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Report by Maureen Schleeper
December 6, 2012
Education is the first step to the prevention and spread of microorganisms within a hospital environment. Understanding the general concepts of bacterial identification and the steps required for the identification of unknown pathogens emphasizes the overall importance of sterile techniques in the clinical setting. This experiment was performed in order to isolate and identify the genus and species of two unknown bacteria. The exercise was executed to test skills and techniques obtained throughout the semester within the laboratory environment.
MATERIALS AND METHODS
The unknown specimen was obtained from the instructor in a tube marked with the number 121. The stock broth contained two bacteria to be identified. The instructor revealed that the specimen contained one Gram-positive bacteria and one Gram-negative bacteria.
The initial procedure of an isolation streak was performed using sterile techniques obtained from the lab manual page 10. The isolation streak was performed on two nutrient agar plates using a flame-sterilized inoculation loop in an attempt to isolate separate colonies. The nutrient agar plates were incubated at 37 degrees C for 48 hours.
Upon inspection of the streak plates, it was observed that two distinct colonies appeared to have grown. In order to obtain a pure culture of each bacterium, two new nutrient agar plates were streaked with each observed colony. These nutrient agar plates were incubated at 37 degrees C for 72 hours.
The growth agar plates were removed from the incubator. Growth on each nutrient agar plate was observed. The first colony morphology was small, off-white colonies labeled Unknown A. The second colony morphology was white, round, and slightly mucoid colonies, which were labeled Unknown B. Gram stains were performed on each of the two cultures. Unknown bacteria A was determined to be a Gram-positive cocci. Unknown bacteria B was determined to be a Gram-negative rod.
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Additional tests performed on unknown A were the nitrate test using the nitrate broth medium (techniques in lab manual page 38) and the mannitol test using mannitol salt agar medium (techniques in lab manual page 16). These tests were chosen from a chart provided by the instructor specifically for determining unknowns. Results are recorded in Table 1.
Additional tests performed on unknown B were the indole test using a tryptone broth medium (techniques in lab manual page 38) and the methyl red test using MR-VP tubes (techniques in lab manual page 36). These tests were also chosen from a chart provided by the instructor specifically for determining unknowns. Results are recorded in Table 2.
Table 1: Test results for unknown bacteria A
|Gram stain||To determine if bacteria is a Gram positive or a Gram negative bacteria||Purple cocci||Gram positive cocci|
|Nitrate test||To test for reduction of nitrate to nitrite or nitrogen gas||No color change after addition of reagents nitrate I, nitrate II, and zinc||Positive for reduction of nitrate|
|Mannitol test||To test for fermentation of the sugar mannitol , producing acid||Color change to yellow||Positive for fermentation of the sugar mannitol|
Table 2: Test results for unknown bacteria B
|Gram stain||To determine if bacteria is a Gram-positive or a Gram-negative bacteria||Pink rods||Gram-negative rods|
|Indole test||To test for production of indole||No color change after the addition of reagent indole||Negative for production of indole|
|Methyl Red Test||To test for acid as final end product of glucose fermentation||No color change to red after the addition of reagent methyl red||Negative for the production of acid|
Nitrate Test (positive)
Positive Negative Staphylococcus aureus Enterococccus faecalis
Mannitol Test (positive)
Staphylococcus aureus Staphylococcus epidermis
Unknown A-Staphylococcus aureus
Indole Test (negative)
Positive Negative Escherichia coli Klebsiella pneumoniae
Proteus vulgaris Enterobacter aerogenes
Methyl Red Test (negative)
Klebsiella pneumoniae Enterobacter aerogenes
Unknown B – Enterobacter aerogenes
A pure culture for unknown A was grown on a nutrient agar plate and used to inoculate all of the tests performed. Unknown bacteria A, being a Gram-positive cocci, was narrowed down to three possible organisms: Staphylococcus aureus, Staphylococcus epidermidis, or Enterococcus faecalis. Two additional tests were performed to differentiate the remaining bacteria. After performing the nitrate test and the mannitol test with clear positive results, the unknown bacteria A was determined to be Staphylococcus aureus. No problems were encountered in this testing process. This identification was verified by the instructor. S. aureus is a spherical bacteria occurring in irregular clusters. It can be found mainly in the nasal passages, skin, oral cavity or gastrointestinal tract. S. aureus is considered a pathogen. One version of the S. aureus species has become resistant to most antibiotics in use today, thus acquiring the name MRSA (Methicillin-resistant Staphylococcus aureus). This resistance occurred due to the overuse and misuse of antibiotics for many years. MRSA occurs in the nosocomial environment as well as in the community.
Nosocomial, or hospital related MRSA, can be spread from patient to patient by contaminated hands or gloves of healthcare workers. Contamination can occur by direct contact with a patients skin, an open sore, dressings on an open sore, or by devices. Patients most vulnerable to MRSA would be those who have weakened immune systems due to illness or surgery, older patients, and those that are hospitalized for long periods of time. Proper hand washing techniques can help prevent patient-to-patient contamination as well as prevent contamination of items or devices in the environment. According to the Centers for Disease Control (CDC), the life-threatening type of MRSA has been on the decline in the hospital environment.
In the community (people not associated with healthcare), MRSA can be spread by direct contact with the skin of another person who is carrying this bacteria on their skin. It can also be spread by touching a surface that has been contaminated such as a towel or a personal care item like a razor. Community related MRSA is common where there is close contact with people such as athletic teams, college dormitories, or daycare facilities. It is also very common to spread MRSA within a household. These infections are normally skin related. Although community-related infections are still on the rise, treatment does not carry many long term problems.
A pure culture for unknown B was also grown on a nutrient agar plate and used to inoculate all of the tests performed. Unknown bacteria B, being a gram negative rod, was narrowed down to five possible organisms: Escherichia coli, Klebsiella pneumonia, Enterobacter aerogenes, Proteus vulgaris, and Pseudomonas aeruginosa. P. aeruginosa was eliminated from this list because it secretes a green pigment when grown on the nutrient agar plate, and this was not observed on the pure culture. Two additional tests were performed to differentiate these gram negative bacteria. The indole test and the methyl red test were completed with clear negative results for both. The unknown bacteria B was determined to be E. aerogenes. This identification was verified by the instructor.
1. Mayo Clinic Staff. “MRSA Infection.” Mayo Clinic. Mayo Foundation for Medical Education and Research, 13 Nov. 2012. Web. 05 Dec. 2012. <http://www.mayoclinic.com/>.
2. “Methicillin-resistant Staphylococcus Aureus (MRSA).” Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 15 Apr. 2011. Web. 05 Dec. 2012. <http://www.cdc.gov/mrsa/>.
3. Chess, Barry, and Kathleen Park. Talaro. Introduction to Microbiology. Vol. 8e. Toronto: McGraw-Hill Ryerson Custom, 2012. Print.