Unknown Determination

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Sosa1 Andrea Sosa Flores BIOL-2420-41003 28 April 2023 Prof. Nikki Goines Unknown Determination There is a multitude of benefits in conducting research to recognize and/or develop more research on explored and non-explored bacteria such as Shigella flexneri. On this research I performed tasks previously learned on class in order to identify a specific microorganism. While conducting research I learned the bacteria’s habitat, growth, and oxygen requirement. Shigella flexneri is named after the American physician Simon Flexner, who extensively studied dysentery in the early 20th century. Shigella flexneri primarily inhabits the intestinal tract of humans and other primates. Typically transmitted through the fecal-oral path, mainly by ingesting contaminated food or water. The bacterium can survive inside the intestines and multiply, causing infection and following symptoms of shigellosis. Shigella flexneri is a facultative aerobe meaning it can grow both in the presence and absence of oxygen. However, it thrives best in low-oxygen conditions, such as the anaerobic environment of the human intestine. The microorganism forms part of the genus Shigella and the species within this genus are Shigella flexneri, Shigella sonnei, Shigella dysenteriae, and Shigella boydii . This group of bacteria can cause gastrointestinal bacterial infections including shigellosis, which primarily affects the intestinal tract and causes symptoms such as diarrhea, abdominal pain, and fever. Shigella infections are primarily associated with humans and can be spread from person to person. However, they can occasionally cause disease in certain animals as well. Unlike some other pathogens, Shigella flexneri does not have a significant environmental reservoir.

Sosa2 Although vaccines have been developed to target Shigella flexneri , their efficacy and availability vary depending on each organism and it has shown that the bacteria developed resistance to various antibiotics over time, which poses a challenge for treatment to control shigellosis. There are some prevention steps such as proper hygiene practices, including regular handwashing and safe food handling. Lastly, there is the taxonomic classification of Shigella flexneri ; Kingdom: Bacteria, Phylum: Proteobacteria , Class: Gammaproteobacteria , Order: Enterobacterales Family: Enterobacteriaceae , Genus: Shigella , Species: Shigella flexneri. Experiment One-Gram Staining Materials: PPE, Inoculating loop, staining rack, microscope slides, slide holder, bibulous paper, deionized water, microscope, lens paper Dyes: Crystal Violet, Iodine Decolorizer, and Safranin Organism: Unknown #19 Procedure: Label the microscope slides with the assigned number, place five slides on the staining tray carefully add DI water to one of the slides placed on the tray, aseptic technique – sterilizing the inoculating instrument by flaming the loop inside the inner cone, wire, loop, wire and repeat the process. Carefully, with the inoculating instrument grab two drops of DI water and spread it on the other microscope slides. Repeat the aseptic technique- sterilize by flaming the loop, wire, loop, and repeat the process. Lift the unknown organism , remove the tube cap, sterilized the mouth of the test tube in the top of the outer cone. Collect sample of the bacteria by insert the loop inside the tube and carefully drag it across the slant, sterilized the tube mouth and carefully cap it. Grab a microscopic slide, place the sample obtained on the slide and mix it with the DI water until a cloudy sample is obtained. Spread the sample evenly over the area. Place the

Sosa3 slide on the staining rack. Allow it to completely air-dry. take the slide holder, with the bacterium facing up, heat fix the smear passing it through the flame two or three times. Repeat the process three more times. Place the slide on the rack and flood with Crystal Violet for 60 seconds. Grab the slide with the slide holder, place the slide at an angle, rinse the smear with water to remove the dye. Grab the slide and cover the bacteria with Iodine for 60 seconds, grab the slide at an angle, and gently rinse with water to remove the additional iodine. Hold the slide at an angle, carefully add three to four drops of decolorizer, add one drop at the time, immediately rinse out with water. Set the slide back on the rack and cover it with Safranin for 60 seconds. Grab the slide with the slide holder, place the slide at an angle, rinse the smear with water to remove the dye. Carefully clean the slide with bibulous paper to absorb the excess of water and dyes. Examine the slide under the microscope, 4x,10x,40x, and 100x. Record data based on oil immersion observations. Dispose the slides under the hood. Results: Organism Observation Results Gram Reaction Unknown # 19 Red color observed. Rod-shaped Shape: bacilles Arrangement: bacillus Gram-negative Experiment Two- Potassium Hydroxide (KOH) Stringing Test Materials: PPE, Staining rack, microscope slide, test tube rack, inoculating loop, slide holder, 3% potassium hydroxide (KOH). Organism: Unknown #19

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Sosa4 Procedure: Label the microscope slide, place the slide on the rack, add a drop of 3% KOH on the slide, aseptic technique- sterilize by flaming the loop, wire, loop, and repeat the process. Lift the unknown organism , remove the tube cap, sterilized the mouth of the test tube in the top of the outer cone. Collect sample of the bacteria by insert the loop inside the tube and carefully drag it across the slant, sterilized the tube mouth and carefully cap it. Grab a microscopic slide, place the sample obtained on the slide slowly mix the organism by moving the inoculating loop in circular pattern, mix for 60 seconds, gradually lift the loop, and look for the “stringy” material. Record data obtained and disposed slides under the hood. Results: Organism Result Interpretation Unknown # 19 Stringy Gram-negative Experiment Three- Streak Plate Isolation Materials: One Mueller-Hinton agar (MHA) plate, inoculating loop, Bunsen burner and PPE. Organism: Unknown #19 Procedure: Grab the MHA plate, draw a line outside of the plate vertically and another one horizontally evenly distribute and label the plate with Q1-Q4. Aseptic technique, sterilize by flaming the loop, wire, loop, and repeat the process. wait for the loop to cool down. Lift the unknown organism , remove the tube cap, sterilize the mouth of the test tube in the top of the outer cone. Collect sample of the bacteria by inserting the loop inside the tube and carefully drag it across the slant, sterilize the tube mouth and carefully cap it. Place the sample obtained on the side of the plate smearing it across the part label Q1. Cover the plate with the lid to prevent air bacteria from adhering to the plate while repeating the aseptic technique- sterilize by flaming the loop, wire, loop, and repeat the process. Carefully remove the lid, touch an area of the first

Sosa5 quadrant with the inoculating loop and drag organisms from the first quadrant into the second quadrant. Place the lid back to the plate and repeat the aseptic technique- sterilize by flaming the loop, wire, loop, and repeat the process. Carefully remove the lid, touch an area of the second quadrant with the inoculating loop and drag organisms from the first quadrant into the third quadrant. Cover the plate and apply aseptic technique- sterilize by flaming the loop, wire, loop, and repeat the process. Remove the lid and touch an area of the third quadrant with the inoculating loop and drag organisms from the first quadrant into the fourth quadrant. Cover the plate and flame the inoculating loop. Close the plate and tape the plate around. Label the tape and place the MHA place into the incubator. incubate the plate for 48 hours in an inverted position. Record the results obtained, dispose the plates properly on the label trash can. Results: 1.What quadrant has the most abundant colonies? Q1 has the most abundant colonies. 2.Which quadrant has the least number of colonies? Q4 has the least number of colonies. 3.Which quadrant(s) have isolated colonies? All quadrants contain isolated colonies. Incubation time: 10:15 am 04/19/23 Removal time: 8:50 am 04/24/23

Sosa6 Experiment Four- Urea Hydrolysis Materials: PPE, Bunsen burner, inoculating loop, flint striker, masking tape, test tube rack, test tube containing urea broth. Organism: Unknown #19 Procedure: Label the test with the unknown number. Lift the unknown organism, r emove the tube cap, sterilize the mouth of the test tube in the top of the outer cone. Collect sample of the bacteria by inserting the loop inside the tube and carefully drag it across the slant, sterilize the tube mouth and carefully cap it. Grab the urea broth tube, remove the cap, and sterilize the mouth of the tube inside the outer cone. Aseptically transfer the bacteria into the urea broth tube in a single line insulation. Sterilize the mouth of the tube inside the outer cone and carefully cap it. Put the test tubes in the rack and place them inside the incubator. incubate the plate for 48 hours. Record the results obtained, dispose the tubes properly under the hood. Results: Organism Observation Results Interpretation of Results Unknown # 19 Orange or yellow liquid observed Negative Results Negative for urease which breaks down urea into ammonia and CO 2 . Incubation time: 9:52 am 04/24/23 Removal time: 8:44 am 04/26/23

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Sosa7 Experiment Five- IMVIC Series: SIM Materials: PPE, Bunsen burner, inoculating loop, flint striker, test tube rack, SIM tube, Kovac’s reagent. Organism: Unknown #19 Procedure: Label the test tubes with the broad material and unknown number, aseptic technique- sterilizing the inoculating instrument by flaming the loop inside the inner cone, wire, loop, repeat process. Lift the unknown organism remove the tube cap, sterilize the mouth of the test tube in the top of the outer cone. Collect sample of the bacteria by insert the loop inside the tube and carefully drag it across the slant, sterilized the tube mouth and carefully cap it. Grab the SIM tube , removed the cap, and sterilize the mouth of the tube inside the outer cone. Aseptically transfer the bacteria into the SIM tube . Place the test tube in the test tube rack and place it in the incubator, incubate the plate for 48 hours. After incubation add 3-5 drops of Kovac’s reagent. Record the results obtained, dispose the tubes properly under the hood. Results: Organism Observation Interpretation of Results Unknown # 19 Red layer of Kovac’s reagent at the top of the tube. Thin layer of growth from inoculating stab line. Media color reminds unchanged. Positive for tryptophanase enzyme and indole production. Negative for motility. Negative for sulfur reduction. Results of Repeated test: Incubation time: 8:52 am 04/24/23 Removal time: 8:44 am 04/26/23 Incubation time: 10:00 am 04/26/23 Removal time: 8:35 am 05/01/23

Sosa8 Organism Observation Interpretation of Results Unknown # 19 No red layer of Kovac’s reagent at the top of the tube. Thick layer of growth from inoculating stab line. Media color reminds unchanged. Negative for tryptophanase enzyme and indole production. Positive for motility. Negative for sulfur reduction. Experiment Six- IMVIC Series: MR-VP Materials: PPE, Bunsen burner, inoculating loop, flint striker, test tube rack, MR-VP broth tube, Methyl red reagent, (VP-A) and (VP-B) reagent, sterile pipet. Organism: Unknown #19 Procedure: Label the test tube with the number of the organism used, aseptic technique- sterilizing the inoculating instrument by flaming the loop inside the inner cone, wire, loop, repeat process. Lift the unknown organism remove the tube cap, sterilized the mouth of the test tube in the top of the outer cone. Collect sample of the bacteria by insert the loop inside the tube and carefully drag it across the slant, sterilized the tube mouth and carefully cap it. Grab the MR-VP tube , removed the cap, and sterilize the mouth of the tube inside the outer cone. Aseptically transfer the bacteria into the MR-VP tube . Place the test tubes in the test tube rack and place it in the incubator, incubate the plate for 48 hours. After incubation split the broth into a clean test

Sosa9 tube using the sterile pipet. Add 5 drops of methyl red to the first tube, to the second tube add 10 drops of VP-A and then add 10 drops of VP-B let it sit for one minute to get better results. Record the results obtained, dispose the tubes properly under the hood. Results: Organism MR Observation VP Observation Interpretation of Results Unknown # 19 Red layer on top of the tube No color change Positive for glucose fermentation to an acid (MR positive, VP negative) Experiment Seven- IMVIC Series: Citrate Materials: PPE, Bunsen burner, inoculating loop, flint striker, test tube rack, Citrate tube. Organism: Unknown #19 Procedure: Label the test tube with unknown organism number, aseptic technique- sterilizing the inoculating instrument by flaming the loop inside the inner cone, wire, loop, repeat process. Lift the unknown organism remove the tube cap, sterilized the mouth of the test tube in the top of the outer cone. Collect sample of the bacteria by insert the loop inside the tube and carefully drag it across the slant, sterilized the tube mouth and carefully cap it. Grab the Citrate tube , removed the cap, and sterilize the mouth of the tube inside the outer cone. Aseptically transfer the bacteria drag it across the slant of the Citrate tube . Place the test tubes in the test tube rack and place it in Incubation time: 8:52 am 04/24/23 Removal time: 8:44 am 04/26/23

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Sosa10 the incubator, incubate the plate for 48 hours. Record the results obtained, dispose the tubes properly under the hood. Results: Organism Observation Interpretation of Results Unknown # 19 Green agar slant Negative for the citrate permease enzyme and citrate utilization as a source of carbon. Experiment Eight- Nitrate Reductase Materials: PPE, Bunsen burner, inoculating loop, flint striker, test tube rack, nitrate broth tubes, nitrate A reagent (Sulfanilic acid), nitrate B reagent (Naphthalamine), Zinc dust. Organism: Unknown #19 Procedure: Label the test tube with the unknown number, aseptic technique-sterilizing the inoculating instrument by flaming the loop inside the inner cone, wire, loop, repeat process. Lift the unknown organism, remove the tube cap, sterilize the mouth of the test tube in the top of the outer cone. Collect sample of the bacteria by inserting the loop inside the tube and carefully drag it across the slant, sterilize the tube mouth and carefully cap it. Grab the nitrate tube , remove the cap, and sterilize the mouth of the tube inside the outer cone. Aseptically transfer the bacteria Incubation time: 8:52 am 04/24/23 Removal time: 8:44 am 04/26/23

Sosa11 into the nitrate tube . Place the test tubes in the test tube rack and place it in the incubator, incubate for 48 hours. Remove from the incubator, look for any indicator of turbidity, add 10 drops of Nitrate A reagent then add 10 drops of Nitrate B reagent. Read results if red appears stop, if there is not color change proceed to the next step. Add a tip full of zinc dust. Record the results obtained, dispose the tubes properly under the hood. Results: Organism Observation after adding A and B Observation after Zinc dust Results Interpretation of Results Unknown # 19 Red observed Positive for nitrate reductase produces nitrate reductase enzyme, nitrate is reduced into NO 2 in a single step. Enteric bacteria. Experiment Nine- Lysine Decarboxylase Materials: PPE, Bunsen burner, inoculating loop, flint striker, test tube rack, lysine broth tubes, mineral oil. Organism: Unknown #19 Incubation time: 9:15 am 04/24/23 Removal time: 8:44 am 04/26/23

Sosa12 Procedure: Label the test tube with the unknown number, aseptic technique- sterilizing the inoculating instrument by flaming the loop inside the inner cone, wire, loop, repeat process. Lift the unknown organism, remove the tube cap, sterilize the mouth of the test tube in the top of the outer cone. Collect sample of the bacteria by inserting the loop inside the tube and carefully drag it across the slant, sterilize the tube mouth and carefully cap it. Grab the lysine broth tube , remove the cap, and sterilize the mouth of the tube inside the outer cone. Aseptically transfer the bacteria into the lysine tube . Add 2-3 drops of mineral oil before and after inoculation. Place the test tubes in the test tube rack and place it in the incubator, incubate the plate for 48 hours. Remove from the incubator. Record the results obtained, dispose the tubes properly under the hood. Results: Organism Observation Result Interpretation of results Unknown # 19 Yellow color observed Negative for decarboxylase Positive for fermentation The unknown organism is negative for the lysine decarboxylase enzyme, an enzyme that removes the carbon group (COOH) results is cadaverine. Positive for fermentation. Experiment Ten- Ornithine Decarboxylase with Combination Media MIO Incubation time: 9:35 am 04/24/23 Removal time: 8:44 am 04/26/23

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Sosa13 Materials: PPE, Bunsen burner, inoculating loop, flint striker, test tube rack, MIO media tubes, mineral oil. Organism: Unknown #19 Procedure: Label the test tube with the unknown number, aseptic technique- sterilizing the inoculating instrument by flaming the loop inside the inner cone, wire, loop, repeat process. Lift the unknown organism, remove the tube cap, sterilize the mouth of the test tube in the top of the outer cone. Collect sample of the bacteria by inserting the loop inside the tube and carefully drag it across the slant, sterilize the tube mouth and carefully cap it. Grab the MIO tube , remove the cap, and sterilize the mouth of the tube inside the outer cone. Aseptically transfer the bacteria into the MIO tube . Add 2-3 drops of mineral oil before and after inoculation. Place the test tubes in the test tube rack and place it in the incubator, incubate the plate for 48 hours. Remove from the incubator. Check for evidence of growth, interpret results for motility and ornithine decarboxylase. Record the results obtained, dispose the tubes properly under the hood. Results: Organism Observation Result Interpretation of results Unknown # 19 Yellow color observed Negative for The negative for the lysine Incubation time: 9:35 am 04/24/23 Removal time: 8:44 am 04/26/23

Sosa14 decarboxylase Positive for fermentation decarboxylase enzyme, an enzyme that removes the carbon group (COOH) results is putrescine. Positive for fermentation. Experiment Eleven- Phenylalanine Deaminase Materials: PPE, Bunsen burner, inoculating loop, flint striker, test tube rack, Pdase media tubes, 12% ferric chloride. Organism: Unknown #19 Procedure: Label the test tube with the unknown number, aseptic technique- sterilizing the inoculating instrument by flaming the loop inside the inner cone, wire, loop, repeat process. Lift the unknown organism, remove the tube cap, sterilize the mouth of the test tube in the top of the outer cone. Collect sample of the bacteria by inserting the loop inside the tube and carefully drag it across the slant, sterilize the tube mouth and carefully cap it. Grab the Pdase media tube , removed the cap, and sterilize the mouth of the tube inside the outer cone. Aseptically transfer the bacteria, drag it across the slant of the Pdase tube. Place the test tubes in the test tube rack and place it in the incubator, incubate the plate for 48 hours. Remove from the incubator and add 2-3 drops of 12% ferric chloride. Record the results obtained, dispose the tubes properly under the hood. Results: Organism Observation Result Interpretation of results Unknown # 19 Color change to yellow Negative does not produce the enzyme phenylalanine deaminase. Phenylpylpyruvic acid present. Other gram – bacillus present. Incubation time: 9:35 am 04/24/23 Removal time: 8:44 am 04/26/23

Sosa15 Experiment Twelve- Phenol Red Fermentation Test Materials: PPE, Bunsen burner, inoculating loop, flint striker, test tube rack, Phenol Red (PR) glucose, lactose, and sucrose broth tubes. Organism: Unknown #19 Procedure: Label the test tube with the unknown number, aseptic technique- sterilizing the inoculating instrument by flaming the loop inside the inner cone, wire, loop, repeat process. Lift the unknown organism, remove the tube cap, sterilize the mouth of the test tube in the top of the outer cone. Collect sample of the bacteria by inserting the loop inside the tube and carefully drag it across the slant, sterilize the tube mouth and carefully cap it. Grab the media tube , remove the cap, and sterilize the mouth of the tube inside the outer cone. Aseptically transfer the bacteria into the Phenol Red (PR) glucose tube . Lift the unknown organism, remove the tube cap, sterilize the mouth of the test tube in the top of the outer cone. Collect sample of the bacteria by inserting the loop inside the tube and carefully drag it across the slant, sterilize the tube mouth and carefully cap it. Grab another media tube , remove the cap, and sterilize the mouth of the tube inside the outer cone. Aseptically transfer the bacteria into the Phenol Red (PR) lactose tube . Lift the unknown organism, remove the tube cap, sterilize the mouth of the test tube in the top of the outer cone. Collect sample of the bacteria by inserting the loop inside the tube and carefully drag it across the slant, sterilize the tube mouth and carefully cap it. Grab another media tube , remove the cap, and sterilize the mouth of the tube inside the outer cone. Aseptically transfer the bacteria into the Phenol Red (PR) sucrose tube . Place the test tubes in the test tube rack and place it in the incubator, incubate the plate for 48 hours. Record the results obtained, dispose the tubes properly under the hood.

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Sosa16 Results: Glucose Organism Observation Color of medium Gas present: Yes, or No? Letter representatio n of results (K, A, G) Results Interpretation of Results Unknown # 19 Yellow broth No, gas present A fermentation of glucose to acid. F or glucose, the microbe is a homolactic fermenter. Lactose Organism Observation Color of medium Gas present: Yes, or No? Letter representatio n of results (K, A, G) Results Interpretation of Results Unknown # 19 Orange broth No, gas present K Gas is not produced and there is not fermentation of lactose to acid. F or lactose, the microbe is not capable of fermenting the sugar into an acid. Sucrose Incubation time: 10:00 am 04/26/23 Removal time: 8:35 am 05/01/23

Sosa17 Organism Observation Color of medium Gas present: Yes, or No? Letter representatio n of results (K, A, G) Results Interpretation of Results Unknown # 19 Orange broth No gas present K Gas is not produced and there is not fermentation of sucrose to acid. F or sucrose, the microbe is not capable of fermenting the sugar into an acid. Experiment Thirteen- Triple Sugar Iron (TSI) Materials: PPE, Bunsen burner, inoculating loop, flint striker, test tube rack, TSI agar tubes. Organism: Unknown #19 Procedure: Label the test tube with the unknown number, aseptic technique- sterilizing the inoculating instrument by flaming the loop inside the inner cone, wire, loop, repeat process. Lift the unknown organism, remove the tube cap, sterilize the mouth of the test tube in the top of the outer cone. Collect sample of the bacteria by inserting the loop inside the tube and carefully drag it across the slant, sterilize the tube mouth and carefully cap it. Grab the media tube , remove the cap, and sterilize the mouth of the tube inside the outer cone. Aseptically transfer the bacteria by stab to the butt of the tube and then streak the slant in a zig-zag fashion into the TSI slant tube . Place the test tubes in the test tube rack and place it in the incubator, incubate the plate for 48 hours. Record the results obtained, dispose the tubes properly under the hood. Results: Organism Observation Results Interpretation of Results Incubation time: 9:52 am 04/24/23 Removal time: 8:44 am 04/26/23

Sosa18 Unknown # 19 Pink (alkaline) / Yellow(acid) butt K/ A Glucose fermentation; protein broken down to alkaline and products Results of Confirmatory test: Organism Observation Results Interpretation of Results Unknown # 19 Pink (alkaline) / Yellow(acid) butt K/ A Glucose fermentation; protein broken down to alkaline and products It was a challenging process to identify my organism; I questioned myself multiple times before taking a step and deciding to reveal the name of the bacteria I had discovered, namely Shigella flexneri . Despite the challenges I encountered, such as obtaining false results for two of my biochemical tests—Nitrate reductase and Indole in the IMViC series category—and the need for repeated and confirmatory tests, I ultimately succeeded in identifying Shigella flexneri . I conducted multiple biochemical tests that guided me to the result I obtained. Some of these tests exhibited a unique response from my organism, such as the Phenol Red tests and TSI, which significantly aided my research. Additionally, I performed several tests that helped in eliminating possible organisms, with the most relevant being the IMViC tests. These tests reduced the probability of selecting the wrong microorganism’s name, along with the Ornithine test, which also played an important role in eliminating possible suspects. To ensure the accuracy of my predictions, I proceeded with multiple confirmatory tests, including TSI, KOH, and Indole. These confirmatory tests were crucial in validating the results I had anticipated. In conclusion, my research led to the successful identification of the bacterium Shigella flexneri . Incubation time: 10:00 am 04/26/23 Removal time: 8:35 am 05/01/23

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Sosa19 Works Cited "Ch. 1 Introduction - Microbiology | OpenStax." OpenStax , openstax.org/books/microbiology/pages/1-introduction.

Sosa20 "Comparison of Chromogenic Biolog Rainbow Agar." ProQuest | Better Research, Better Learning, Better Insights , www.proquest.com/docview/748817618? parentSessionId=LTKWOaIXl%2FyWaPL1Ux73abr%2BsMzC1AFI9622yxMvT5I %3D&pq-origsite=primo&accountid=7079. "Shigella - Medical Microbiology - NCBI Bookshelf." National Center for Biotechnology Information , www.ncbi.nlm.nih.gov/books/NBK8038/. "Shigella Flexneri Infection: Pathogenesis and Vaccine Development." OUP Academic , 1 Feb. 2004, academic.oup.com/femsre/article/28/1/43/635550. "Top Hat." Top Hat , app.tophat.com/e/282569/assigned/item/985169::33b27742-4e66-4d96- af45-91c0ecd19f8c.

Unknown Determination

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