Unknown Lab report (REV)

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Nov 24, 2024

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1 Lab Report on Unknown Katie Barnett Professor Johnasha Stuart Tuesday & Thursday 2:30-3:50pm Enterobacter aerogenes #5

2 Gram Stain Gram staining is a simple procedure but requires timely keenness when conducting due to the short time required for reagent reaction. Several reagents are needed, including crystal violet, a primary stain; gram's iodine solution, which is the mordant; acetone/ethanol, which is the decolorizer, 0.1% basic fuchsin solution, which is the counterstain; and water. Moreover, the procedure is facilitated by equipment such as a Bunsen burner, an alcohol-cleaned microscope slide, a slide rack, and a microscope. The procedure was conducted in a few steps that started with transferring a drop of suspended culture to an alcohol-cleaned microscope slide using an inoculation loop, aseptic technique was employed to ensure safety. A small circle was made using the inoculation loop, a diameter of about 15mm. The slide was then dried over a gentle heat for faster drying to prevent culture loss during rinsing and help in cell adhesion. Crystal violet stain was then added to the fixed culture, and after about sixty seconds, the stain was washed off using water. Iodine stain was used purposefully to cover the smear for about sixty seconds before being washed off using water. A few drops of ethanol/methanol mixture (decolorizer) were added to the slide for five seconds before being washed off with water. Moreover, after each staining step, aseptic technique was utilized to ensure the safety standards in the lab were upheld. The smear was then counterstained with fuschin solution for about sixty seconds and washed off with water. The slide was then air-dried and ready for microscopic examination. Through the microscopic observation, it was evident that the organism was gram-negative staining—moreover, the organism presented with a rod-shaped morphology spread over the slide.

3 Figure 1; a picture of the gram stain prepared in the lab showing a gram rod-negative pink stain. Materials and Methods Establishing the organism required a procedural flow of methods using specific materials and reagents. The procedure followed a three-day event encompassing the transfer of unknown bacteria to the sterile TSA slant from the culture plate using a sterile loop. The contents of the slant were then labeled 'R' for proper identification, and aseptic procedures were fully exploited. The TSA slant was then incubated for 24 hours at 37 °C, allowing a reading time in the next lab session. The technique used to read the test was a direct observation of the color change and presence of gas. Carbohydrate fermentation was also conducted as one of the tests during the first-day procedures. A sterile loop was used to transfer a considerable amount of the unknown bacteria into the test tubes that contained phenol red broth for the three constituents of carbohydrates, which include glucose, lactose, and sucrose. The red phenol indicator was the primary reagent in the carbohydrate fermentation procedure. The tubes were then incubated at 37

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4 °C for 24 hours. The technique used to read the test was direct observation for a specific color change, red that would have indicated no acid production, and yellow designated acid production. Moreover, gas production was also checked during the observation. An additional sulfide-indole-motility (SIM) test was also conducted to find the unknown bacteria accurately. The inoculation technique used a loop to introduce the unknown bacteria to the bottom of the tube. The tubes were then incubated at 37 °C for 24 hours. Two reagents were used in the SIM test, sodium thiosulfate and Kovac's reagent. Sodium thiosulfate was added in the SIM medium for detecting hydrogen sulfide production, and Kovac's reagent for detecting indole. The motility test was done through direct observation to identify diffusion of growth in comparison to the stab line. Results

5 Figure 2; a positive result of the carbohydrate test, showing yellow color as an indication of acid production and gas production.

6 Figure3; pictures showing a SIM test result. Test Result Interpretation Phenol red broth Glucose The yellow color was observed. Presence of gas bubble production. The bacteria fermented glucose with the production of gas. Phenol red broth Sucrose The yellow color was observed. Presence of gas bubble The bacteria fermented sucrose with the production of gas.

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7 production. Phenol red broth lactose The yellow color was observed. Presence of gas bubble production. The bacteria fermented lactose with the production of gas. Sulfide No black precipitate in the SIM media. No production of hydrogen sulfide. Indole No red color after adding Kovac’s reagent. An organism incapable of producing indole from tryptophan. Motility No detectable diffuse growth away from the stab line. No motility detected. Discussion The gram stain result was negative, only narrowing the unknown organism to the gram- negative organisms. The gram stain was challenging, hence running it three times without success, but a positive result was achieved in the fourth round. The positive carbohydrate test provided a basis for eliminating non-lactose fermenters, which are gram-negative such as Salmonella and Proteus, which are non-glucose fermenters and gram-negative. Other bacteria such as Shigella was eliminated because they ferment glucose but no gas production. Pseudomonas aeruginosa was a close candidate for the selection and was eliminated by being a non-lactose fermenter, yet it was gram-negative. After the scrutiny and elimination, it was established that the unknown organism was Enterobacter aerogenes based on the characteristic SIM test result (sulfide and indole negative), also carbohydrate test came positive with gas production and was gram-negative. However, motility test did not achieve a positive result despite Enterobacter aerogenes being motile.

8 Enterobacter aerogenes belong to the family Enterobacteriaceae and is a facultative anaerobe hence can grow in the presence or absence of oxygen. Enterobacter aerogenes habitat is majorly in the intestinal tract of the human, soil, and water. Moreover, Enterobacter aerogenes is an opportunistic nosocomial pathogen acquired since it colonizes medical equipment surfaces ( Davin-Regli et al., 2019) . Enterobacter aerogenes majorly infect the urinary tract, cause wound infections and sepsis if left untreated, and may cause pneumonia. The bacteria is also challenging to treat due to antibiotic resistance.

9 Reference Davin-Regli, A., Lavigne, J. P., & Pagès, J. M. (2019). Enterobacter spp.: update on taxonomy, clinical aspects, and emerging antimicrobial resistance. Clinical microbiology reviews , 32 (4), e00002-19. https://doi.org/10.1128/CMR.00002-19 Tripathi, N., & Sapra, A. (2022). Gram Staining. In StatPearls . StatPearls Publishing. Retrieved from- https://pubmed.ncbi.nlm.nih.gov/32965827/

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