MBIO Lab report 1

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Jan 9, 2024

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Patel 1 Siddhi Patel Ex 8-11 “Soil Microbial Count” Lab Report MBIO 3812-002 September 22, 2023 Differences in Microbial Growth & Diversity Based on Soil Sample Location Abstract Soil samples were collected from two University of Oklahoma Norman campus locations. The standard dilution techniques and standard plate isolation method determined the total colony forming units per mL (CFU/mL). The range of CFU/mL included 1.0 x 10^2 CFU/mL as the lowest and 2.7 x 10^4 CFU/mL as the highest. In soil samples one and two, the trial with the highest microbial count was the simple soil bacteria and the lowest was the fungi. However, sample two had higher counts than sample one. Similar cell morphology was observed in both soil samples. The experiment enabled students to understand essential concepts of microbiology, such as cell morphology, and important lab components used in real-life situations, such as colony isolation, aseptic techniques, and serial dilutions. Introduction Why are soil microbes a vital topic? To start, the microbes found in the soil are ubiquitous. A handful of soil can contain millions, if not billions, of different microbes. The types of microbes found in the soil are bacteria, filamentous fungi, protozoans, and nematodes. These microbes found in the soil play an important role in many different biogeological cycles, such as the nitrogen cycle, sulfur cycle, and carbon cycle, and they are also a key player in the soil food web. Though a handful of soil contains about millions of different types of microbes, only 5% of the soil is composed of microorganisms and macroorganisms. The other components that make up the soil are air and H2O (50%), inorganic material (40%), and organic matter (5%) (1). Many factors influence the type of microbes found in soil, such as temperature, location, soil type, and nutrient availability. Among these factors, the most prominent are soil type and location because they factor into which microbes will grow. Organic matter in the soil will directly correlate to the bacteria in the soil sample, and the highest concentration of organic matter is found in the top twelve inches of soil (2). In this lab exercise, it is hypothesized that the CFC/mL will be more significant in soil sample #2 than in soil sample #1 because of the different conditions of the collection sites. Soil sample #2 was taken from a moist spot with various plant vegetation and animal species. Sample #1 was taken from a construction site that has little to no plant vegetation and no visible animal life present. These factors will be critical in the number of microbial colonies formed in each sample. These microbes will be enumerated from the soil samples using the spread plate methods, also known as serial dilution plates. Serial dilution reduces the concentration of the original soil sample so that we can isolate colonies and calculate the CFU/mL (3). Materials and Methods

Patel 2 The experimental procedure 8-11, “Soil Microbial Count,” was performed as outlined in the Leboffe and Pierce laboratory manual (4) with modifications detailed in the procedural document posted in Canvas (5). For the experiment, the necessary materials included a micropipette (p-1000 uL), microtubes, a microtube rack, a box of sterile tips, a tube of sterile saline, a metal spreader, ethanol, a Bunsen burner, a striker, a sharpie, and soil samples one and two. Soil sample #1 was collected on August 20, 2023, when the temperature was 37.8 Celsius. It was obtained from a construction site with a 4-6 inches depth. Soil Sample #2 was obtained from the OU Duck Pond on August 15, 2023, when the temperature was 24.4 Celsius, and the depth from which it was collected was 4-6 inches. Each sample was then plated on three different plate mediums for different microbes to grow. Nutrient agar was used to promote the growth of simple soil bacteria. In order to promote the growth of Actinobacteria , glucose yeast extract (GYE) agar was used, and sabouraud dextrose (SD) agar was used to support fungi growth. For this exercise, the lab was separated into six bench groups. Three of the six bench groups had soil sample one, each with a different agar medium to conduct the exercise. The other three groups had soil sample two, each with a different agar medium. Before starting the experiment, a few setup steps need to be completed. For example, properly labeling 48 agar plates, 8 for each sample and different agar medium; this also includes the 48 microtubes that go along with it (8 for each sample and different agar ). For each trial, one agar plate and one microtube will serve as a control that needs to be labeled. Each microtube will have 0.9mL of sterile saline aseptically added by each group using the micropipette with a new tip for each one. Once completed, each group will mix 0.1mL of their respective soil sample into the labeled microtube T1 with a brand-new tip. Once microtube T1 has been mixed using a new tip, each group will transfer 0.1 mL of the solution to microtube T2. This step will continue until each group reaches microtube T7 while using a new tip, with each transfer completing a serial dilution. The eighth microtube is the control and will only have 0.9 mL of sterile saline and no soil sample. The following steps will involve the agar plates; each bench group works with a different agar medium. Each microtube will be aseptically transferred to the corresponding labeled agar plate. For example, the control microtube will be transferred into the agar plate labeled control. In between each transfer, the groups will spread the sample to cover the entire surface of the agar plate. This will be done using a metal spreader, a Bunsen burner, and ethanol. The metal spreader will be dipped into the ethanol and set aflame over the Bunsen burner. Hence, the alcohol ignites and, in the process, sterilizes the spreader to avoid any contamination. Once the metal spreader has cooled down, spread the solution until it covers the entire agar plate. Allowing the agar plates to solidify, each group will invert them and allow them to incubate at 25 Celsius for eight days. Once the incubation period has ended, each group will collect the plates they prepared, count the countable colonies, and calculate the CFU/mL, which will be recorded in the datasheet. Each group will pick four prominent colony types, describe the morphology of each one, and record it in the datasheet.

Patel 3 Results Table 1: Morphology descriptions of four prominent colony types in samples one and two agar plates with countable colonies. SOIL SAMPLE 1 Morphological Descriptions of the Four Predominant Colony Types Simple Soil Bacteria 1. flat, raised elevation, small, yellow, irregular margins, circular colony 2. raised, spreading edge elevation, small, brown, round margin, and colony shape 3. convex elevation, large, white, round margin, and colony shape 4. convex elevation, extremely small, opaque with a round margin and colony shape Actinobacteria species 1. raised elevation, filamentous margins, lobate whole colony shape, white 2. convex elevation, smooth margin, smooth whole colony shape, off-white 3. Umbonate elevation, rhizoid margin, smooth whole colony shape, while/off-while 4. flat elevation filamentous margins, irregular whole colony shape, white Fungi 1. White, filamentous, fuzzy, round surface, flat top 2. White in the middle, brown on the edges, round 3. Yellow, small, round, smooth 4. N/A SOIL SAMPLE 2 Morphological Descriptions of the Three Predominant Colony Types Simple Soil Bacteria 1. Flat elevation, smooth margins, round shape, small size, and red color 2. Plateau elevation, filamentous margins, filamentous shape, white but opaque, and big in size 3. Flat elevation, smooth margins, round shape, medium size, and black color 4. Flat elevation, smooth margins, round shape, medium size, and yellow color Actinobacteria species 1. Convex elevation, smooth margins, round shape, medium/large size, and yellow color 2. Flat elevation, smooth margins, round shape, small size, and orange color 3. Raised, spreading edges elevation, rhizoid margin and colonies, large size, and yellow color 4. Flat elevation, smooth margins, round shape, small size, and white/yellow color Fungi 1. circular colony, fuzzy, dark green with white rim, medium/large size 2. small, circular colony, bright yellow, shiny, convex elevation, smooth

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Patel 4 3. small/medium size, circular colony, off-white color, raised, filamentous center, shiny 4. medium/large size, white/cloudy, irregular margin, fuzzy, raised elevation Table 2: Colony results of agar plates containing serially diluted samples of microbes in favored agar mediums. NOTE: * The control plate was switched with plate #7. Also, for soil sample two Fungi trials the tips were not changed after each dilution.

Patel 5 Figure 1: The Concentrations of Simple Soil Bacteria, Actinobacteria , and Fungi growth in two different soil samples. Soil Sample one was taken from a construction site whereas Soil Sample two was taken near the OU Duck Pond. Two of six control inoculations were found, with growth visible. This is because of a mix-up in labeling and new tips not being used in between each dilution, leading to contamination. Each group counted the colonies present on the plates, and then the data was used to calculate the CFU/mL. The formula used is (# of colonies)/ ((dilution factor) x (0.1ml)). Discussion/Conclusion Soil microbes thrive better in soil full of nutrients and closer to water (6). Soil sample two was obtained from an environment with water and more nutrient access than soil sample one. When referred to Figure 1, there was a higher CFU/mL for simple soil bacteria in sample two than in sample one. Whereas the CFU/mL for fungi and Actinobacteria was the opposite. It shows that the difference in amounts of CFU/ml in soil sample one and soil sample two has some discrepancies. This outcome is due to human errors made during the experimental laboratory exercise. These errors include not changing micropipette tips between serial dilutions and mislabeling agar plates. In conclusion, the expected results were not achieved because there were differences in soil samples one and two that needed more consistency. The expected result from this exercise was that the CFU/mL in soil sample two would be greater than in soil sample one. In this experiment, the fungi CFU/mL is not a good representative because no agar plates met the counting rule for colonies and, therefore, are insignificant to the exercise. The data collected did not support the hypothesis. As stated in the introduction, soil sample two would have had more

Patel 6 nutrients due to the conditions from which they were collected, allowing microbial species to thrive. References 1. Meysick K. 2023. Lab #4 Module, Ex 8-11 Soil Microbial Count [Powerpoint]. Retrieved from University of Oklahoma MBIO 3812 Course website https://canvas.ou.edu/courses/291647 . 2. Abdul Rahman NSN, Abdul Hamid NW, Nadarajah K. Effects of Abiotic Stress on Soil Microbiome. Int J Mol Sci. 2021 Aug 21;22(16):9036. doi: 10.3390/ijms22169036. PMID: 34445742; PMCID: PMC8396473. 3. Jove. Culturing and Enumerating Bacteria from Soil Samples. https://www.jove.com/v/10099/culturing-and-enumerating-bacteria-from-soil- samples#:~:text=Soil%20bacteria%20are%20enumerated%2C%20and,resulting%20colo nies%20are%20then%20counted . Website accessed Sep. 19, 2023. 4. Leboffe, Michael, J. and Pierce, Burton, E. 2016, Soil Microbial Count 641-643 in Martins, Marta, R. and Bailey, Rayna, S., Microbiology Laboratory Theory and Application, Morton Publishing Company, USA. 5. Meysick K. 2023. Lab #4 Module, Ex 8-11 Bench Assignment and Procedure [PDF]. Retrieved from University of Oklahoma MBIO 3812 Course website https://canvas.ou.edu/courses/291647 . 6. Miransari, M., 2013. Soil microbes and the availability of soil nutrients. Acta Physiol Plant 35, 3075–3084 (2013). https://doi.org/10.1007/s11738-013-1338-2

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