Identification of an Unknown Gram-Negative Bacterium: E. coli

Abstract

An unknown Gram-negative bacterium was randomly obtained from the sole of a shoe in this study to identify potential microorganisms. Several biochemical tests were conducted using standard procedures to determine the identity of the bacterium among many possible candidates. These tests included Triple-Sugar Iron Agar (TSIA), Sulfur Indole Motility (SIM), Citrate, Urease, Gelatinase, Methyl Red (MR), and Voges-Proskauer (VP) tests. To characterize the properties of the microorganism, the sample was first isolated using a T-streak technique, and the resulting colonies were Gram-stained to reveal their shape and morphology.

Subsequently, the isolated colonies were inoculated into various media corresponding to the appropriate biochemical tests. Data were collected to identify the unknown bacterium after allowing sufficient incubation time for each biochemical test. In this investigation, the bacterium was determined to be Escherichia coli.

Introduction

All living organisms are categorized into three domains: Bacteria, Archaea, and Eukarya. Bacteria belong to the domain of life where organisms are primarily prokaryotic. While the existence of bacteria and archaea shares similarities, archaea are closer in evolutionary terms to eukarya (Madigan et al.

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, 2016). The ability to adapt to a wide range of environments is a key characteristic that explains why prokaryotes are the most abundant life forms on Earth. Fundamental traits of prokaryotes include the absence of a nucleus, circular DNA, and the absence of membrane-bound organelles. The cell wall, which maintains cell shape and provides physical protection, is a prominent feature of nearly all prokaryotic cells. Most bacterial cell walls contain peptidoglycan, a cross-linked structure of modified sugar polymers with short polypeptides.

While all known pathogenic bacteria fall under prokaryotes, not all pathogenic organisms are bacteria (Madigan et al., 2016).

Bacteria can be classified into two groups through a differential staining procedure: Gram-positive and Gram-negative. Gram-positive bacteria have a thick peptidoglycan cell wall, whereas Gram-negative bacteria possess a thinner peptidoglycan layer with an outer membrane containing lipopolysaccharides, making them more complex. Gram staining is a valuable tool for determining whether an unknown bacterium is Gram-negative or Gram-positive. In this procedure, samples are initially stained with crystal violet and iodine, followed by alcohol wash, and finally, safranin. Crystal violet binds to the peptidoglycan layer, staining it violet in the case of Gram-positive bacteria. Due to the thinner peptidoglycan layer, Gram-negative cells appear pink or red, as crystal violet is easily removed from them (Leboffe, 2016).

The diversity among microorganisms in this domain makes it challenging to identify them without utilizing staining and biochemical tests. Biochemical tests can assess various bacteria-specific characteristics, such as shape, morphology, and more. Tests for glucose fermentation, such as the Methyl Red and Voges-Proskauer tests, are used. The Citrate test is a carbon source utilization test that determines whether a microorganism produces the enzyme citrate-permease to utilize citrate as its sole carbon source. The Urease test is employed to determine if the organism can hydrolyze urea. The Gelatinase test indicates whether a bacterium contains the enzyme capable of hydrolyzing proteins, allowing the conversion of gelatin into a solid when cooled.

Biochemical tests were carried out using two types of combination differential media: Sulfur Indole Motility (SIM) and Triple Sugar Iron Agar (TSIA). SIM provides three test results: sulfur reduction, determining whether a bacterium can use enzymes to reduce sulfur to hydrogen sulfide; indole production, used to determine if the microorganism can hydrolyze tryptophan to pyruvate and ammonia; and motility, indicating whether a mobile bacterium can deviate from the central line in the semisolid media. TSIA is designed to differentiate bacteria based on glucose fermentation, lactose fermentation, sucrose fermentation, and sulfur reduction (Cowan, 2009).

SIM provides three test results: sulfur reduction, determining whether a bacterium can use enzymes to reduce sulfur to hydrogen sulfide; indole production, used to determine if the microorganism can hydrolyze tryptophan to pyruvate and ammonia; and motility, indicating whether a mobile bacterium can deviate from the central line in the semisolid media. TSIA is designed to differentiate bacteria based on glucose fermentation, lactose fermentation, sucrose fermentation, and sulfur reduction (Leboffe and Pierce, 2016).

Materials and Methods

All procedures were carried out with meticulous care to ensure accuracy and prevent errors. Aseptic techniques were employed throughout the experiments, including flame sterilization of the loop between uses on the Bunsen burner and flaming the test tube opening before introducing the microorganisms into the culture.

Unknown microorganisms were streaked onto both a Tryptic Soy Agar (TSA) plate and a TSA slant for the purpose of isolating pure individual colonies. The plates were then incubated at 37 degrees Celsius for 24 hours before being transferred to a cold room at 4 degrees Celsius.

Gram staining was performed on the isolated single colonies after incubation to confirm whether the unknown microorganism was indeed a Gram-negative organism. A small drop of sterile water was used to spread the organism onto a glass slide. The smear was allowed to air-dry and then heat-fixed to immobilize the bacteria on the slide. Staining commenced with crystal violet, which was applied to completely cover the slide for one minute, followed by rinsing with DI water for approximately 10 seconds. The slide was then blot-dried with bibulous paper, and under 100x oil immersion, the microscope was used to observe the bacteria's color, shape, and morphology, which were recorded.

The first test conducted was the oxidase test, which was used to check for the presence of cytochrome c oxidase. A small amount of the bacterial culture was transferred to a filter paper strip, and a drop of DI water was added. Data were recorded within 20 seconds.

The second test performed was the Triple Sugar Iron Agar (TSIA) test. This test determines whether starch can be fermented and whether gas and hydrogen sulfide are produced. The bacterium was inoculated onto a needle, stabbed two-thirds down into the TSIA medium, and then streaked on the slant of TSIA. The tube was incubated in a hot room for 24 hours. After 24 hours, the TSIA and SIM test media were examined for results.

For the TSIA test, both the butt and the slant were evaluated for changes in color. A yellow color indicated a positive result for acid production, black precipitate indicated sulfur reduction, and gas production indicated glucose and lactose fermentation.

To assess fermentation, indole production, and motility of the bacterium, the Sulfur Indole Motility (SIM) test was performed. The semisolid medium contained sodium thiosulfate, casein, amino acids, an iron-containing compound, and sulfur. The SIM tube was initially observed for sulfur reduction, indicated by darkening around the stab line, and for motility by examining the growth pattern away from the stab line. Kovac's reagent was added to the medium with a depth of 2-3 mm. A red color change in the alcohol layer indicated a positive result for indole production.

After 48 hours, the MR and VP test media were examined for results. These tests determine the bacterium's ability to ferment glucose. Both tests included peptone, glucose, and a phosphate buffer. Three drops of methyl red were added to the MR test tube. A positive MR result would turn red immediately as the solution became more acidic, while negative results would not result in a color change. For the VP test, 15 drops of reagent A and 5 drops of reagent B were added to the tube. The tube was observed for an hour for any color change. A red color change indicated a positive VP test, while no color change indicated a negative result.

The citrate utilization test was employed to determine the bacterium's ability to use citrate as its carbon source. After 24 and 48 hours, the citrate test was examined for changes in color. If the bacterium survived in the medium and utilized citrate, it would convert ammonium phosphate into ammonia and ammonium hydroxide, both of which tend to alkalize the agar. Even a slight change in the blue color indicated citrate utilization.

The Gelatinase test was used to determine whether the organism produced gelatinase, an extracellular enzyme that some microorganisms release to hydrolyze gelatin, causing the medium to remain liquid even after being chilled. The Gelatin media were placed in a 4-degree Celsius room for one week. After 24 hours, the Gelatin media were checked for solidification. If the medium solidified, it indicated the absence of gelatinase. If it remained liquid, the incubation process was repeated for eight days. If the medium still remained liquid after eight days, the test was considered negative (Leboffe and Pierce, 2016).

The Urease test was used to differentiate organisms with urease enzyme based on their ability to hydrolyze urea. The Urea test was examined for changes in color every two hours over one week. A change from orange/yellow to pink indicated urea production or hydrolysis. If no color change to pink occurred in the initial observation, it was considered a positive result, and the test was repeated in the hot room for eight days. If there was still no change, the test was deemed negative (Lab Manual).

Results

The results of the biochemical tests and their interpretations for the unknown bacterium are summarized below:

Following 24 hours of incubation, the TSA plate showed small, white, and minute individual colonies. The bacteria appeared pink and rod-shaped when observed under the microscope. After 24 hours, TSIA media exhibited both a yellow slant and butt, indicating acid production from glucose and lactose fermentation. Following a 24-hour incubation period, the SIM test showed no change in the color of the slant, but there was cloudiness in the medium. The citrate test results, with the addition of bromthymol blue indicator, were negative.

The MR and VP tests were evaluated after 48 hours. The MR test was positive, as indicated by the quick change to a red color, signifying mixed acid fermentation. However, the VP test showed no change in color after the addition of reagents. Following one week of incubation, the urease test remained negative, with no color change. The gelatin test concluded as negative since the media remained solid even after one week of incubation in the hot room.

Discussion (Continued)

The Gram stain confirmed that the unknown bacterium was Gram-negative. This result was corroborated by a series of tests and observations that consistently indicated a Gram-negative nature. The initial observation of small, white, and rod-shaped colonies on the TSA plate suggested the possibility of Klebsiella pneumoniae. However, the subsequent biochemical tests provided further insights.

The TSIA test, which evaluates fermentation capabilities, hydrogen sulfide production, and gas production, revealed that the bacterium could ferment both glucose and lactose, as indicated by the yellow slant and butt. This result is characteristic of Escherichia coli, which is known for its ability to ferment these sugars.

The SIM test showed some cloudiness near the stab line, which indicated motility. Additionally, the change in color of Kovac's reagent to red confirmed the bacterium's ability to produce indole by hydrolyzing tryptophan. These results further supported the identification of Escherichia coli.

However, the Citrate test yielded a negative result due to the absence of a color change. This indicated that the bacterium did not utilize citrate as its carbon source, consistent with the characteristics of Escherichia coli.

The MR test resulted in a positive outcome, suggesting mixed acid fermentation, which is a common metabolic pathway in Escherichia coli. On the other hand, the VP test showed no color change, indicating the inability to convert acidic by-products into acetoin, further supporting the identification of Escherichia coli.

The Gelatinase test was negative, as the media remained solid even after one week of incubation, indicating the absence of gelatinase. Lastly, the Urease test did not exhibit a color change, suggesting that the bacterium did not possess urease activity.

In conclusion, based on the data obtained from the biochemical tests, colony morphology, shape, and Gram-negative staining, it can be confidently concluded that the unknown bacterium is Escherichia coli. While some test results shared similarities with other Gram-negative organisms, the combination of results and the consistency of characteristics with Escherichia coli led to this identification.

Further Considerations

Escherichia coli is a diverse bacterium with both beneficial and harmful strains. In the context of the gastrointestinal (GI) tract, E. coli can be beneficial, aiding in digestion and maintaining a healthy gut flora. It plays a crucial role in breaking down food and preventing the overgrowth of harmful bacteria. However, the presence of certain pathogenic strains of E. coli can lead to gastrointestinal illnesses, urinary tract infections, and other health issues if they migrate to other parts of the body.

It's important to note that the impact of E. coli on the body can vary depending on the specific strain and its characteristics. Beneficial strains are important for digestive health, while harmful strains can cause illnesses and infections.