Microbiology Lab Unknown Project

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Microbiology Lab Unknown Project Identifying Unknown Bacteria in a Liquid Culture Nicole Maribona

Introduction The purpose of this experiment was to be able to identify given, unknown bacteria in a liquid culture tube. Using the procedures and methods learned thus far, we should be able to complete a successful trial in determining the three different bacteria. By completing a series of tests such as, gram stains, acid fast stains, EMB or Mannitol salt plate test, and protein or fermentation tests, the bacteria should be identified using the appropriate tests. Once isolated, a Gram stain should allow the researcher to identify and classify the bacteria. The results of the Gram stain allow the bacteria to be categorized and therefore further identifiable. If the bacterium is stained purple, it can be categorized as Gram positive, whereas a pink stain would indicate a Gram negative bacterium. A positive bacterium would result in the possibility of several bacteria such as Bacillus subtilis or Staphylococcus aureus . A negative bacterium would most likely mean Pseudomonas aeruginosa , Enterobacter aerogenes , E. Coli , or Serratia marcescens . The only other possible bacteria listed in this experiment, Mycobacterium phlei , is not commonly found using a Gram stain. The only appropriate to correctly identify this bacterium is with an Acid-fast stain. Every bacterium has specific tests that can be completed to further aid in identifying each unknown bacterium. Some bacteria produce acid and gas production from carbohydrates. These bacteria are easily identifiable using fermentation tests and protein tests such as glucose test, lactose test, sucrose test, citrate agar slants, urea test, gelatin test, and peptone iron deeps. In fermentation tests there are inverted tubes called Durham tubes that trapped produced gas. Fermentation tests can have a main ingredient called phenol red which is an acid base indicator. When the test is not inoculated its color is red, however if the bacteria produce acid and gas, the pH of the phenol red will drop, turning the indicator yellow. Protein tests help identify bacteria

due to their ability to hydrolyze polypeptide and peptide bonds to release amino acids. These newly released amino acids are then used as the energy source whereas some bacteria use carbohydrates. In many protein tests, amino acids are decarboxylated to produce products that could be used to manufacture other products. Protein tests not only change color due to pH changes but can also produce a precipitate along with the color change. Different results indicate different meanings and each one can help identify an unknown bacterium. Therefore, since I obtained three different sets of results, I can hypothesize that I have isolated my three different types of bacteria which are Pseudomonas aeruginosa , Enterobacter aerogenes , and Bacillus subtilis .

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Materials For Acid Fast Stains x Carbolfuchsin x Methylene Blue x Acid-alcohol x Slide x Distilled water x Hot plate x Beaker with water x Bunsen Burner x Starter x Hose x Slide holder x Inoculation loop x Sharpie x Silver staining container x Bibulous paper x Bacteria x Microscope For Gram Stain x Crystal violet x Gram¶s iodine x Ethanol x Safranin x Slide x Distilled water x Bunsen burner x Starter x Hose x Slide holder x Inoculation loop x Sharpie x Silver staining container x Bibulous paper x Bacteria x Microscope

For Nutrient Broth x Culture tube x 0.4 g Nutrient Agar broth powder x 50 mL of distilled water x Autoclave x Inoculation loop x Transfer pipet x Autoclave tape x Labeling tape For Nutrient Agar Plates x Petri dish x 3.45 g Nutrient Agar powder x 150 mL of distilled water x 250 mL Erlenmeyer flask x Autoclave tape x Autoclave x Labeling tape For Urea slant test inoculation x Bunsen burner x Hose x Starter x Inoculation loop x Bacteria x Large test tube with Urea slant test x Cap x Autoclave For Sucrose & Lactose test inoculation x Bunsen burner x Hose x Starter x Inoculation loop x Bacteria x Large test tube with sucrose test x Large test tube with lactose test x 2 Durham tubes x 2 Caps x Autoclave EMB Plate x EMB plate x Inoculation loop x Bunsen burner x Hose x Starter x Bactria x Autoclave

For Peptone Iron x Erlenmeyer flask x Peptone iron powder 5.4 g x 150 mL of distilled water x Autoclave x Autoclave tape x Labeling tape x Aluminum foil For Phenol Red Dextrose Broth x Erlenmeyer flask x Dextrose powder 3 g x 150 mL of distilled water x Autoclave x Autoclave tape x Labeling tape x Aluminum foil For Nutrient Agar x Erlenmeyer flask x Nutrient agar powder 3.45 g x 150 mL of distilled water x Autoclave x Autoclave tape x Labeling tape x Aluminum foil Already made supplies x EMB plate x Mannitol salt plate x Urea slant test x Lactose test x Sucrose test

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Methods Obtain a randomly selected bacteria and record your tube number. Conduct an acid-fast stain with methylene blue, carbolfuchsin, water, acid alcohol, and a steaming water bath in a specific order. The acid-fast stain eliminates the option of Mycobacteria is all the bacteria is blue when viewed under a microscope. Record your observations. Then complete an isolation streak plate with your original bacteria culture obtained from the professor. Make sure to label the plate as you might need it for replicating tests. Using an inoculation loop and a Bunsen burner, sterilize your loop before starting. Streak your plate carefully as to gain successful isolated colonies. Place in an incubator and leave overnight for the best results. If done correctly, there should be a clear morphological difference between the tree types of colonies that grow. Record the morphological differences. Take a colony of each type of bacteria and inoculated them in a nutrient broth liquid culture and place in the incubator for 24 hours. Label each of the three culture tubes as Bacteria 1, Bacteria 2, and Bacteria 3. Sometimes for the best results it is best to leave the culture tubes over a second 24 hours. These cultures can now be used to complete each test separately. Using the newly separated bacteria, we can Gram stain without any contamination from the two other bacteria. The Gram stain uses crystal violet, safranin, water, and ethanol in a specific order to classify bacteria as Gram negative (pink) or Gram positive (purple). If your bacteria have the result of a Gram negative stain (pink rods) and Gram positive (purple cocci), you can proceed to conduct test specific for those types of bacteria. The first test being selective and differential medias such as EMB and mannitol salt plates. Since I only had two Gram negative bacterium, there were two corresponding growth quadrants on my EMB plate. With only one Gram positive bacterium, I had one corresponding growth quadrant on the mannitol salt plate. With these results, I can now complete additional tests to further distinguish each bacterium. I inoculated the bacteria in a urea test for Bacteria 1, a sucrose test for Bacteria 2, and a lactose test for Bacteria 3, and left it in the incubator over the Thanksgiving holiday to allow for the best results. Record your observations and findings. Medias are used to provide cultures the bacteria can grow on while also providing us a way to distinguish what bacteria can grow on them. Read the label to find the g in mL ratio for the correct measurements. Use the following table in results to see the calculations on how to make Dextrose, Nutrient Agar, and Peptone iron. An autoclave is used for making medias, so make sure to correctly label your contents and place a piece of autoclave tape on your flask. The environment of the autoclave should be 121 ႏ for 15 minXtes at 15psi . Use caution to remove the medias from the autoclave and immediately pour/ pipet them into their appropriate tubes/plates to prevent hardening.

Results Figure 1.1: Culture tube #17 (original bacteria) Figure 1.2: Isolation streak plate of original bacteria Figure 1.3: Gram Negative stain of unknown Bacteria 1 (pink rods & excess purple stain) Figure 1.4: Gram Negative stain of unknown Bacteria 3 (pink rods & excess purple stain) Figure 1.5: Gram Positive stain of unknown Bacteria 3 (purple cocci & excess pink stain)

Figure 1.6: Mannitol salt plate of Bacteria 1, Bacteria 2, & Bacteria 3 Figure 1.7: Nutrient Agar Broth culture tubes inoculated with Bacteria 1, Bacteria 2, & Bacteria 3 Figure 1.8: Urea test positive for Pseudomonas Figure 1.9: Sucrose test positive for Staphylococcus Figure 2.1 : Lactose test positive for Enterobacter

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A colony of Bacteria 1 was taken from the original streak plate (see Figure 1.2) and grown in a culture tube. From the culture tube it was Gram stained and showed pink rods (see Figure 1.3). Being Gram negative, Bacteria 1 was grown on an EMB plate in quadrant B1 and was positive for growth. Bacteria 1 was then tested on a Urea test and left in the incubator. After the Urea test was taken out of the incubator, the peachy color of the test turned pink (Figure 1.8). The only bacteria to test positive for a Urea test is Pseudomonas . Due to these the positive results in a Gram stain, EMB plate, and a urea test, Bacteria 1 in my unknown culture #17 is therefore Pseudomonas aeruginosa. Bacteria 2 was collected from the isolation streak plate and Gram stained. The bacteria, when placed under a microscope, appeared to look like purple cocci. In this microscope slide, the original bacteria culture was used to find the results. The purple cocci are clearly visible amid multiple pink rods (see Figure 1.5). Knowing that the bacteria was Gram negative cocci the unknown Bacteria 2 must be Staphylococcus aureus. The Bacteria was confirmed due to the positive yellow growth on the mannitol salt plate clearly seen in Figure 1.6. Bacteria 2 also tested positive for a sucrose test (Figure 1.9) producing a yellow color with no gas. Due to the positive results in a Gram stain, mannitol salt plate, and a sucrose test, Bacteria 2 in my unknown culture #17 is therefore Staphylococcus aureus . The third unknown bacteria were tested using a colony from the isolation plate. Bacteria 3 was then viewed under a microscope after being Gram stained. The bacteria viewed as Gram negative pink rods (Figure 1.4) and was then grown on an EMB plate in quadrant B3. This quadrant showed colorless growth which would exclude Serratia and E. Coli, leaving me to decide between Enterobacter and Pseudomonas. Bacteria 3 was then inoculated onto a Lactose test. Bacteria 3 showed results of a positive yellow color change and gas (see figure 2.1). Due to the positive results in the Gram stain, EMB plate, and a lactose test, Bacteria 3 in my unknown culture #17 is therefore Enterobacter aerogenes . Phenol Red Dextrose Nutrient Agar Peptone Iron 20g in 1,000 mL 23g in 1,000 mL 36g in 1,000 mL 20g x ଵହ଴ ௠௅ ଵ,଴଴଴ ௠௅ = 3g 23g x ଵହ଴ ௠௅ ଵ,଴଴଴ ௠௅ = 3.45g 36g x ଵହ଴ ௠௅ ଵ,଴଴଴ ௠௅ = 5.4g 3g diluted in 150 mL of DI water 3.45g diluted in 150 mL of DI water 5.4g diluted in 150 mL of DI water Table 1.1: Calculations showing specifics for media making

Discussion Based on the three tests (Gram stain, EMB plate, Urea test) I performed on Bacteria 1, I conclude that I have Pseudomonas aeruginosa bacteria. I arrived at this conclusion by first conducting a streak plate. Since none of my bacteria grew pink, I could therefore cancel out Serratia. Using the isolated colony, designated Bacteria 1, I then Gram stained my colony to find out whether it was Gram + or Gram -. Since my bacteria was Gram - rods under a microscope, it would grow on an EMB plate but not a mannitol salt plate. So, the second test I conducted was to streak my bacteria on an EMB plate and a mannitol salt plate to test for growth. As expected, my bacteria only grew on the EMB and not the mannitol. The growth on the EMB plate was colorless and not a metallic green or purple as E. coli demonstrates. This reasoning leaves me only with two bacteria to test for: Pseudomonas and Enterobacter. The third and final test I conducted for Bacteria 1 was a urea test. If my bacteria are Pseudomonas, the test would turn from a peach color to a pink color. If my bacteria are Enterobacter it would produce a negative result. After taking out the test from the incubator, the conclusion was confirmed that Bacteria 1 in culture tube #17 is Pseudomonas aeruginosa due to the pink color of the urea test. Based on the three tests (Gram stain, Mannitol salt plate, Sucrose test) I performed on Bacteria 2, I conclude that I have Staphylococcus aureus bacteria. I arrived at this conclusion by first conducting a streak plate. From the previous paragraph, I know the bacteria it cannot be (Pseudomonas, Serratia, E. coli,). Using the respective colony for Bacteria 2, I then conducted a Gram stain which produced a Gram + cocci (which excludes Bacillus). Knowing this, I tested Bacteria 2 on both a mannitol salt plate and an EMB knowing it would only grow on mannitol. Since Bacteria 2 has a Gram + identity, the mannitol salt plate correlated with the result and tested positive for growth. The bacteria changed the media to a yellow color while also producing yellow colonies confirming my suspicions of Staphylococcus. Due to this outcome the final step in confirming Bacteria 2 was to perform a sucrose test. After taking out the test from the incubator, the conclusion was confirmed that Bacteria 2 in culture tube #17 is Staphylococcus aureus due to the red to yellow color change with no gas production. Based on the three tests (Gram stain, EMB plate, Lactose test) I performed on Bacteria 3, I conclude that I have Enterobacter aerogenes bacteria. I arrived at this conclusion by first conducting a streak plate and taking a colony from there that represented Bacteria 3. Using this designated colony, I performed a Gram stain. The Gram stain testes for Gram ± pink rods. From the previous paragraphs the only reasonable bacteria left to consider is Enterobacter (excluding E. coli, Pseudomonas, Serratia). Knowing the Gram identity allows me to conduct a EMB plate and mannitol salt plate to test for growth. Bacteria 3 test for a positive colorless growth on EMB and negative for growth on mannitol salt. To confirm my hypothesis, I conducted a lactose test. After taking out the test from the incubator, the conclusion was confirmed that Bacteria 3 in

culture tube #17 is Enterobacter aerogenes due to the red to yellow color change with gas production.

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References Johnson, T. R., & Case, C. L. (2019). Laboratory experiments in microbiology (12th ed.). NY, NY: Pearson. Olms, M. (2013, December 8). Microbiology Unknown Project Report: Bacillus cereus. Retrieved December 11, 2019, from https://acls-bls-memphis.com/microbiology- unknown-project-report-bacillus-cereus/. Tortora, G. J., Funke, B. R., & Case, C. L. (2019). Microbiology an introduction (12th ed.). Boston: Pearson.

Microbiology Lab Unknown Project

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