livestrong) Bacteremia-related Enterococcus faecalis usually happens from infections following other sites such as wound infections. According to Dr. Elmer Koneman, it is associated with 80 to 90 percent of human enterococcal infections. Enterococci account for approximately 110,000 urinary tract infections, 25,000 cases of bacteremia, 40,000 wound infections, and 1,100 cases of endocarditis annually in the United States. (Emerging Infectious Disease, Vol. 4)Enterococcus faecalis and other Enterococci are becoming more and more dangerous as they have resistance capabilities to a wide variety of antibiotics. Most antibiotics that are used to treat infections and remain effective against other strains of Gram-positive coccus are becoming more and more ineffective against Enterococci faecalis. Enterococcus faecalis is among the most antibiotic-resistant bacteria known. It contains many natural antibiotic resistances, along with several acquired immunities transferred in R-plasmids among promiscuous bacteria. More than 25% of the Enterococcus faecalis genome is exogenously acquired, leading to its resistance to the strongest antibiotics and in some cases all antibiotics. It is also considered a carrier of vancomycin resistance for other genera of bacteria. (web.mst.edu) Antibiotic resistance organisms are becoming a huge problem in the world today. Partly because many physicians are too free with their prescriptions and often prescribe something to make the patient happy when indeed it isn’t necessarily in the best interest of the patient. The thought of multiple drug-resistant strains of any type of bacteria scares me, especially thinking of the future and what that may look like if this trend of drug-resistant bacteria continues.
1. Talaro, Chess. Foundations of Microbiology, 8th edition, McGraw-Hill Companies, Inc., 2012
4. Emerging Infectious Diseases, Vol 4, No 2, April-June 1998