Bacterial Identification Laboratory: Unraveling the Mystery of Staphylococcus aureus and Proteus vulgaris

Identifying bacteria is akin to unraveling a mystery, requiring the interpretation of various clues. Bacteria, among the earliest life forms on Earth, inhabit diverse environments, ranging from the depths of the ocean to the human body. Despite their minuscule size, bacteria can be scrutinized using a basic light microscope. The distinctive characteristics of bacteria play a vital role in their identification (Hogan, 2010).

Various laboratory techniques, such as streak isolation, Gram staining, Catalase, or Simmon's Citrate, serve to identify bacteria. Gram staining, for instance, unveils the cell wall composition, categorizing bacteria as either Gram-positive or Gram-negative.

Tests like Mannitol Salt Agar (MSA) and Eosin Methylene Blue (EMB) indicate specific bacterial conditions based on color changes. Through a series of tests, each providing results, the process leads to the discovery of the bacteria type.

The objective of this report is to identify two unknown bacteria using a sequence of tests while eliminating improbable choices. A test tub

Streak isolation on a nutrient agar plate was executed to separate a bacterial mixture into pure cultures.

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Following incubation at 37°C for 48 hours, the isolation plate exhibited two colonies with distinct colors: a white colony and an orange colony. Each colony was meticulously transferred to a nutrient agar plate, then incubated at 37°C for 48 hours to cultivate pure cultures. Subsequently, a Gram stain procedure was conducted. The white colony, identified as Gram-positive staphylococci based on morphology, eliminated Gram-positive Bacillus cereus and Bacillus subtilis due to their rod-shaped characteristics.

The orange colony underwent Gram staining, and under the light microscope, it appeared pink and purple, signifying a mixed culture.

As the Gram-positive bacteria were isolated, MacConkey Agar (MAC) and Eosin Methylene Blue (EMB) tests were chosen to isolate the Gram-negative bacteria. These selective media inhibit the growth of Gram-positive bacteria. After incubation at 37°C for 48 hours, a Gram stain procedure was performed, revealing Gram-negative rods. With the isolation of a pure bacterial colony accomplished, a series of tests were conducted to identify the bacteria. For Gram-positive bacteria, the Mannitol Salt Agar (MSA) test was executed. MSA, a selective and differential media with high salt concentration, promotes the growth of specific bacteria. The isolated Gram-positive bacteria were streaked on an MSA plate and incubated at 37°C, with results observed and recorded. A significant color change from pink to yellow indicated a positive MSA test, while no change signified a negative result.

Bacterial identification involves a systematic approach utilizing various laboratory tests. In this laboratory, the goal is to identify two unknown bacteria from a mixture by employing a series of tests that eliminate unlikely choices. The provided test tube contains a mix of Gram-positive and Gram-negative bacteria, with a list of ten possible bacteria, and the objective is to identify the unknown species through a rigorous testing protocol.

Materials and Methods:

Streak isolation on a nutrient agar plate was initiated to separate the bacterial mixture into pure cultures. Incubation at 37°C for 48 hours yielded two distinct colonies - a white colony and an orange colony. The colonies were transferred to nutrient agar plates for further isolation. The Gram stain procedure was then conducted, revealing the white colony as Gram-positive staphylococci. The orange colony, still exhibiting mixed culture under the light microscope, underwent MacConkey Agar (MAC) and Eosin Methylene Blue (EMB) tests to isolate the Gram-negative bacteria. The subsequent Gram stain confirmed the Gram-negative rods, enabling further identification.

For Gram-positive bacteria, Catalase, Blood Agar, and Spirit Blue Agar tests were performed. The Catalase test determined the presence of catalase, an enzyme breaking down hydrogen peroxide to water and oxygen. The Blood Agar test, focusing on beta hemolysis, assessed the bacterium's ability to lyse red blood cells, while the Spirit Blue Agar test examined lipase activity. Results were observed, and conclusions drawn based on the presence or absence of specific characteristics.

For Gram-negative bacteria, a Glucose Fermentation test, Simmon's Citrate test, Nitrate Reduction test, and Sulfur test were conducted. The Glucose Fermentation test illustrated the bacterium's ability to use glucose as an energy source. Simmon's Citrate test determined the production of citrase, while the Nitrate Reduction test assessed nitrate to nitrite conversion. The Sulfur test in SIM medium detected the reduction of sulfur to hydrogen sulfide. Observations were recorded for each test, aiding in the comprehensive identification of the Gram-negative bacteria.

Results and Discussion:

The Catalase test for Gram-positive bacteria exhibited the presence or absence of gas bubbles, indicating catalase activity. The Blood Agar test, focused on beta hemolysis, resulted in visible clearing of the agar if red blood cells were completely lysed. The Spirit Blue Agar test showed a positive result if there was visible clearing around bacterial growth, indicating lipase activity.

For Gram-negative bacteria, the Glucose Fermentation test demonstrated a color change from pink to yellow if fermentation occurred. The Simmon's Citrate test turned the medium blue if citrase was produced, indicating a positive result. The Nitrate Reduction test showed a red color if nitrate was converted to nitrite, and the addition of zinc powder was used to confirm negative results. The Sulfur test in SIM medium resulted in a black color if sulfur was reduced to hydrogen sulfide.

Calculations and Formulas:

No specific calculations or formulas were required for the tests conducted in this laboratory. The identification process relied on qualitative observations and characteristic changes in the media or reactions.

Tables:

This laboratory successfully identified the unknown bacteria through a systematic series of tests. The Gram-positive and Gram-negative bacteria were subjected to specific assays, revealing distinct characteristics that facilitated their identification. The results obtained from the Catalase, Blood Agar, Spirit Blue Agar, Glucose Fermentation, Simmon's Citrate, Nitrate Reduction, and Sulfur tests provided a comprehensive profile for each bacterial type. The laboratory protocol showcased the importance of a methodical approach in bacterial identification.

Following a series of tests, the identity of the unknown bacteria has been determined to be Staphylococcus aureus and Proteus vulgaris. The Gram-positive nature of one of the bacteria was established through mannitol, catalase, Blood agar, and Spirit Blue agar tests. With five potential Gram-positive bacteria, the Gram stain technique had already ruled out Gram-positive bacillus due to observed clusters with round shapes. The positive result in the mannitol test further eliminated Staphylococcus epidermidis. Subsequent positive results in the catalase, Blood agar, and Spirit Blue agar tests confirmed the presence of Staphylococcus aureus.

For Gram-negative bacteria, the possibilities were Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, Proteus vulgaris, and Pseudomonas aeruginosa. The positive result in the Glucose fermentation test excluded Pseudomonas aeruginosa. A negative result in the Citrate test eliminated Klebsiella pneumoniae and Enterobacter aerogenes. This narrowed down the Gram-negative bacteria to Proteus vulgaris and Escherichia coli. The positive result in the Nitrate reduction test confirmed Proteus vulgaris, with further positive results in the catalase and SIM tests supporting this conclusion.

Despite challenges in isolating the bacteria mixture initially, the use of EMB and MAC tests successfully yielded a pure culture for subsequent testing. The identification process was comprehensive, and additional tests facilitated the accurate determination of the unknown bacteria.

Proteus vulgaris is a rod-shaped, Gram-negative bacterium commonly found in soil, polluted water, raw meat, and the gastrointestinal tract of animals. It is known to cause urinary tract infections in humans and is particularly prevalent in sewage, where it thrives in a favorable medium. Without such a medium, Proteus vulgaris has a survival span of 1-2 days. The species exhibits high resistance to antibiotics, making infections challenging to treat.

Proteus vulgaris possesses an extracytoplasmic outer membrane and obtains energy and electrons from organic molecules. It ferments glucose and sucrose but not lactose, and it curdles milk through acid production. While it does not have a specific temperature range for growth, optimal growth occurs between 20°C and 30°C. The bacterium is highly motile and can swarm across the surface of an agar plate. During a growth cycle, distinct colonies with zonation patterns are formed.