Abstract

 Enterococcus faecalis is a cocci-shaped, gram positive microbe and is classified as one of the most prevalent antibiotic drug resistant nosocomial pathogens worldwide. Enterococcus faecaliscan be found in soil, water and plants, however, some strains are found in the human intestinal system as facultative anaerobes. The bacteria doesn’t normally cause infection in the gut microbiome, but if it grows in large amounts or it spreads to other regions of the body, it can cause serious infections. A gram stain, as well as two biochemical tests, the catalase test and the blood agar test, were performed to identify the unknown bacteria. The gram stain was identified as a gram positive cocci shaped bacterial cell. The catalase test was negative for bubble formation and the blood agar test displayed gamma hemolysis. Based off the results of the two tests as well as the gram-positive gram stain, it can be concluded that the unknown microbe is Enterococcus faecalis.

Introduction

 Enterococcus faecalis is a cocci-shaped, gram positive microbe that has the ability to colonize very harsh environments such as the gastrointestinal tract of both humans and animals [1]. In order to do so, this bacteria must be able to have a flexible metabolism to survive many different environmental challenges [2]. Enterococcus faecalis is associated with both community and nosocomial obtained infections that can either be local or systemic, including but not limited to the urinary tract, abdominal infections, bacteremia and endocarditis [2].

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 In this experiment, a gram stain and two tests were performed to correctly identify the microbe: catalase test and blood agar hemolysis. The gram stain is performed by smearing the unknown bacteria on a microscope slide and staining the smear with the gram stain procedure [4]. Bacteria that appear purple are gram positive and have a thick cell wall composed of peptidoglycan that resist the decolorization from the alcohol and therefore retain the crystal violet. Those that appear red are gram negative and have a thin peptidoglycan layer and a lipid-bilayer that is known as the outer membrane that is easily disrupted by decolorization, therefore absorbing the red counter-stain, safranin [5].

 The catalase test is used to find out if a particular bacterium can produce the catalase enzyme [6]. To test for this enzyme, a small inoculate of bacterial cells are placed on a clean microscope slide and then H2O2 is added to the smear. Rapid bubbling indicates a positive result and the lack of catalase is indicated by no bubble formation. Catalase is an enzyme that is produced by aerobic microorganisms to neutralize toxic forms of oxygen metabolites in order to stop the bactericidal effects. Catalase mediates the breakdown of hydrogen peroxide, H2O2, into H2O and O2 gas and differentiates Staphylococcus species, which are the catalase producers, and Streptococcus species, which are not. Both of these species are gram-positive bacteria [6].

 The blood agar plate is a differential media that is useful for determining the hemolytic potentiality of an organism. Bacteria can be observed on that plate as three different hemolysis types [7]. Beta-hemolysis breaks down the blood cells in the agar completely, leaving a clear zone around the streak. Alpha-hemolysis breaks down the red blood cells in the agar partially, leaving a greenish color behind. Gamma hemolysis occurs if there is no clearing or greenish color change around the streak, indicating the organism did not break down the blood cells [7].

 The purpose of this study was to carry out and analyze the different biochemical tests used to identify unknown bacteria in the lab. Microorganisms are unique and distinctive with regard to what tests they react positively and negatively with, therefore their respective tests are able to be analyzed outside of the lab setting due to the prior knowledge that was gained earlier in the lab, as well as studying the flow chart that was provided.

Materials and Methods

 To begin our experimental process, a master plate was streaked with an unknown organism and was given at the beginning of the study to use for streaking the working plate, and to have a plate that is uncontaminated. Using this master plate, a working plate was made by obtaining bacteria with a sterile, flamed loop and streaking it on a new plate. The working plate was then incubated at 37oC for 24 hours, at which point it was placed in the class refrigerator. A gram stain and two biochemical tests were then performed in a specific order based on results from the previous tests.

Gram Staining

 The gram staining process began with creating a smear by adding a drop of deionized water to a clean microscope slide, followed by an inoculated loop full of unknown bacterial cells, then mixing them together so as to spread the bacteria amongst the slide [4]. The smear was allowed to air dry, then heat fixed in order to kill the bacteria. Further, the smear was stained with crystal violet for two minutes. After those two minutes had passed, the smear was rinsed with deionized water, which I will now refer to as DI water, and then stained with Gram’s iodine for one minute. After one minute, the smear was cleansed with DI water again and 4-5 drops of the alcohol decolorizer was added to the slide. More decolorizer could be used until the crystal violet no longer ran off the bottom of the slide [5]. Next, the smear was rinsed carefully with DI water and then counter stained with safranin for 30 seconds. Lastly, after 30 seconds, the smear was rinsed with DI water and blotted dry. Finally, the slide was viewed under a microscope to identify the morphology and gram stain culture of the unknown bacteria [5].

Catalase test

 A clean microscope slide was inoculated with theunknown bacteria from the working plate by a sterile loop that was flamed above a Bunsen burner [7]. Then 2-3 drops of hydrogen peroxide, H2O2,  was obtained from the refrigerator and then were added to the clump of bacterial cells to observe either the formation of bubbles due to O2 gas or no bubble formation [7,8]. The results are immediate, so the observations were noted and the slide was then tossed in the sharp biohazard boxes.

Blood agar test

 A plate containing blood agar was provided after completion of the catalase test and the plate was then inoculated with a sterile loop full of unknown bacteria. The loop was sterilized by placing it under the flame of a bunsen burner and then a decent amount of the unknown bacteria was transferred from the working plate to the blood agar [9]. The plate was then stored in the incubator at 37oC for 24 hours. After the 24 hours, the plate was observed for the hemolysis type and the unknown bacteria was identified [10].

Results

Table 1: shows the type of test that was performed on the unknown bacteria and the results and the observations of those said tests. A picture of the gram stain and the blood agar test were also included.

Type of test

Results

Observations

Pictures

Gram Stain

Gram Positive Cocci

Bacterial cells appear to be in clumps and are cocci. purple color.

Catalase Test

Negative

no change in bacterial smear, no bubble formation

a picture was not obtained.

Blood Agar Test

Gamma hemolysis

no color change, no clearing around the bacterial streak, growth but nothing out of the ordinary

After the gram staining procedure was performed and then viewed under a microscope, Enterococcus faecalis appeared to be a purple, cocci shaped microorganism.

When the catalase test was performed and the H2O2 was dropped on the unidentified bacteria on the microscope slide, there was no bubble formation and further, no change in the bacterial cells that were on the slide.

After the blood agar plate was inoculated with the unknown microbe and incubated for 24 hours, the bacterial colony showed no change on the plate except for growth. There was no clearing around the bacteria and no color change around the colony.

Discussion

The outcome from both the gram stain and the two biochemical tests that were performed showed that the unknown microbe I was assigned was identified as Enterococcus faecalis. The results of the tests that were performed were crucial in the determination of the identity of the unknown bacteria due to the exact chemical nature and response to biochemical tests that are unique to each microbe in the bacteria domain. E. faecalis was a gram positive cocci-shaped organism when gram stained, negative when tested for catalase and displayed gamma hemolysis when tested on the blood agar plate.

Gram Staining

 After the gram stain was performed and then observed under the microscope, the Enterococcus faecalis cells appeared as purple, cocci shaped cells which also appeared to be in clumps. The cells appeared to be purple in color because the bacterial cells have a thick cell wall consisting of several layers of peptidoglycan that resisted the decolorization from the alcohol and therefore retain the crystal violet, allowing us to observe the purple color [4,5].

Catalase test

 Further, the catalase test was performed directly after the gram stain and viewed immediately. It was observed that there was no bubble formation and there was no change in the bacterial smear. This meant that the bacteria does not have the catalase enzyme and is likely an anaerobic species [8]. When the hydrogen peroxide was applied, there was no reaction that took place due to the absence of catalase, therefore also determining the microbe is a streptococcus species [8].

Blood Agar test

 Lastly, when the blood agar test was performed and observed after being placed in the 37oC incubator for 24 hours, the species showed gamma hemolysis. This means that the bacteria does not produce hemolysins, and therefore does not induce hemolysis thus the agar under the colony is completely unchanged [9]. This proved that the microbe is non-hemolytic, as the bacteria did not break down any of the red blood cells in the agar [9,10].

 Overall, the results of the unknown bacterial study was a success and there was not any problems encountered during the process of each of the experiments. However, some possible sources of error include most obviously contamination. There is a possibility that at any step during the process of identifying our unknowns, our media could become contaminated, thus causing the rest of the experiment to be off. Furthermore, there could always be a source of error by our peers. Since there were so many people running tests at one time, there is a possibility that someone picked up the wrong media and described that for their experiment thus throwing off their flow chart. However, by all counts, and with proven results, the unidentified microbe I was given is Enterococcus faecalis based on the bacteria’s unique reaction to each of the biochemical tests performed as well as the gram stain.

Works Cited

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5)     Kaplan, M. L., & Kaplan, L. (1933, March 1). The Gram Stain and Differential Staining. Retrieved October 18, 2019

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7)     Baureder, M., & Hederstedt, L. (2012). Genes important for catalase activity in Enterococcus faecalis. PloS one, 7(5), e36725. doi:10.1371/journal.pone.0036725

8)     Iwase, T., Tajima, A., Sugimoto, S., Okuda, K., Hironaka, I., Kamata, Y., … Mizunoe, Y. (2013). A simple assay for measuring catalase activity: a visual approach. Scientific reports, 3, 3081. doi:10.1038/srep03081

9)     Yeh, E., Pinsky, B. A., Banaei, N., & Baron, E. J. (2009). Hair sheep blood, citrated or defibrinated, fulfills all requirements of blood agar for diagnostic microbiology laboratory tests. PloS one, 4(7), e6141. doi:10.1371/journal.pone.0006141

10) Hanson, C. W., & Martin, W. J. (1976). Evaluation of enrichment, storage, and age of blood agar medium in relation to its ability to support growth of anaerobic bacteria. Journal of clinical microbiology, 4(5), 394–399.