MBIO Lab Report #2

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The University of Oklahoma *

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3812

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Biology

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Jan 9, 2024

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Siddhi Patel MBIO 3812-002 October 27, 2023 Vibrio natriegens: Growth Conditions Under Different Osmotic Pressures Abstract Different bacterial species thrive in various environmental conditions. Environmental conditions are impacted by changes in pH, temperature, and osmotic pressure. It is crucial to understand a bacteria's optimal environment condition to know how to control the growth of bacteria species. A closed-system growth method was used with media consisting of brain heart infusion broth (BMI) and four different salt concentrations inoculated with Vibrio natriegens. The absorbance (A) was measured using a spectrophotometer at 600 nm. Then, McFarland standards generated in the first part of the experiment helped determine the bacterial concentration. Results pointed to the optimal growth condition for Vibrio natriegens at BMI with 250 mM salt at 37 Celsius. The experiment enabled students to understand essential concepts of microbiology, such as reading closed-system growth curves, and important lab components used in real-life situations, such as bacterial cell concentration calculations, aseptic techniques, and generation time calculations. Introduction There are many types of bacteria, those that are pathogenic and those that are not. Many species of bacteria in the world replicate using binary fission, allowing them to generate copies of themselves quickly (5). The doubling time of bacteria varies from twenty minutes to an hour, depending on their environmental conditions (5). Bacteria are ubiquitous and can be found in different habitats and, therefore, can survive and grow under various environmental conditions. Environmental conditions such as pH, temperature, and osmotic pressures can affect the growth of bacteria (1). It is essential to know what conditions bacteria prefer to stop the spread of bacteria. In this experiment, we will observe Vibrio natriegens, a non-pathogenic bacterium known to be a moderate halophilic (3). Halophilic means it can grow in or tolerate saline conditions (8). When a bacterium is halophilic, it cannot thrive in any salt-concentrated condition. Only specific concentrations are favored. Osmotic pressure is what can be affected by the concentration of salt, which then can affect the bacteria’s growth and survival because the salt induces a level of stress that most bacteria cannot deal with (1). This experiment will use Vibrio natriegens to monitor the effects of osmotic pressure in a closed-system growth. A closed-system growth is when there is no addition of nutrients or subtraction of waste from the system itself. When conducting a closed-system growth, four distinct phases can be observed. These four phases are lag, log, stationary, and death, conveying a different stage of the bacteria’s life cycle. With the four phases noticed, we can use the data to determine the optimal conditions for the generation time of a bacteria, in this case, Vibrio natriegens. It is hypothesized that in this exercise, Vibrio natriegens’ optimal growth condition will be observed in BHI with 250 mM salt concentration at 37 Celsius. This is because Vibrio natriegens is a moderate halophile, which leads to the assumption that it can tolerate salt conditions but does not prefer it; therefore, 250 mM is closer to the bacterium’s favored environmental condition.

Materials and Methods The experimental procedure 6-4, “Closed-System Growth,” was performed as outlined in the Leboffe and Pierce laboratory manual (2) with modifications detailed in the procedural document posted in Canvas (6). Along with the procedure outline in the handout received in the lab for the supplemental exercise “McFarland Standards and the Spectrophotometer” was followed. For the experiment, the necessary equipment included a micropipette (p-1000 uL), a box of sterile tips, a spectrophotometer, cuvettes, a cuvette filled with water, and cuvettes with the McFarland Standards, an incubator set at 37 Celsius shaking, and Vibrio natriegens in 50 mL brain heart infusion broth (BMI) with four different salt concentrations. The four different salt concentrations were 0 mM, 50 mM, 250 mM, and 1000 mM. To begin the experiment, obtaining the McFarland Standard Curve was necessary. Each pair went and set the spectrophotometer to measure the absorbance (A) at 600 nm and then blanked the spectrophotometer with a cuvette filled with water until the machine read 0.0 before moving on. Each person then continues to measure the A600 of each McFarland standard and record it as raw data. Once completed, the pair averages the readings for each McFarland standard and creates a standard curve. The second part of the experiment is performed as a whole class with their partners. The entire class was divided into four groups and assigned a different BMI plus salt concentration. The BMI flasks were assembled before the experiment and inoculated with 1.5 mL of turbid culture of Vibrio natriegens and its designated salt concentrations. The Vibrio natriegens flask, and its assigned salt concentration were incubated at 37 Celsius, shaking every twenty minutes to keep it turbid. Every five minutes, each pair would go to the back and blank the spectrophotometer with regular BMI broth. Once completed, 1 mL of the culture would be transferred into the cuvette, and the reading would be recorded on the data sheet provided for the whole class.

Results Figure 1: McFarland standard curve graph generated using values from Table 1. These standards were used in the part 2 of the experiment for calculating bacterial concentrations for each sample and it corresponding time. # Bacteria/mL Average A 600 1.5 x10 8 0.085 3.0 x10 8 0.135 6.0 x10 8 0.255 9.0 x10 8 0.29 1.2 x10 9 0.46 Table 1: Average A 600 obtained from McFarland standard numbers at different # Bacteria/mL. y = 3E-10x + 0.0337 R² = 0.9646 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.00E+00 2.00E+08 4.00E+08 6.00E+08 8.00E+08 1.00E+09 1.20E+09 1.40E+09 Average Absorbance (A) at 600 nm Bacterial Concentration (# bacteria/mL) McFarland Standard Curve

Time (min) OD 600 # Bacterial cell/mL 0 0.03 1.12 x10 8 20 0.02 1.12 x10 8 40 0.01 1.12 x10 8 60 0.02 1.12 x10 8 80 0.01 1.12 x10 8 100 0.04 1.12 x10 8 120 0.02 1.12 x10 8 140 0.02 1.12 x10 8 160 0.01 1.12 x10 8 180 0.04 1.12 x10 8 200 0.04 1.12 x10 8 220 0.03 1.12 x10 8 240 0.03 1.12 x10 8 Table 2: Absorbance (A) reading of Vibrio natriegens in BMI broth with 0 mM salt at 37 Celsius and calculated bacterial concentrations of each sample. Time (min) OD 600 # Bacterial cell/mL 0 0.02 1.12 x10 8 20 0.01 1.12 x10 8 40 0.05 1.12 x10 8 60 0.06 1.12 x10 8 80 0.03 1.12 x10 8 100 0.03 1.12 x10 8 120 0.02 1.12 x10 8 140 0.01 1.12 x10 8 160 0.01 1.12 x10 8 180 0.01 1.12 x10 8 200 0 1.12 x10 8 220 0 1.12 x10 8 240 0.01 1.12 x10 8 Table 3: Absorbance (A) reading of Vibrio natriegens in BMI broth with 50 mM salt at 37 Celsius and calculated bacterial concentrations of each sample.

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