Pius Lab 4 growing bacteria in a slant

Lab 4 Creating a pure culture by transferring the single, pure colony of bacteria that was previously Gram-stained on an agar slant. Purpose: The purpose of this lab is to aseptically transfer pure bacteria from a colony we grew in the petri dish into an agar slant where they will be generated, tested, isolated and preserved for a longer amount of time (Petersen & McLaughlin, 2018). The purpose of growing bacteria in a slant test tube is to maintain growth of bacteria for long as the cap will maintain moisture preventing it from drying out. (Lehman, 2019) The purpose of using the Zigzag method is to reproduce the pure culture and increase the quantity. Hypothesis: In this lab I expect to successfully transfer pure colony from a petri dish using the Zigzag method to a slant to spread the bacteria. I predict the bacteria will thrive well as Agar slants provide nutrients and wider surface area for bacteria to colonize within a test tube, allowing for the storage of pure cultures for a relatively long period of time. It is possible to retain the pink color of the individual colony, as the bacteria will remain pure. Introduction Agar slant tubes are a type of culture tube that is partially filled with agar, typically nutrient agar. The tube is then allowed to cool, with the agar laid at an angle. This allows for a wide surface area to be utilized for the propagation of a culture (Caprette, 2017). Agar is a gelatinous   substance drawn out from red algae mainly used as a culture medium   for microbiological   work. Nutrient are added into agar to intensify growth. Agar medium is in liquid form when it is put in the test-tube and positioned at an angle to allow agar to cool and solidify resulting to a slanted surface (Lehman, 2018). In order to produce agar slants, it is boiled until it reaches the boiling point. The sample is poured into the test tube. The agar is set on the side of the test tube prior to cooling and solidifying. The test tube can then be stored upright after cooling, and allowed to harden vertically (agar deep) or slant vertically (agar slant) (Lehman, 2018). Agar slants provide a wider surface area for bacteria to colonize within a test tube, allowing for the storage of pure cultures for a relatively long period of time. The medium drying out of agar allows for minimal contamination or loss of culture due to limited exposure to external air, such as dust and other particulate matter, due to the limited volume of air within the tube and the narrow opening, which limits water loss (Caprette,2017). We transfer one single bacterial colony to a slant to obtain a pure culture. We sterile inoculation loop to transfer bacteria to the slant and use zig zag method to spread bacteria in the slant. You can keep the clean slant in the fridge or at room temperature, and it'll be ready to use as soon as the agar hardens (Lehman, 2018). Once the slants have been inoculated, they're put in an incubator to grow (Ahern, 2019). Storing a slant in the fridge can help stop or slow down the growth of bacteria (Laboratory Methods, n.d.) Methods:

Materials Required: 1. Pure bacteria in the Petri Dish (Hughes, 2020, “Simulated Lab Growing Bacteria in a Slant”). 2. Micro incinerator (Hughes, 2020, “Simulated Lab Growing Bacteria in a Slant”). 3. Inoculation loop (Hughes, 2020, “Simulated Lab Growing Bacteria in a Slant”). 4. Masking tape (Hughes, 2020, “Simulated Lab Growing Bacteria in a Slant”). 5. Slant test tube (Hughes, 2020, “Simulated Lab Growing Bacteria in a Slant”). 6. Paper towel. 7. Water. 8. Soap. 9. Spraying bleach solution. 10. Hibiclens. 11. Marker. 12. Latex gloves 13. Mask Procedures : 1. When entering the lab ensure you have your all your hair is 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 hand and 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 slant, pure bacteria in a petri dish from the refrigerator, inoculating loop, micro incinerator from the storage table. 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. 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. 7. Sterilize the slant by taking the cap off the slant tube and holding the cap on your pinky finger pressed against your palm and hold it in that position. Hold the mouth of the slant

incubator to grow. 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. Would your results be different if you did not sterilize your inoculating loop before making the transfer? Why or why not? It's possible that the outcome will different and we can’t get pure colonies. There will be contamination from other microbes in the loop that could mess with the sample. Q2. Why is the agar in the slant, slanted? What functions/purpose does that serve? Agar slants provide a wider surface area for bacteria to colonize within a test tube, allowing for the storage of pure cultures for a relatively long period of time. The medium drying out of agar allows for minimal contamination or loss of culture due to limited exposure to external air, such as dust and other particulate matter, due to the limited volume of air within the tube and the narrow opening, which limits water loss (Caprette, 2017).

References Caprette, D. (2017).  Agar slant tubes . Rice.edu. https://www.ruf.rice.edu/~bioslabs/BIOC318/agar_slants.asp Laboratory Methods . (n.d.). Www.phys.ksu.edu. https://www.phys.ksu.edu/gene/g1.html Lehman, C. (2018).  Five Steps to Prepare Agar Slants . Sciencing. https://sciencing.com/five- steps-prepare-agar-slants-12070149.html Lehman, C. (2018).  What Are Agar Slants?  Sciencing. https://sciencing.com/agar-slants- 8538817.html Lehman, C. (2018).  What Are Agar Slants?  Sciencing. https://sciencing.com/agar-slants- 8538817.html Michelle Hughes. (2021, February 2).  Simulated Lab Growing Bacteria in a Slant  Video  [Video]. YouTube. Retrieved September 18, 2023, from  https://www.youtube.com/watch?v=hQ2P_A2260s Petersen, J., & McLaughlin, S. (2018, December 18).  2.1: Introduction . Biology LibreTexts. https://bio.libretexts.org/Learning_Objects/Laboratory_Experiments/ Microbiology_Labs/Book %3A_Laboratory_Exercises_in_Microbiology_(McLaughlin_and_Petersen)/ 02%3A_Introduction_to_Aseptic_Techniques_and_Growth_Media/ 2.01%3A_Introduction Petersen, J., & McLaughlin, S. (2021, April 29).  2.1: Introduction Growth Media . Biology LibreTexts. https://bio.libretexts.org/Courses/North_Carolina_State_University/ MB352_General_Microbiology_Laboratory_2021_(Lee)/ 02%3A_Cultivation_of_Microbes/2.01%3A_Introduction_Growth_Media