Pius Lab 7 Lactose Lab

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Lab 7 media lab that is selective and different. Purpose: The purpose of this lab is to perform lactose test to separate lactose-fermenting from lactose- nonfermenting gram-negative bacteria and the isolation of gram-negative bacteria (Allen, 2005). The purpose of completing lactose lab is to determine whether the microorganism can use lactose, a sugar, as a carbon source for fermentation (Lactose Fermentation Test - Virtual Microbiology Lab Simulator Software, 2022). A selective and differentiating medium called MacConkey Agar (MAC) is used to extract and separate different types of enterics according to how well they can ferment lactose. Gram positive organisms can't grow in the presence of bile salts or crystal violet. Differentiation is made possible by the source of fermentable carbohydrates that lactose offers (Welcome to Microbugz - MacConkey Agar, n.d.). Hypothesis: In this lab I expect to see a gram-negative MacConkey’s agar thus inhibit the growth of Gram- positive microbes. I expect unknown A sample to be Escherichia coli because it generates colonies that appear to be red or pink because lactose fermentation produces acid, which changes the color of the medium. I expect unknown B sample to be Salmonella because in the absence of lactose fermentation, colonies will appear white/clear since the pH won't change. Introduction Bacteria were transfered using the zigzag approach in the lactose lab. Only gram-negative bacterial species can grow on MacConkey agar, which is a selective and differentiating agar that may further distinguish the gram-negative organisms based on their lactose fermentation. Those bacteria that can ferment and those that cannot (Jung & Hoilat, 2021). The crystal violet dye, bile salts, lactose, and neutral red are important components of the MacConkey medium (Jung & Hoilat, 2021). Acidic waste product is produced when fermentation take place Organic acids, notably lactic acid, are produced by lactose-fermenting microbes, bringing the pH down as the pH falls below 6.8, it changes from off-white to a bright red or pink color. (Jung & Hoilat, 2021). pH indicator that changes color to pink in an acidic environment therefore Lactose-fermenting are gram-negatives (Jung & Hoilat, 2021). Escherichia coli is a gram-negative, facultative anaerobic bacillus that can ferment lactose to create hydrogen sulfide (Yaratha et al., 2017). On MacConkey's agar, it will turn pink and, possibly, take on a deeper color around the colony as a result of bile salt excretion (Escherichia Coli (E Coli) Infections Workup: Laboratory Studies, Imaging Studies, Other Tests, n.d.). Colonies that develop colorless colonies are home to gram-negative bacteria that do not ferment lactose (Jung & Hoilat, 2021). Colonies of non-lactose fermenting bacteria, such as Shigella spp., Salmonella spp., Pseudomonas aeruginosa, Proteus spp., and others, are translucent, whitish, or yellowish in color (VetBact, 2021). The difference in color depends on the medium's color and how much time has passed; it has nothing to do with the differential reaction.

Pseudomonas putida colonies on MacConkey agar have a rounded, flat appearance. The organism is a lactose non-fermenter; hence the media's color will be colorless (Sapkota, 2022). The gram-positive bacteria's growth is stopped by bile salts and the color crystal violet (Jung & Hoilat, 2021). If there is no growth in the lactose lab, the result is labeled lactose unknown and is presumed to be gram positive. Methods: Materials Required: 1. 2X Petri Dish with MacConkey agar (Hughes, 2019, “Lactose Lab”). 2. Bacteria unknown A &B (Hughes, 2019, “Lactose Lab”). 3. Micro incinerator (Hughes, 2019, “Lactose Lab”). 4. Inoculation loop (Hughes, 2019, “Lactose Lab”). 5. Marker 6. Paper towel. 7. Water. 8. Soap. 9. Spraying bleach solution. 10. Hibiclens. 11. Latex gloves. 12. 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, Micro incinerator from the storage, inoculation loop 5. Turn on the Micro incinerator by plugging it on to the power using the power cable and switch it on at the on/off button which will turn the light on at the switch to show it is on and let it warm for 10 mins. You know it is ready to use when the inside has an orange glow. 6. Using a marker label the petri dish from the outside to read 1-2, 2-3,3-4 and 4- 41/2 in a square like shape at the far corners of the Petri dish with the numbers following each

other at the corners (i.e. label 1 on the upper left corner, 2 at the left lower corner, 3 on the right lower corner and 4 on the upper right corner). 7. Sterilize your inoculating loop by inserting it at the Micro incinerator without touching any sides for 10 second until the wire turn orange then remove the inoculation loop from the micro incinerator and let it cool down for 30 seconds. 8. Open petri dish that has the bacteria unknown A insert your sterile inoculating loop in the petri dish and scoop a small sample, close it return to the incubator. Transfer the scooped bacteria to the labeled petri dish using the zig zag technique. Apply the scooped bacteria at the labeled petri dish number 1 along the line to number 2 using thin zigzag movements. 9. When done drawing the zigzags from 1 to 2, sterilize inoculating loop by inserting it to the micro incinerator for 10 seconds until the wire turn orange and remove it to allow it to cool down for 30 seconds. 10. Using the sterile inoculating loop, dilute the bacteria by drawing a zigzag movement from number 2 to 3 by touching where the zigzag ended at the number 2 corner and draw along the line to corner number 3. Sterilize inoculating loop by inserting it to the micro incinerator for 10 seconds until the wire turn orange and remove it to allow it to cool down for 30 seconds. 11. Using the sterile inoculating loop, dilute the bacteria further by drawing a zigzag movement from number 3 to 4 by touching where the zigzag ended at the number 3 corner and draw along the line to corner number 4. Sterilize inoculating loop by inserting it to the micro incinerator for 10 seconds until the wire turn orange and remove it to allow it to cool down for 30 seconds. 12. Using the sterile inoculating loop, dilute the bacteria by drawing a zigzag movement from number 4 to half way towards number 1 by touching where the zigzag ended at the number 4 corner and draw along the line midway between 4 and 1 to avoid contamination of the diluted bacteria and we will have achieved colonies. Sterilize inoculating loop by inserting it to the micro incinerator for 10 seconds until the wire turn orange and remove it to allow it to cool down for 30 seconds. 13. Label the petri dish unknown A wit P.K initials and place in the incubator facing upside down agar up for 48 hours (Hughes, 2019, “Lactose Lab”). 14. Using a marker label the second petri dish from the outside to read 1-2, 2-3,3-4 and 4- 41/2 in a square like shape at the far corners of the Petri dish with the numbers following each other at the corners. 15. Sterilize your inoculating loop by inserting it at the Micro incinerator without touching any sides for 10 second until the wire turn orange then remove the inoculation loop from the micro incinerator and let it cool down for 30 seconds. 16. Open petri dish that has the bacteria unknown B insert your sterile inoculating loop in the petri dish and scoop a small sample, close it return to the incubator. Transfer the scooped bacteria to the labeled petri dish using the zig zag technique. Apply the scooped bacteria at the labeled petri dish number 1 along the line to number 2 using thin zigzag movements. 17. When done drawing the zigzags from 1 to 2, sterilize inoculating loop by inserting it to the micro incinerator for 10 seconds until the wire turn orange and remove it to allow it to cool down for 30 seconds.

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18. Using the sterile inoculating loop, dilute the bacteria by drawing a zigzag movement from number 2 to 3 by touching where the zigzag ended at the number 2 corner and draw along the line to corner number 3. Sterilize inoculating loop by inserting it to the micro incinerator for 10 seconds until the wire turn orange and remove it to allow it to cool down for 30 seconds. 19. Using the sterile inoculating loop, dilute the bacteria further by drawing a zigzag movement from number 3 to 4 by touching where the zigzag ended at the number 3 corner and draw along the line to corner number 4. Sterilize inoculating loop by inserting it to the micro incinerator for 10 seconds until the wire turn orange and remove it to allow it to cool down for 30 seconds. 20. Using the sterile inoculating loop, dilute the bacteria by drawing a zigzag movement from number 4 to half way towards number 1 by touching where the zigzag ended at the number 4 corner and draw along the line midway between 4 and 1 to avoid contamination of the diluted bacteria and we will have achieved colonies. Sterilize inoculating loop by inserting it to the micro incinerator for 10 seconds until the wire turn orange and remove it to allow it to cool down for 30 seconds. 21. Label the petri dish unknown B with P.K initials and place in the incubator facing upside down agar up for 48 hours (Hughes, 2019, “Lactose Lab”). 22. When done, switch off the micro incinerator and remove power cable from the socket putting it away to storage table. Discard appropriately 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. 23. 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. Wait until Label the petri dish # Unknown A&B in a square like way i.e. 1,2,3,4, 41/2 at the 4 corners. Transfer the bacteria from the sample specimen using a sterilized inoculating loop along 1 to 2, 2 to 3, 3 to 4 and from 4 towards 1 but leave it halfway between 1 and 4 using the zigzag technique. Remember to sterilize the inoculating loop in the micro incinerator allowing it to cool as you

draw the zigzag along the labeled numeric lines. When completed place the petri dish upside down in the incubator and sterilize the inoculating loop that was in use. 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. Is MacConkey agar selective and/or differential? If you chose one or both answers, why is it selective and/or differential? Only gram-negative bacterial species can grow on MacConkey agar, which is a selective and differentiating agar that can also distinguish gram-negative organisms based on their lactose metabolism (Jung & Hoilat, 2021). Q2 What happens on the agar when lactose ferments into lactic acid (explain what the reaction is and what happens to the bacterium itself) The pH will decrease as a result of the lactic acid produced by lactose-fermenting microbes. A pH indicator called neutral red changes color from off-white to bright red or pink as the pH falls below 6.8 (Jung & Hoilat, 2022). References

Allen, M. (2005). MacConkey Agar Plates Protocols. American Society for Microbiology. https://asm.org/ASM/media/Protocol-Images/MacConkey-Agar-Plates-Protocols.pdf? ext=.pdf Escherichia coli (E coli) Infections Workup: Laboratory Studies, Imaging Studies, Other Tests. (n.d.). Emedicine.medscape.com. Retrieved October 22, 2023, from https://emedicine.medscape.com/article/217485-workup#:~:text=Most%20E%20coli %20strains%20are Hughes,M.(2019) Lactose Lab Video Www.youtube.com. https://www.youtube.com/watch? v=tIGLHJMx184 Jung, B., & Hoilat, G. J. (2021). MacConkey Medium . PubMed; StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK557394/#:~:text=MacConkey%20agar%20is %20a%20selective Sapkota, A. (2022, July 3). Pseudomonas putida- An Overview. Microbe Notes. https://microbenotes.com/pseudomonas-putida/ VetBact. (2021). Vetbact.org. https://www.vetbact.org/?displayextinfo=57#:~:text=Non %2Dlactose%20fermenting%20bacteria%20(Proteus Welcome to Microbugz - MacConkey Agar . (n.d.). Www.austincc.edu. https://www.austincc.edu/microbugz/macconkey_agar.php#:~:text=MacConkey%20Agar %20(MAC)%20is%20a Yaratha, G., Perloff, S., & Changala, K. (2017). Lactose vs Non-Lactose Fermenting E. coli: Epidemiology, Clinical Outcomes, and Resistance. Open Forum Infectious Diseases, 4(Suppl 1), S589–S590. https://doi.org/10.1093/ofid/ofx163.1546

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Pius Lab 7 Lactose Lab

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