Antibiotic Resistance

Microbiology BIO 3302

Unknown #7 Lab Report

Prof. Nasreen Haque

Stephanie Denis

Unknown #7 Lab Report
Stephanie Denis
MB Lab

Introduction: Microorganisms, bacteria, are extremely diverse and live in almost every type of environment. Microbes are essential to human life and serve in different ways like food fermentation, biotechnology and bioremediation and on the other end of the spectrum can be very pathogenic to the human body by causing disease. Identification of bacteria is beneficial to healthcare for many reasons. The ability to isolate, culture and identify bacteria is essential to determine the disease-causing microorganism and the specific drug to fight against it. The techniques and applications taught in microbiology lab for the identification of the unknown bacteria.
Methods and Materials: An unknown mixture, labeled 7, was given out to me by the lab professor. The first procedure was to perform a gram stain test, to detect not only whether the unknown was gram positive or negative but the shape of the bacteria. The next step was to isolate the bacterium on a solid medium to get isolated colonies, this was done by using the isolation streak plate method on to a nutrient agar plate at 37º C for two days. At the same time as the isolation streak, the unknown bacteria were inoculated on to multiple agar plates: blood agar, Mannitol salt, Phenylethyl Alcohol Agar (PEA), and MacConkey plates. All were done using the streak method and cultivated for two days at 37º Centigrade.
-All the following tests were performed on the unknown bacteria:
1. Methyl red/Voges-Proskauer
2. Sulfur Indole Motility (SIM)
3. Urease
4. Nitrate
Test Purpose Reagents Observation Results
Gram Stain To determine the gram reaction of the bacteria. Crystal violet, Iodine, Decolorizer, Safranin Pink rods Gram negative rods
Nutrient agar The primary isolation of microorganisms. None White colonies Inoculation occurred.
PEA Used to cultivate gram positive organisms. Inhibit or reduce growth of gram negative organisms by interfering with DNA synthesis None Very sparse, individual white colonies A gram-positive grows.
MacConkey agar Designed to isolate and differentiate enteric bacteria based on ability to ferment lactose. Crystal Violet Pink/purple colonies Lactose fermenter
Mannitol salt Agar Selective and differentiates with respect to mannitol fermentation. Phenol red No color change, small white dots Negative for halophilic bacteria
Blood agar Cultivates fastidious organisms and determining hemolytic capabilities of an organism. None White colonies, no encapsulating color Gamma hemolytic
SIM Test ability to: reduce sulfur, produce indole, and swim through the agar (motility) Kovac’s reagent No color change, semisolid turbidity extending from stab line throughout the medium. Negative for H2S production and indole, positive for motility.
Nitrate Determine the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) using nitrate reductase Nitrate A & B reagents red Positive for nitrite (NO2)
Urease Determine the ability to produce urease, wgich hydrolyze urea. None No color change Negative
Methyl red
Used to identify organisms producing stable acids by way of mixed acid fermentation of glucose. Methyl red reagent Yellow (no color change) Negative
Voges-Proskauer Used to detect acetoin in bacterial broth culture. VPAA reagent Yellow (no color change) Negative

After performing several biochemical tests, the results lead to two correct identification of both microorganisms. However, significant problems were during the process. I was able to properly get a Gram stain of the gram-negative bacteria, but what I should’ve did to better identify the gram-positive bacteria was to isolate the bacteria from the (PEA) plate and perform a Gram stain on it since I knew that it was a gram-positive bacterium growing on it. It was established that the unknown gram-negative is E. aerogenes and the gram positive is S. epidermidis. The (VP) results confused me because it was negative and E. aerogenes would’ve yielded positive results and the only other bacteria that fit the criteria was P. aeruginosa. However, I knew that the unknown was lactose fermenter from the MacConkey plate results that is also selective for gram-negative bacterium, which P. aeruginosa was not, and concluded the bacteria is E. aerogenes. Taking a careful look at all my results, I knew that the bacteria couldn’t be P. aeruginosa so I looked at the results of the agar plates, specifically MacConkey since it was the most comprehensible. To determine the identification of the gram-positive bacterium, I looked at the flow chart of the biochemical tests that coincided with the negative results of E. aerogenes and determined it was S. epidermidis.
Staphylococcus epidermidis belongs to the genus group Staphylococcus. It’s usually found in colonizing the human skin and mucosa and is a part of the human’s normal flora. Most strains are nonpathogenic but can be spread by skin to skin or during medical procedures. S. epidermidis can cause infections like meningitis, urinary tract infection, conjunctivitis, and endocarditis. People with compromised immune systems are most at risk to develop infections caused by S. epidermidis.
Enterobacter aerogenes belong to the genus Enterobacter, is generally found in the human gastrointestinal tract. And doesn’t normally cause disease in healthy individual, however E. aerogenes causes opportunistic infections. Some infections result from specific antibiotic treatments, surgical procedures. Its found in various wastes, hygienic chemicals, and soil. And has some commercial significance with the production of hydrogen gas.

Cowan, M.K. et al. (2016). Microbiology Fundamentals: A Clinical Approach, Second ed. New York,
NY. McGraw Hill Education.
Finazzo, S. & Obenauf S. (2017). Microbiology Fundamentals: A Clinical Approach (Lab manual). New
York, NY: McGraw Hill Education.