Purpose of the Practical
The purpose of the practical is to identify the unknown microorganism in the culture by determining its shape and morphology. The step-by-step procedures done in order to identify the microorganism are preparation of slide smear to make a permanent slide; gram staining of the smear is done to differentiate gram-negative from Gram-Positive Organisms by utilizing the composition of their cell walls. Microscopy is then done on the smear to visualize the microorganisms. The last portion of the lab work done is the IMViC test, i.e., Indole production test, Methyl red, Voges-Proskauer test, and citrate utilization. Hydrogen sulfide production test is also done to further distinguish the microorganisms.
Gram staining is a method used to broadly classify the bacteria based on the cell wall composition into gram-positive and gram-negative. Gram staining steps involve the preparation of a slide smear and staining it with a primary dye, fixing it with a mordant dye, the decolorizing the smear, and applying a counterstain (Mai-Prochnow et al., 2016). The difference in composition of the cell wall of a bacterium is what differentiates the gram-positive from gram-negative bacteria. Gram-positive bacteria have a thick peptidoglycan layer and thus retain the primary dye, while the gram-negative ones have a thin peptidoglycan layer and thus do not retain the primary dye, only stained by the counterstaining (Cappuccino et al., 2017).
Staphylococcus saprophyticus, Staphylococcus epidermidis, and Staphylococcus capitis, Streptococcus salivarius, and Enterococcus durans are among the gram-positive bacteria under investigation. Citrobacter freundii, Escherichia coli, and Enterobacter aerogenes are the gram-negative bacteria under investigation.
To begin the experiment, label two slides with the microorganism’s number as well as the experimenter’s initials. After that, a drop of saline is poured on each slide. Using an inoculation loop, spread the culture on the slide evenly and allow it to dry. When dry, heat-fix it to the slide by passing it through a flame at least five times. The bacteria attach to the culture after it has been heat-fixed to the slide. The slide is then sprayed with crystal violet and left to sit for one minute. The slide is cleansed with water after one minute. The slide is additionally treated with a mordant (iodine) for one minute before being rinsed with water. Ethanol is then added until the slide is almost clear and there is no longer any violet tint. Then the slide is rinsed with water and safranin is added and allowed to sit for 45 seconds (Tripathi & Sapra 2020). Washing with water and preparing for microscopy are the final steps of Gram staining. To assess if the culture is gram-positive or gram-negative, microscopy is used. The 5th number is pink rod-shaped bacteria, thus a gram-negative microbe.
To further classify and identify the gram-negative microbe IMViC and hydrogen sulfide tests were carried out.
Indole production test: this is the initial test done using tryptophan as a substrate. The media is SIM agar, while the enzyme tested is tryptophanase. SIM agar is a differential media testing for the production of indole, hydrogen sulfide gas, and motility of the organisms. The indicator in the media is Kovac’s reagent. Positive results with the production of indole is pink ring color. No color change indicates negative results (Kohlerschmidt et al., 2021).
Gram Staining and Bacterial Classification
Methyl red test: the substrate in this media is glucose. Media used is Methyl red-Voges-Proskauer. This media is a differential media. Tests utilization of glucose by the microorganism to produce acids (Kohlerschmidt et al., 2021). The indicator is methyl red, which turns red when acids are produced, indicating a positive result. In a negative result, the media remains yellow.
Voges Proskauer test: a substrate for this media is glucose, and the media used is MR-VP broth. This is a differential media. Significant products are alcohols, and the indicator used is Barrit’s reagent. This reagent has alcoholic α-naphthol and 40% KOH. Positive results are the formation of a pink ring. No color change for a negative result (Kohlerschmidt et al., 2021).
Citrate test utilization: this is a selective and differential media. The substrate is Simmons citrate; it is used to determine the Utilization of citrate by the microorganisms as a source of carbon (Van et al., 2018). The product following the breakdown of citrate is carbon dioxide; the indicator used is Bromothymol blue. Positive results following the Utilization of the citrate is a color change to blue, negative results- media remains green.
Hydrogen sulfide test: Thiosulfate and cysteine are the substrates, and the enzymes involved are thiosulfate reductase and cysteine desulfurase. SIM agar is the medium which is a differential media for detecting hydrogen sulfide gas (H2S), indole and motility. Positive motility occurs when the growth is not restricted to the inoculation line but extends across the entire test tube (Kohlerschmidt et al., 2021). Negative motility occurs when growth is restricted to the inoculation line. Hydrogen sulfide gas is a substantial byproduct (H2S). The presence of a black precipitate, as well as motility throughout the test tube, indicates a positive growth and motility test result. The presence of a black participant with no motility suggests a positive growth result but a poor motility outcome. It is a poor result for growth and motility if there is no black precipitation or motility. Triple sugar agar is used to assess the microorganism utilization of sugars and production of hydrogen sulfide gas.
- The Indole Production Test- there was no color change to pink; thus, the indole test is negative.
- The Methyl Red Test shows no color change; thus, the microorganism does not utilize glucose as a source of energy.
- The Voges Proskauer Test result indicates a pink ring on the media, indicating the test is positive. This indicates the organisms utilize glucose to produce acetylmethyl carbinol that is converted by presence of ∝- naphthol to diacetyl thus color change
- The Citrate Utilization Test done turned blue in color thus, the microorganism utilizes citrate as a source of carbon during metabolism.
- The final test is the Hydrogen Sulfide Test which tests for gas production and motility of the microorganism. The media showed no dark color change, thus no hydrogen sulfur gas production.
- Motility test of the organism on the triple sugar iron slant, produced a yellow butt and slant indicating motility nature of the bacteria.
Gram staining was done to know whether the organism was gram-positive or negative. The results turned pink, thus indicating that the microorganism of interest is a gram-negative bacterium. To narrow down the identification of the gram-negative microorganism, further tests done include; indole production test (Kohlerschmidt et al., 2021). This test is done to note whether the bacteria utilize tryptophan as its substrate. Some gram-negative bacteria that are indole positive ones on the list of interest are E. coli and Enterobacter aerogenes. The other gram-negative in the list are eliminated at this stage. The next test done is methyl red test. This is done to demonstrate glucose utilization by the bacteria to produce acids. All the gram-negative organisms on the list turned the media red except Enterobacter aeruginosa remained yellow. The MR-VP test done was showed a red ring; thus, MR-VP was positive for E. aeruginosa. Citrate utilization tests done on the gram-negative bacteria; some turned negative; this was E coli while Enterobacter aerogene and C fruendii are citrate positive. To distinguish the two citrate-positive microorganisms, the H2S test and motility is done. H2S is produced by both E. coli, while Enterobacter aerogenes is hydrogen sulfide negative. Thus, Enterobacter aeruginosa is confirmed.
aeruginosa has been identified as a gram-negative rod-shaped bacterium belonging to the Enterobacteriaceae family (Cappuccino & Welsh, 2017). It is a normal microbiota and may become an opportunistic pathogen when immunity fails. It has been implicated as a cause of nosocomial infections for patients in prolonged intensive care and those on mechanical ventilation (I??k et al., 2019). These bacteria are not only prevalent in human intestines, but they are also important to the ecosystem, as they may survive in soil, water, sewage, and even food sources. These bacteria, in particular, cause urinary tract infections (UTIs), respiratory infections, and adult meningitis, which are particularly common in people with compromised immune systems in hospital settings. This bacterium also produces a variety of symptoms including, cough, fever, tachypnea, tachycardia, and purulent sputum (I??k et al., 2019). Pain, fevers, frequency, and urgency to urinate are all symptoms of UTIs. E. aeruginosa causes chest pain, difficulty breathing, and persistent cough in those who have respiratory illnesses. Finally, these bacteria can cause adult meningitis i.e., inflammation and infection of the brain’s meninges or layers. Seizures, photophobia, neck stiffness, and vomiting are some of the disease’s symptoms (Ramirez & Giron 2021).
Antibiotics are one of the therapies used to eradicate E. aeruginosa. Cephalosporins, fluoroquinolones, and carbapenems are the most popular antibiotics used to treat these infections (Lavigne et al., 2018). For antibiotics to effectively work, the treatment plan is usually based on the microorganism’s drug susceptibility and sensitivity, as well as the infection site. Has a poor prognosis if not managed properly.
References:
Cappuccino, J. G., & Welsh, C. T. (2017). Microbiology: A laboratory manual. Pearson.
I??k, D. U., Bas, A. Y., Kulali, F., Ozcan, B., Unal, S., Yucel, H., & Demirel, N. (2019). Nosocomial infection outbreak with Enterobacter aerogenes at a neonatal intensive care unit and its outcomes. Journal of Pediatric Infectious Diseases, 14(05), 223-227.
Kohlerschmidt, D. J., Mingle, L. A., Dumas, N. B., & Nattanmai, G. (2021). Identification of aerobic Gram-negative bacteria. In Practical Handbook of microbiology (pp. 59-70). CRC Press.
Lavigne, J. P., Sotto, A., Nicolas-Chanoine, M. H., Bouziges, N., Pagès, J. M., & Davin-Regli, A. (2018). An adaptive response of Enterobacter aerogenes to imipenem: regulation of porin balance in clinical isolates. International journal of antimicrobial agents, 41(2), 130-136.
Mai-Prochnow, A., Clauson, M., Hong, J., & Murphy, A. B. (2016). Gram positive and Gram-negative bacteria differ in their sensitivity to cold plasma. Scientific reports, 6(1), 1-11.
Ramirez, D., & Giron, M. (2021). Enterobacter infections. In StatPearls [Internet]. StatPearls Publishing.
Tripathi, N., & Sapra, A. (2020). Gram staining.
Van Mastrigt, O., Mager, E. E., Jamin, C., Abee, T., & Smid, E. J. (2018). Citrate, low pH and amino acid limitation induce citrate utilization in Lactococcus lactis biovar diacetylactis. Microbial Biotechnology, 11(2), 369-380.
Order an Essay Now & Get These Features For Free:
Turnitin Report
Formatting
Title Page
Citation
Outline
