ABSTRACT:
The Microbiology & Immunology Laboratory Unknown Identification Project is designed to test the students’ skills and knowledge of performing biochemical tests learned during the course of the lab. Students receive a numbered tube containing a mixed culture of two unknown organisms and are required to determine the identity of each microorganism by performing a series of biochemical tests of their choice. Unknown test tube #25 was obtained and streaked onto two agar plates to obtain individually isolated colonies. The gram-staining procedure was used to determine organismal morphology, resulting in a gram-positive cocci and gram-negative bacilli. A sucrose utilization test was performed and narrowed the possibilities of the gram-negative bacilli to Proteus vulgaris and Enterobacter cloacae. Then a Methyl Red and Voges-Proskauer (MR-VP) test was done to confirm the identity of the gram-negative bacilli as Enterobacter cloacae. A catalase test for gram-positive cocci eliminated Enterococcus faecalis and an MR-VP test confirmed the identity of the gram-positive cocci as Bacillus cereus. These biochemical tests led to the identification of these organisms as Enterobacter cloacae and Bacillus cereus but the organisms in test tube #25 were Enterobacter cloacae and Micrococcus luteus. INTRODUCTION: Enterobacter cloacae is part of the Enterobacter genus which is of the family Enterobacteriaceae and also includes Enterobacter aerogenes. …show more content…
E. cloacae is a gram-negative, rod-shaped (bacilli) bacterium. It possesses flagella for motility and is a facultative anaerobic microorganism that is able to obtain energy from both aerobic and anaerobic environments (Britannica). E. cloacae is a nosocomial pathogen, more commonly contracted in the ICU. Enterobacter are ubiquitous in nature. Animals contain Enterobacter in their intestinal tracts allowing for further transmission of the bacteria in soil, water and sewage (Britannica). In humans, pathogenic Enterobacter, such as E. cloacae, result in bacteremia, urinary tract infections (UTIs), intra-abdominal infections, endocarditis, lower respiratory infections, septic arthritis, and CNS and ophthalmic infections (Medscape). The management of these diseases is difficult due to the organism’s resistance to multiple antibiotics. Economically, E. cloacae reduces soluble, toxic selenite that can bio-accumulate in the food chain to elemental selenite that is non-toxic and insoluble (Microbwiki). Additionally, E. cloacae has been used as a biological control for plant disease (Microbwiki). Micrococcus luteus is a species that belongs to the Micrococcaceae family. M. luteus is a gram-positive cocci, commonly arranged in purple tetrads or irregular clusters. M. luteus is a non-motile anaerobe and forms yellow colonies on nutrient agar in the lab. M. luteus is usually non-pathogenic as it is part of the skin’s bacterial flora and is present in water, dust and soil. It also colonizes the human mouth, mucosal linings of the upper pharynx and respiratory tract (CDC). The optimal temperature for growth is 37°C and on human skin, it breaks down compounds in sweat into compounds with bad odor (Microbwiki). Infections caused by M. luteus occurs in immunocompromised patients, such as those with HIV+. M. luteus can result in recurrent bacteremia, septic shock/arthritis, endocarditis, meningitis and pneumonia in these patients (Microbwiki). METHODS: The biochemical tests utilized in this project were a carbohydrate utilization test, specifically sucrose, a Methyl Red and Voges-Proskauer (MR-VP) test, and a catalase test. The sucrose test was used to help determine the gram-negative bacilli microorganism. The purpose of the sucrose test is to test the microorganism’s ability to ferment sucrose as a carbon source and examine if acid and/or gas products are formed. Gas is collected in a Durham tube and a pH indicator, the chemical phenol red, indicates acidity, a positive result, if the medium turns from red to yellow. An inoculum of the unknown gram-negative bacteria was transferred aseptically to a sterile tube of sucrose medium and incubated at 35-37°C for 24-48 hours. Possible results after incubation are either acid or gas, neither or both end products. The Voges-Proskauer test results in the formation of a neutral product, Acetylmethylcarbinol (AMC) or acetoin that occurs under the conversion of acid products by some bacterial species. Presence of acetoin results in a red medium and constitutes a positive test. The methyl red (MR) test alone is used to test for acid production and a positive test constitutes a red color. The MR-VP test can be performed on the same medium and both are important in the
The Microbiology & Immunology Laboratory Unknown Identification Project is designed to test the students’ skills and knowledge of performing biochemical tests learned during the course of the lab. Students receive a numbered tube containing a mixed culture of two unknown organisms and are required to determine the identity of each microorganism by performing a series of biochemical tests of their choice. Unknown test tube #25 was obtained and streaked onto two agar plates to obtain individually isolated colonies. The gram-staining procedure was used to determine organismal morphology, resulting in a gram-positive cocci and gram-negative bacilli. A sucrose utilization test was performed and narrowed the possibilities of the gram-negative bacilli to Proteus vulgaris and Enterobacter cloacae. Then a Methyl Red and Voges-Proskauer (MR-VP) test was done to confirm the identity of the gram-negative bacilli as Enterobacter cloacae. A catalase test for gram-positive cocci eliminated Enterococcus faecalis and an MR-VP test confirmed the identity of the gram-positive cocci as Bacillus cereus. These biochemical tests led to the identification of these organisms as Enterobacter cloacae and Bacillus cereus but the organisms in test tube #25 were Enterobacter cloacae and Micrococcus luteus. INTRODUCTION: Enterobacter cloacae is part of the Enterobacter genus which is of the family Enterobacteriaceae and also includes Enterobacter aerogenes. …show more content…
E. cloacae is a gram-negative, rod-shaped (bacilli) bacterium. It possesses flagella for motility and is a facultative anaerobic microorganism that is able to obtain energy from both aerobic and anaerobic environments (Britannica). E. cloacae is a nosocomial pathogen, more commonly contracted in the ICU. Enterobacter are ubiquitous in nature. Animals contain Enterobacter in their intestinal tracts allowing for further transmission of the bacteria in soil, water and sewage (Britannica). In humans, pathogenic Enterobacter, such as E. cloacae, result in bacteremia, urinary tract infections (UTIs), intra-abdominal infections, endocarditis, lower respiratory infections, septic arthritis, and CNS and ophthalmic infections (Medscape). The management of these diseases is difficult due to the organism’s resistance to multiple antibiotics. Economically, E. cloacae reduces soluble, toxic selenite that can bio-accumulate in the food chain to elemental selenite that is non-toxic and insoluble (Microbwiki). Additionally, E. cloacae has been used as a biological control for plant disease (Microbwiki). Micrococcus luteus is a species that belongs to the Micrococcaceae family. M. luteus is a gram-positive cocci, commonly arranged in purple tetrads or irregular clusters. M. luteus is a non-motile anaerobe and forms yellow colonies on nutrient agar in the lab. M. luteus is usually non-pathogenic as it is part of the skin’s bacterial flora and is present in water, dust and soil. It also colonizes the human mouth, mucosal linings of the upper pharynx and respiratory tract (CDC). The optimal temperature for growth is 37°C and on human skin, it breaks down compounds in sweat into compounds with bad odor (Microbwiki). Infections caused by M. luteus occurs in immunocompromised patients, such as those with HIV+. M. luteus can result in recurrent bacteremia, septic shock/arthritis, endocarditis, meningitis and pneumonia in these patients (Microbwiki). METHODS: The biochemical tests utilized in this project were a carbohydrate utilization test, specifically sucrose, a Methyl Red and Voges-Proskauer (MR-VP) test, and a catalase test. The sucrose test was used to help determine the gram-negative bacilli microorganism. The purpose of the sucrose test is to test the microorganism’s ability to ferment sucrose as a carbon source and examine if acid and/or gas products are formed. Gas is collected in a Durham tube and a pH indicator, the chemical phenol red, indicates acidity, a positive result, if the medium turns from red to yellow. An inoculum of the unknown gram-negative bacteria was transferred aseptically to a sterile tube of sucrose medium and incubated at 35-37°C for 24-48 hours. Possible results after incubation are either acid or gas, neither or both end products. The Voges-Proskauer test results in the formation of a neutral product, Acetylmethylcarbinol (AMC) or acetoin that occurs under the conversion of acid products by some bacterial species. Presence of acetoin results in a red medium and constitutes a positive test. The methyl red (MR) test alone is used to test for acid production and a positive test constitutes a red color. The MR-VP test can be performed on the same medium and both are important in the