Lab 8 Particular & Distinctive Analyzing Urine Cultures and Catalase .

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Lab 8 Particular & Distinctive Analyzing Urine Cultures and Catalase. Purpose: The purpose of this lab is to look for urinary tract infections. Bacteria can enter your urethra and cause a UTI. The purpose of completing lab is to determine if a urinary tract infection (UTI) is being caused by bacteria or yeast. An antibiotic sensitivity test can determine which antibiotic is most likely to kill specific bacteria if they multiply (Cleveland Clinic, 2021). The catalase test makes it easier to find this enzyme in bacteria. It is necessary to distinguish between catalase- positive and catalase-negative Micrococcaceae and Streptococcaceae . It is helpful in the speciation of some gram positives, but its main application is in the differentiation of genera (American Society for Microbiology, 2010). Perfect for identifying infections of the urinary tract. EMB can distinguish between lactose fermenters and non-lactose fermenters and is selective for gram negative bacteria. Most species of Candida will also grow on EMB (Blood Agar 5%/Eosin Methylene Blue (EMB) Agar, n.d.). Gram-positive organisms, especially cocci, can grow on phenylethyl alcohol (PEA) agar, however the majority of gram-negative bacteria and fungi cannot. In particular, gram-positive Staphylococcus and streptococcus species are isolated from clinical specimens using PEA agar, a selective medium (Phenylethyl Alcohol (PEA) Agar, n.d.). Hypothesis: In this lab I expect to see the Eosin Methylene Blue Agar retain its maroon color since the urine is sterile thus I don’t expect to see any bacteria. E coli causes spontaneous bacterial peritonitis strain when hydrogen peroxide is applied, the Phenylethyl Alcohol Agar will show catalase positive for Staphylococcus , that Couse Staphylococcal food poisoning strain which will bubble. Introduction Infected urinary tracts (UTI) are the most common infection in humans in all parts of the urinary system, especially the bladder and urethra, are susceptible to these diseases when bacteria enter and grow. UTIs are mostly caused by the bacteria Escherichia coli . Symptoms include urine with a burning feeling, blood in it, and a strong odor (Common Bacterial Diseases, 2022). Human adult pee is sterile. Unidentified pathways may exist between the local bacterial community and urinary health and illness. Two important complimentary methods for scientific investigation in urologic research are enlarged urine cultures and bacterial genomic sequencing (Wolfe & Brubaker, 2015). Urinary microbiome sample collection can be problematic because the most popular and noninvasive method, midstream urine sampling, can become contaminated from surrounding skin, genital tract, and urethra areas. This can lead to genitourinary/urogenital microbiota for microbial communities obtained from female voiding urine samples, and can result in unreliable/contaminated taxonomic profiles (Čeprnja et al., 2023). Urinary tract bacteria that do not exhibit symptoms of infection are rather frequent and have been linked to antibiotic abuse, which in turn fosters resistance (Bacteria in Urine Doesn’t Always Indicate Infection, n.d.). Gram-negative bacteria can only be grown on EMB agar. Gram-positive bacteria are prevented from growing by the medium's methylene blue dye (Lal & Cheeptham, 2007). When lactose or sucrose is fermented, eosin dye and methylene-blue indicator react to alter color. The amide

bonding of the dyes in an acidic environment results in blue-black colonies with a green metallic sheen in lactose-fermenting coliforms like Escherichia coli (Tankeshwar, 2016). Escherichia coli colonies grow with a metallic sheen and a dark center, Aerobacter aerogenes colonies have a brown center, and non-lactose-fermenting are colorless, gram-negative bacteria look pink on EMB agar. These organisms are part of the colon-typhoid-dysentery group (Eosin-Methylene Blue Agar Plates Protocol, n.d.). Gram-negative bacteria are the only ones that grow on EMB agar. The medium contains methylene blue dye, which effectively stops most gram-positive bacteria from growing in modest concentrations (Lal & Cheeptham, 2007). A selective medium called phenylethyl alcohol agar (PEA) is used to grow Gram-positive organisms. By interfering with DNA synthesis, the active component, phenylethyl alcohol, inhibits or significantly lowers the growth of Gram-negative organisms PEA also stops Proteus species from congregating on the agar's surface. (Welcome to Microbugz - Phenyethyl Alcohol Agar, n.d.). Phenylethyl alcohol (PEA) agar allows growth of gram-positive organisms, particularly cocci, while inhibiting most gram-negative bacteria and fungi. Specifically, PEA agar is a selective medium that is used for the isolation of gram-positive Staphylococcus species and Streptococcus species (Phenylethyl Alcohol (PEA) Agar, n.d.). When separating catalase- positive staphylococci from streptococci (catalase-negative), the catalase test is crucial. Several drops of 3% hydrogen peroxide are flooded in order to complete the test (Foster, 1996). The majority of oxygen-consuming bacteria make hydrogen peroxide, which, if it built up, would be harmful to the cell (Mahaseth & Kuzminov, 2017). The body produces the enzyme catalase to defend itself by breaking down hydrogen peroxide before it can produce hydroxyl radicals (Schwarcz, 2017). Catalase catalyzes the process that turns hydrogen peroxide into oxygen and water (Group, n.d.). Breakdown into water and oxygen gas, this enzyme detoxifies hydrogen peroxide. An obvious sign of a catalase positive result are the bubbles created by the generation of oxygen gas (Catalase Test | Summary of Biochemical Tests | Additional Info | Molb 2021 | College of Agriculture and Natural Sciences, n.d.). When bubbles form as a result of oxygen gas generation, the bacteria are catalase positive. The bacteria are catalase negative if no bubbles form. Catalase is present in Staphylococcus species , but not in Streptococcus species (Rapid Tests | Lab10 | Virtual Edge | Molb 2021 | College of Agriculture and Natural Sciences, n.d.). Materials Required: Materials according to (Hughes, 2019, “Selective and Differential Urine Culture Analysis and Catalase Lab,”). 1. Petri Dish with Eosin Methylene Blue (EMB). 2. Petri Dish with Phenylethyl alcohol (PEA). 3. 6 sterile swabs. 4. Urine sterile culture specimen bottle with lid 5. Urine 6. Marker. 7. Paper towel. 8. Water. 9. Soap.

10. Spraying bleach solution. 11. Hibiclens. 12. Latex gloves. 13. Mask. 14. Camera. Procedure : 1. When entering the lab ensure you have all your hair tied up, closed shoes to cover entire foot and have a protective clothing on top of your home cloths. Sterilize the working table by spraying bleach solution and wipe with a paper towel. 2. Remove jewelry that are on your hands to the wrist, roll up your sleeves to perform hand washing. Turn on the faucet and adjust the water temperature to your comfortable temperature, wet your hand with water and apply anti bacteria hand wash soap and scrub your hands to include 4 inch above the wrist. Scrubbing to remove the dirt for at least 1 minute then rinse the soap with running water. If you touch the sink or anything repeat the whole hand washing process and dry your hands with a paper towel and discard it to the correct bin and pick other clean paper towels and shut the faucet off and dispose the paper towels in the disposal bin. Do not touch your face, eye and mouth while working in microbiology lab. 3. Carefully put on your face mask and wear latex gloves. 4. Gather all the materials and instruments needed for the lab work i.e. petri dish from the refrigerator. 5. Pick a sterile culture bottle and ensure it has a lid on it. In the bathroom, using your sterile urine culture bottle open the lid and put urine in it. The urine does not have to be from a sterile stream. When done putting the urine in the sterile urine bottle, close it with the lid immediately (Hughes, 2019, “Selective and Differential Urine Culture Analysis and Catalase Lab,”). 6. In this lab we will be using 2 different types of agar in each petri dish. The first petri dish will contain EMB which has a maroon color to it before any bacteria is put on it. The second one will have the PEA which looks whitish just like the nutritional agar used earlier with gram staining lab. We will also be using 2 different swabs each swab for each agar-containing petri dish (Hughes, 2019, “Selective and Differential Urine Culture Analysis and Catalase Lab,”). 7. Remove the lid from the culture bottle that contains the urine specimen dip the 1 st sterile swab to collect the urine specimen and close the culture bottle with the lid (Hughes, 2019, “Selective and Differential Urine Culture Analysis and Catalase Lab,”). 8. Open petri dish that containing the EMB agar using the zig zag technique draw onto the petri dish in zigzag movements from top to bottom a third of the dish with the 1 st swab containing the urine specimen (Hughes, 2019, “Selective and Differential Urine Culture Analysis and Catalase Lab,”). 9. When done drawing the first zigzags from top to bottom, discard the swab in a biohazards bin. 10. Remove the lid from the culture bottle that contains the urine specimen dip the 2 nd swab to collect the urine specimen and close the culture bottle with the lid (Hughes, 2019, “Selective and Differential Urine Culture Analysis and Catalase Lab,”).

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11. Open petri dish that containing the EMB agar using the zig zag technique draw onto the petri dish in zigzag movements from top to bottom a third of the dish with the 2 nd swab containing the urine specimen (Hughes, 2019, “Selective and Differential Urine Culture Analysis and Catalase Lab,”). 12. When done drawing the first zigzags from top to bottom, discard it in a biohazards bin. 13. Remove the lid from the culture bottle that contains the urine specimen dip the 3 rd swab to collect the urine specimen and close the culture bottle with the lid (Hughes, 2019, “Selective and Differential Urine Culture Analysis and Catalase Lab,”). 14. Open petri dish that containing the EMB agar using the zig zag technique draw onto the petri dish in zigzag movements from top to bottom a third of the dish with the 3 rd swab containing the urine specimen (Hughes, 2019, “Selective and Differential Urine Culture Analysis and Catalase Lab,”). 15. When done drawing the first zigzags from top to bottom, discard it in a biohazards bin. 16. Put the lid on the petri dish and allow it to dry for 5 minutes, invert the petri dish so as to have the agar up, clearly label the petri Dish with the EMB agar with initials PK, 301 and put it in the incubator (Hughes, 2019, “Selective and Differential Urine Culture Analysis and Catalase Lab,”). 17. Remove the lid from the culture bottle that contains the urine specimen dip the 4 th swab to collect the urine specimen and close the culture bottle with the lid (Hughes, 2019, “Selective and Differential Urine Culture Analysis and Catalase Lab,”). 18. Open petri dish that containing the second agar (PEA) using the zig zag technique draw onto the petri dish in zigzag movements from top to bottom a third of the dish with the 4 th swab containing the urine specimen (Hughes, 2019, “Selective and Differential Urine Culture Analysis and Catalase Lab,”). 19. Discard it after drawing the zigzag in a trash can. 20. Remove the lid from the culture bottle that contains the urine specimen dip the 5 th swab to collect the urine specimen and close the culture bottle with the lid (Hughes, 2019, “Selective and Differential Urine Culture Analysis and Catalase Lab,”). 21. Open petri dish that containing the second agar (PEA) using the zig zag technique draw onto the petri dish in zigzag movements from top to bottom a third of the dish with the 5 th swab containing the urine specimen (Hughes, 2019, “Selective and Differential Urine Culture Analysis and Catalase Lab,”). 22. Discard it after drawing the zigzag in a biohazards bin 23. Remove the lid from the culture bottle that contains the urine specimen dip the 6 th swab to collect the urine specimen and close the culture bottle with the lid (Hughes, 2019, “Selective and Differential Urine Culture Analysis and Catalase Lab,”). 24. Open petri dish that containing the second agar (PEA) using the zig zag technique draw onto the petri dish in zigzag movements from top to bottom a third of the dish with the 6th swab containing the urine specimen (Hughes, 2019, “Selective and Differential Urine Culture Analysis and Catalase Lab,”). 25. Discard the swab after drawing the zigzag in a trash can. 26. Put the lid on the petri dish and allow it to dry for 5 minutes, invert the petri dish so as to have the agar up, clearly label the Dish with the PEA agar label petri dish with the initials PK, 301 and put it in the incubator Leave both petri dishes in the incubator for 48 days to

grow (Hughes, 2019, “ Selective and Differential Urine Culture Analysis and Catalase Lab,”). 27. When done, empty the urine in urine Collection Container Sterile Sample Specimen bottle in the toilet and flush discard swabs and empty urine Collection Container Sterile Sample Specimen bottle in the trash can discard any disposable waste materials from the lab work and put away other materials and equipment to appropriate storage at the end of the lab work. Then disinfect the working table by spraying bleach solution and wipe with a paper towel. 28. Remove the gloves and face mask and discard in the appropriate disposal bin and perform hand washing. Turn on the faucet wet your hand with water and apply anti bacteria hand wash soap and scrub up to 4 inch above the wrist, scrubbing thoroughly for at least 1 minute then rinse the soap with running water and apply a dime size amount of hibiclens, rub hands for 30 seconds then rinse it off. If you touch the sink or anything repeat the whole hand washing process and dry your hands with a paper towel and discard it in to the correct disposal bin. Use another paper towel to shut the faucet and dispose it. Roll down your sleeves and can leave the lab when done. Catalase test Materials Required: a. Petri Dish with Phenylethyl alcohol (PEA). b. Hydrogen peroxide (H2O2) (1.18: Catalase Test, 2022). c. Paper towel. d. Water. e. Soap. f. Spraying bleach solution. g. Hibiclens. h. Latex gloves. i. Mask. Procedure: 1. When entering the lab ensure you have all your hair tied up, closed shoes to cover entire foot and have a protective clothing on top of your home cloths. Sterilize the working table by spraying bleach solution and wipe with a paper towel. 2. Remove jewelry that are on your hands to the wrist, roll up your sleeves to perform hand washing. Turn on the faucet and adjust the water temperature to your comfortable temperature, wet your hand with water and apply anti bacteria hand wash soap and scrub your hands to include 4 inch above the wrist. Scrubbing to remove the dirt for at least 1 minute then rinse the soap with running water. If you touch the sink or anything repeat the whole hand washing process and dry your hands with a paper towel and discard it to the correct bin and pick other clean paper towels and shut the faucet off and dispose the paper towels in the disposal bin. Do not touch your face, eye and mouth while working in microbiology lab. 3. Carefully put on your face mask and wear latex gloves. 4. Gather all the materials and instruments needed for the lab work i.e. petri dish from the refrigerator. 5. Open the petri dish with the specimen to be tested

6. Open the hydrogen peroxide bottle and collect some drops of the hydrogen peroxide using a Pasteur Pipette. Recap the hydrogen peroxide (1.18: Catalase Test, 2022). 7. Put some 2-3 drops of the hydrogen peroxide and observe for any bubbling of fizzing as soon as the hydrogen peroxide drops are poured (1.18: Catalase Test, 2022). 8. If it does bubble, it means it catalase positive and if it does not bubble, it means it is catalase negative (1.18: Catalase Test, 2022). 9. When done discard appropriately any disposable waste materials from the lab work and put away other materials Hydrogen peroxide and petri dish to appropriate storage at the end of the lab work. Then disinfect the working table by spraying bleach solution and wipe with a paper towel. 10. Remove the gloves and face mask and discard in the appropriate disposal bin and perform hand washing. Turn on the faucet wet your hand with water and apply anti bacteria hand wash soap and scrub up to 4 inch above the wrist, scrubbing thoroughly for at least 1 minute then rinse the soap with running water and apply a dime size amount of hibiclens, rub hands for 30 seconds then rinse it off. If you touch the sink or anything repeat the whole hand washing process and dry your hands with a paper towel and discard it in to the correct disposal bin. Use another paper towel to shut the faucet and dispose it. Roll down your sleeves and can leave the lab when done. Results: Conclusion: Restate: The purpose of this lab is to perform lactose test to separate lactose-fermenting from lactose-nonfermenting gram-negative bacteria Explain: In the lab we will begin by preparing the working area and self. Sterilize the working area with bleach, roll up sleeves and remove jewelry and perform hand hygiene. When hands are dry wear on gloves and mask and gather all the materials and equipment’s to be used. To have Micro incinerator on, plug in the power cable to the electric switch and turn it on. Pick a sterile culture bottle and ensure it has a lid on it. In the bathroom, using your sterile urine culture bottle open the lid and put urine in it. When done putting the urine in the sterile urine bottle, close it with the lid immediately in this lab we will be using 2 different types of agar in each petri dish. The first petri dish will contain EMB. The second one will have the PEA We will also be using 6 different swabs each swab for each agar-containing petri dish. Inoculate the 1 st swab. Remove the lid of the culture bottle that contains the urine specimen dip the inoculated swab to collect the urine specimen and close the culture bottle with the lid. Using a zigzag movement, draw onto the petri dish containing the EMB agar from top to bottom a third of the dish with the swab containing the urine specimen. Pick the 2 nd swab and open the culture bottle, dip it into the urine specimen. When you have obtained a specimen, remove the swab from the culture bottle and put the lid in it. Repeat the same zigzag movement on the same EMB dish from top to bottom a third of the dish next to the first one done. pick 3 rd swab and open the culture bottle, dip it into the urine

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specimen. When you have obtained a specimen, remove the swab from the culture bottle and put the lid in it. Repeat the same zigzag movement on the same EMB dish from top to bottom a third of the dish next to the second one. Put the lid on the petri dish and allow it to dry for 5 minutes, invert the petri dish so as to have the agar up, clearly label the Dish with the EMB agar label the initials PK,301 and put it in the incubator. On the second agar (PEA), we will repeat the same procedure as we did on the EMB but this time using the 4 th swab. Remove the lid from the culture bottle that contains the urine specimen dip the inoculated swab to collect the urine specimen and close the culture bottle with the lid. Using a zigzag movement, draw onto the petri dish containing the PEA agar from top to bottom a third of the dish with the swab containing the urine specimen. Pick the 5 th swab and open the culture bottle, dip it into the urine specimen. When you have obtained a specimen, remove the swab from the culture bottle and put the lid in it. Repeat the same zigzag movement on the same PEA dish from top to bottom a third of the dish next to the first one done. Pick 6 th swab and open the culture bottle, dip it into the urine specimen. When you have obtained a specimen, remove the swab from the culture bottle and put the lid in it. Repeat the same zigzag movement on the same PEA dish from top to bottom a third of the dish next to the second one. Put the lid on the petri dish and allow it to dry for 5 minutes, invert the petri dish so as to have the agar up, clearly label the Dish with the PEA agar with initials PK,301 and put it in the incubator. Leave both petri dishes in the incubator for 48 days. Put away the instruments and materials in the correct storage and finish by disinfecting the work surface with bleach and remove the gloves, and dispose them off at the correct bin. Perform hand washing after lab work. Turn on the faucet wet your hand with water and apply anti bacteria hand wash soap and scrub 4 inch above the wrist, scrubbing for at least 60 seconds then rinse the soap with running water. If you touch the sink or anything repeat the whole hand washing process, apply Hibiclens, rub hands for 30 seconds then rinse it off and dry your hands with a paper towel. Use the paper towel to shut the faucet and dispose it. Do not touch your face, eye and mouth while working in microbiology lab. After you are done leave the lab. Results Uncertainty Q1. What results would you expect to see in the EMB agar if the female patient always wiped back to front after urination? Why would you expect these results? I would expect to see E. coli , Klebsiella , and Enterobacter on the EMB agar. Certain germs from the anus can seriously compromise the health of urethra and vagina if they get the chance. Q2 What type of bacteria does PA agar inhibit? How does it inhibit this particular bacterium (be thorough)? What type of bacteria does EMB agar inhibit? How does it inhibit this particular bacterium (be thorough)? Gram positive organisms are cultivated on phenylethyl alcohol agar (PEA), a selective medium. Phenylethyl alcohol, the active component, interferes with DNA synthesis to either significantly limit or stop the growth of Gram-negative organisms. PEA additionally keeps Proteus species from swarming over the agar's surface (Welcome to Microbugz - Phenyethyl Alcohol Agar, n.d.). Gram-negative bacteria are only present on EMB agar. Gram-positive bacterial growth is

inhibited by the medium's methylene blue dye, which works well to stop most gram-positive bacterial growth in tiny levels (Lal & Cheeptham, 2007). References

American Society for Microbiology. (2010, November 11). Catalase Test. ASM.org. https://asm.org/Protocols/Catalase-Test-Protocol#:~:text=The%20catalase%20test %20facilitates%20the Bacteria in Urine Doesn’t Always Indicate Infection. (n.d.). Www.idsociety.org. https://www.idsociety.org/news--publications-new/articles/2019/BACTERIA-IN- URINE-DOESNT-ALWAYS-INDICATE-INFECTION/#:~:text=Clinical%20Infectious %20Diseases.- Catalase Test | Summary of Biochemical Tests | Additional Info | Molb 2021 | College of Agriculture and Natural Sciences. (n.d.). UWYO. https://www.uwyo.edu/molb2021/additional_info/summ_biochem/catalase.html#:~:text= This%20test%20is%20used%20to Catalase Test. (2022, January 15). Biology LibreTexts. https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology_Laboratory_Manual_( Hartline)/01%3A_Labs/1.18%3A_Catalase_Test#:~:text=Laboratory%20Instructions Čeprnja, M., Edin Hadžić, Oros, D., Melvan, E., Starčević, A., & Jurica Žučko. (2023). Current Viewpoint on Female Urogenital Microbiome—The Cause or the Consequence? Microorganisms, 11(5), 1207–1207. https://doi.org/10.3390/microorganisms11051207 Cleveland Clinic. (2021, May 11). Urine Culture: Purpose, Results & What To Expect. Cleveland Clinic. https://my.clevelandclinic.org/health/diagnostics/22126-urine-culture Common bacterial diseases. (2022, May 20). Imb.uq.edu.au. https://imb.uq.edu.au/common- bacterial-diseases#:~:text=Urinary%20tract%20infections%20(UTI)&text=UTIs%20are %20mainly%20caused%20by

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Eosin-Methylene Blue Agar Plates Protocol. (n.d.). Eosin-Methylene Blue Agar Plates Protocol. https://asm.org/Protocols/Eosin-Methylene-Blue-Agar-Plates-Protocol Foster, T. (1996). Staphylococcus (S. Baron, Ed.). PubMed; University of Texas Medical Branch at Galveston. https://www.ncbi.nlm.nih.gov/books/NBK8448/#:~:text=The%20catalase %20test%20is%20important Group, T. (n.d.). M-CSA Mechanism and Catalytic Site Atlas. Www.ebi.ac.uk. https://www.ebi.ac.uk/thornton-srv/m-csa/entry/573/#:~:text=Catalase%20is%20a %20heme%20containing Hughes,M.(2019) Lab 8 Selective and Differential Urine Culture Analysis and Catalase Lab. Www.youtube.com. https://www.youtube.com/watch?v=GF564kbMImU Lal, A., & Cheeptham, N. (2007). Eosin-Methylene Blue Agar Plates Protocol. American Society for Microbiology. https://asm.org/ASM/media/Protocol-Images/Eosin- Methylene-Blue-Agar-Plates-Protocol.pdf?ext=.pdf Mahaseth, T., & Kuzminov, A. (2017). Potentiation of hydrogen peroxide toxicity: From catalase inhibition to stable DNA-iron complexes. Mutation Research, 773(PMC5607474), 274– 281. https://doi.org/10.1016/j.mrrev.2016.08.006 Phenylethyl Alcohol (PEA) Agar. (n.d.). ASM.org. https://asm.org/Protocols/Phenylethyl- Alcohol-Agar-Protocol#:~:text=Phenylethyl%20alcohol%20(PEA)%20agar%20allows Rapid Tests | Lab10 | Virtual Edge | Molb 2021 | College of Agriculture and Natural Sciences. (n.d.). UWYO. https://www.uwyo.edu/molb2021/virtual-edge/lab10/lab10_rapid.html#:~:text=If %20bubbles%20appear%20(due%20to

Schwarcz, J. (2017, January 18). Hydrogen Peroxide: the body’s best defence system. Office for Science and Society; McGill University. https://www.mcgill.ca/oss/article/general- science-you-asked/hydrogen-peroxide-bodys-best-defence-system#:~:text=To%20protect %20itself%2C%20the%20body Tankeshwar, A. (2016, October 21). Eosin Methylene Blue (EMB) Agar : Composition, uses and colony characteristics - Microbeonline. Microbeonline. https://microbeonline.com/eosin- methylene-blue-emb-agar-composition-uses-colony-characteristics/ Welcome to Microbugz - Phenyethyl Alcohol Agar. (n.d.). Www.austincc.edu. https://www.austincc.edu/microbugz/phenyethyl_alcohol_agar.php#:~:text=Phenylethyl %20alcohol%20agar%20(PEA)%20is Wolfe, A. J., & Brubaker, L. (2015). “Sterile Urine” and the Presence of Bacteria. European Urology, 68(2), 173–174. https://doi.org/10.1016/j.eururo.2015.02.041

Lab 8 Particular & Distinctive Analyzing Urine Cultures and Catalase .

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