Introduction
There are a large number of identification techniques and procedures that help differentiate the question of identifying an unknown bacterial specimen. The clinical and academic value of such processes helps to determine a particular specimen’s pathogenic, biochemical, and morphological features, identify their danger to public health, and subsequently implement preventive control measures when necessary (Hartline, 2023a). A good approach to identification procedures involves a top-down pathway in which the field of potential bacterial variants for the role of the unknown specimen is gradually narrowed until only one variant can be identified with high accuracy.
In the present laboratory, qualitative bacterial identification of the unknown specimen was performed. For this purpose, Gram-staining techniques, biochemical metabolic tests, and microscopy were used. The work is a laboratory report of the results and a discussion of the results of the identified bacterial species.
Methods and Materials
Laboratory staining techniques and the performance of metabolic tests were used for bacterial identification of the unknown specimen. Two unknown specimens were obtained on red blood agar, the primary purpose of which was to determine the structure of the bacterial envelope. Since Gram-negative bacteria are known not to possess a broad layer of peptidoglycan but have a thin layer protected by an outer membrane, their Gram staining does not result in the fixation of crystal violet. Specifically, samples were treated with crystal violet, iodine solution as a mordant, ethyl alcohol as a decolorizing agent, and safranin as a contrasting dye; between the addition of each subsequent phase, excess amounts were removed by washing with distilled water.
Microscopic examination was then applied to examine each sample’s morphology and colony arrangement patterns. Gram-positive bacteria were sent for metabolic tests, including Catalase, NaCl, Bile Esculin Agar, Optochin, Bacitracin, SXT DISC, CAMP, and Hemolysis. Based on the results, it was assumed with high accuracy that bacterial species might have been used as the grape-positive variant.
Bacterial Wall Composition
The Gram staining results showed a purple stain for the first sample and a red stain for the second sample. This implied that the first sample could be classified as Gram-positive because the broad peptidoglycan layer within the bacterial wall could fix crystal violet molecules. Microscopic observation showed that the Gram-positive bacterium had a coccus morphology arranged in associated chains, in contrast to the loose arrangement of rods for the Gram-negative specimen.
Qualitative Identification Using Tests
The Gram-positive bacterial species was sent for several additional tests, the purpose of which was to determine the metabolic characteristics of the sample. First, the motility test demonstrated that the bacteria could not actively move independently. Table 1 summarizes the observations and results of the eight biochemical tests performed. It shows that the bacteria were catalase-negative, which means that they could not synthesize catalase because they did not form vesicles (Hartline, 2023b).
The NaCl test showed a lack of activity and, thus, a general intolerance of the bacterium to high salt concentrations. Bile Esculin Agar results also showed an adverse reaction, indicating an inability to grow on agar medium and hydrolyze esculin in bile’s presence. The negative results of the Optochin test proved the resistance of the bacterial species to the antibiotic action of Optochin and Bacitracin in the corresponding test (Aryal, 2022).
The SXT DISC assay also demonstrated resistance to sulfamethoxazole and trimethoprim. The results of the CAMP Test indicated the possibility of synthesis of a specific enzymatic protein and, thus, the presence of group B Streptococcus in the sample. Finally, a negative Hemolysis Test showed that the bacterial species could not destroy erythrocytes, meaning its pathogenic properties were not based on hemolytic processes. Based on the consistent narrowing of the framework, Streptococcus agalactiae is a potential option for the role of a Gram-positive bacterium.
Table 1. Results of the bacterial identification tests performed
Discussion and Conclusions
The present laboratory work aimed to identify the bacterial species in situ of Gram-positive bacteria using staining, performance tests, and inductive narrowing procedures. Consistent results showed that Streptococcus agalactiae was the species sought because it responded to each test or staining.
Clinically, Streptococcus agalactiae is a nonpathogenic bacterium that inhabits the urogenital tract of a woman or man and the gut (Yoshida et al., 2021). When the microflora is disrupted or opportunistic infections occur, Streptococcus agalactiae can lead to infection of these organs and organ systems, especially in immunocompromised patients. Such infections result in meningitis, urinary tract infections, and invasive infections in pregnant women (Joyce et al., 2022).
The main routes of transmission are direct contact with bodily fluids, such as in childbirth and sexual contact. Preventive measures to reduce the risk of infection include regular diagnostic tests, hygiene and sanitation, and antibiotic drugs if necessary. Antibiotics are also used as the primary treatment, but overuse can lead to the development of antibiotic resistance in the bacterium.
The laboratory work can be completed, as all key milestones have been passed and the results are satisfactory. The initial goal of identifying the bacterial species has been fully achieved. Nevertheless, there are several areas where the work can be improved and expanded upon subsequently. First, the number of repetitions should be increased to reduce the probability of errors. Second, it is acceptable to use more identification tests to prevent any chance of making a detection error.
References
Aryal, S. (2022). Optochin susceptibility test for the identification of Streptococcus pneumoniae. Microbiology Info. Web.
Hartline, R. (2023a). Unknown bacteria identification project. LibreTexts Biology. Web.
Hartline, R. (2023b). Catalase test. LibreTexts Biology. Web.
Joyce, L. R., Manzer, H. S., da C. Mendonça, J., Villarreal, R., Nagao, P. E., Doran, K. S., & Guan, Z. (2022). Identification of a novel cationic glycolipid in Streptococcus agalactiae that contributes to brain entry and meningitis. PLoS Biology, 20(2), 1-15. Web.
Yoshida, H., Goto, M., Takahiro, M., Fukushima, Y., Fujita, T., Tsuyuki, Y., & Takahashi, T. (2021). Intracellular invasion ability of Streptococcus agalactiae among non-invasive isolates from human adults and companion animals in Japan. Journal of Infection and Chemotherapy, 27(7), 999-1004. Web.