Experiments Meetings

Experiments Day 1

Agenda 1 : Practicing with Micropipettes

Experimental Principle

• Accuracy in micropipette practice is crucial for making the medium correctly without any mistakes.

Materials Needed

1. Micropipette
2. Pipette tips
3. Micro test tubes
4. 600 mL Beaker filled with distilled water
5. Disposal Bin

Procedure

1. Add the correct pipette tip onto the micropipette. Ensure that it is put on tightly and does not fall.
2. Fully press the button on the top of the micropipette and place it into the beaker. Make sure that only the tip comes in contact with the water.
3. Release the button to absorb the liquid. Confirm that the correct amount of water has been absorbed.
4. Take the micropipette out of the water and place it over the micro test tube.
5. Press the button on the top of the micropipette once more and make sure all the water is released.
6. Release the tip of the micropipette into a disposal bin by pressing the button on the side.

Agenda 2 : Preparation of Stock Solutions

Experimental Principle

• Concentrated stock solutions are prepared to create the final modified BG11 medium.
• Nitrogen and phosphorus sources will be eliminated from the sources to ensure PHB production in bacteria.

Materials Needed

1. NaNO3 (Sodium Nitrate)
2. K2HPO4 (Dipotassium Phosphate)
3. MgSO4·7H2O (Magnesium Sulfate Heptahydrate)
4. CaCl2·2H2O (Calcium Chloride Dihydrate)
5. Citric Acid·H2O
6. Ferric Ammonium Citrate
7. Na2EDTA·2H2O (Disodium EDTA)
8. Na2CO3 (Sodium Carbonate)
9. Electronic balance
10. Spatulas
11. Weighing boats
12. 50 mL screw-capped tubes
13. Distilled water
14. 100 mL graduated cylinder
15. Disposal Bin
16. Test tube rack

Procedure

• For each chemical:

1. Add the weighing boat into an electronic balance and zero it.
   
   [Figure 2: Korea-HS Member Getting the Correct Amount of Mass of the Powder Using the Electronic Balance]
2. Add the correct amount of powder onto the weighing boat using a spatula. Make sure the weight is as close to the desired value as possible. If there is any excess material, place it in the disposal bin.
3. Empty all of the powder on the weighing boat into a 50 mL tube.
   
   [Figure 3: Korea-HS Member Emptying Out the Powder Using a Spatula]
4. Label the chemical using a Sharpie. Include the date, name of the chemical, and final concentration.
5. Get 50 mL of distilled water from the water distiller using a 100 mL graduated cylinder.
   
   [Figure 4: Korea-HS Member Getting Distilled Water from the Water Distiller]
6. Pour all of the distilled water in the graduated cylinder into the 50 mL tubes.
   
   [Figure 5: Korea-HS Member Adding 50 mL of Distilled Water into the Test Tube]
7. Close the lid of the 50 mL tube, then constantly flip it for one minute to ensure that it is thoroughly mixed.
8. Place the tube into a test tube rack. Make sure the labeled side is faced forward.
   
   [Figure 6: All of the Stock Solutions Labeled and Placed onto the Test Tube Rack]

Agenda 3: Creating of Modified BG11 Medium

Experimental Principle

• Stock solutions are combined in proportions to create the final culture medium.
• The medium will be prepared without nitrogen and phosphorus to promote PHB production.

Materials Needed

1. Prepared stock solutions
2. Pipette aid
3. Pipette aid tips
4. 500 mL bottle
5. Graduated Cylinder
6. Second test tube rack

Procedure

1. For each stock solution:
   1. From the test tube rack, get one of the stock solutions.
   2. Get a pipette aid, and place a new pipette aid tip onto the edge.
   3.      Using the pipette aid, add 5 mL of the stock solution into the 500 mL bottle.
      
      [Figure 7: Korea-HS Member Getting 5 mL of the Stock Solution using a Pipette Aid]
   4. Replace the pipette tip to avoid contamination.
   5. Place the used test tube onto a separate rack to avoid confusion.
2. Once all the stock solutions are correctly added, fill the rest of the container with distilled water.
3. Label the bottle as “modified BG11 medium.”

Agenda 4 : Measurement of pH

Experimental Principle

• pH is critical for bacterial growth, and therefore we must maintain the pH of the medium at the desired level.

Materials Needed

1. pH meter with probe
2. Calibration solutions
3. NaOH solution
4. Distilled water (for rinsing)

Procedure

1. Remove the pH probe from the electrode storage bottle.
2. Rinse the probe gently with distilled water and use a paper towel to wipe the probe dry.
3. Place the probe into a 4.0 pH calibration solution, press the calibration button on the pH meter, and wait.
4. Once the meter reads 4.0, remove the probe from the solution and repeat step 2.
5. Place the probe into a 7.0 pH calibration solution, press the calibration button on the pH meter, and wait.
6. Once the meter reads 7.0, remove the probe from the solution and repeat step 2.
7. Place the probe into a 10.0 pH calibration solution, press the calibration button on the pH meter, and wait.
8. Once the meter reads 10.0, remove the probe from the solution and repeat step 2.
9. Prepare the liquid-based medium and place the probe inside the medium.
   
   [Figure 9: Image of the pH Probe Placed Inside of the Liquid Medium]
10. Once the pH stabilizes, take note of the pH, and then repeat step 2
    
    [Figure 10: Image of the Final pH of our Liquid Medium Shown on the pH Meter]
11. Place the pH probe back into the electrode storage bottle.

Experiments Day 2

Agenda 5: CO₂ Capture and Conversion using Synechocystis and RuBisCo

Experimental Principle

• Direct Capture and Conversion of CO₂ from the air into 3-PGA using bacteria and the enzyme RuBisCo in the Calvin Cycle.
  • Cultivate the bacteria at a high pH
  • Prepare a solid culture plate
  • Apply the cultured synechocystis
  • Use the streaking method to spread the bacteria to the agar plate with a modified BG11 medium

Materials Needed

1. 1 electronic weighing balance
2. 1 spatula
3. 2 weighing boats
4. 1 200mL measuring cylinder
5. 1 microwave
6. 10 sterile petri plates (1 per person)
7. 1 alcohol lamp
8. 1 timer
9. 2.8g of nutrient agar powder
10. 1 200mL of BG11 solution
11. 1 200μL pipette
12. 1 glass rod
13. 1 ethanol spray
14. 1 glass bottle
15. 10 tissue papers
16. 1 400mL of distilled water
17. 1 centrifuge
18. 1 shaking incubator
19. 1 500mL conical flask

Procedure

1. Agar-BG11 mixture Preparation
   1. Measure 2.8g of agar powder using an electronic weighing balance.
      1. If there are excesses in case of spillage/loss when pouring, discard the excess agar powder after measuring mass into an empty weighing boat.
   2. Pour the 2.8g of agar powder into a separate glass bottle using a spatula to mix.
   3. Measure 200mL of Bg11 medium using a measuring cylinder.
   4. Pour 200mL of the Bg11 medium into the glass bottle that contains the agar powder.
   5. Mix the solution until it seems hazy and shows a yellowish mixture.
   6. Seal the top of the glass bottle with foil (make sure the top is completely covered).
   7. Microwave the solution for 2-3 minutes, stop immediately if the solution boils or overflows.
      1. Repeat this step by stirring the solution in the middle until the solution becomes clear.
   
   [Figure 11: Korea-HS Team Member Pouring 200mL of Modified Bg11 into the Glass Bottle with 2.8g of Agar Powder]
2. Agar Plates Preparation
   1. Label each sterile petri plate with the name, date, colony number, and the start date of culture on the bottom edge.
   2. Wrap the glass bottle with 2-3 tissue papers as it is very hot and to avoid burns.
   3. Pour approximately 15mL (till when the solution fills the whole plate) of molten agar-Bg11 mixture into each sterile Petri plate.
      1. Pour it very carefully so that you avoid creating bubbles.
   4. Partially close the lid (half) and leave it for 15 minutes to solidify under a sterile environment with the alcohol lamp.
      
      [Figure 12: Korea-HS TeamMmember Pouring 15mL of Molten Agar-Bg11 Mixture into the Sterile Petri Plate]
3. Colony Centrifuge
   1. First, place the bacterial culture tubes into a centrifuge at 3500 rpm for 5 minutes to concentrate the bacteria.
   2. Use a pipette set to 200μL to pump the bacteria culture and distribute them evenly by pipetting up and down 5-10 times.
      
      [Figure 13: Korea-HS Team Member Pumping Bacteria Culture with a Pipette to Distribute Them Evenly]
   3. After that, pull out the solution and drop it on the surface of the plate (draw like a circle).
      1. Make sure to remove the pipette tip after use.
   4. Sanitize the glass rod by spraying ethanol 2-3 times and wiping it with tissue paper.
      1. Repeat this every time you use it once for each bacterial culture.
   5. Use a glass rod for streaking, and rub it against the surface of the plate.
      1. Rotate the plate while streaking to ensure even distribution of the bacteria.
      
      [Figure 14: Korea-HS Team Member Rubbing the Glass Rod Against the Surface of the Plate for Streaking]
   6. When complete, close the lid and flip the plate.
      1. Incubate at 30ºC for 1 week - 3-4 plates stacked at once, place tube inside shaking incubator.
      
      [Figure 15: All Sterile Petri Plates Placed in an Incubator Maintained at 30ºC]

Experiments Day 3

Agenda 6 : Development and Optimization of Synechocystis species Strain

Experimental Principle

• Familiarizing with Microbiological Techniques
  • Micropipetting
  • Colony picking
  • Cell imaging
• The primary objective of this experiment is to gain proficiency in the essential microbiological techniques listed above. The attained skills will facilitate the development and optimization of a strain of Synechocystis that can efficiently capture and convert CO₂ from the atmosphere.

Materials Needed

1. Cell Imaging Microscope
2. Bacteria Culture Kit
3. Micropipette
4. Modified BG11 Medium

Procedure

1. Insert a tip at the front of the micropipette.
2. Put the solution in the tube for the bacteria sample.
3. Poke the bacteria 3-4 times and soak it in and out inside the tube using the micropipette to ensure the bacteria is in the tube.
   
   [Figure 16: Kor-HS Team Member Poking the Bacteria with a Micropipette]
4. Inoculate Synechocystis cells into the modified BG11 medium, designed to sustain high pH levels.
5. Periodically sample and streak cells onto solid BG11 medium to isolate single colonies.
6. Select colonies exhibiting altered coloration or other phenotypic changes indicative of potential adaptive mutations.
   
   [Figure 17: Kor-HS Team Member Creating Colonies with a Micropipette]
7. Select the strains that show increased PHB production, identifiable by increased granule formation within cells or using chemical assays that quantify PHB content.
8. Image selected Synechocystis species that can grow in high pH and produce PHB efficiently .
   
   [Figure 18: Kor-HS Team Member Looking into the Microscope to See Synechocystis sp.]

Experiments Day 4

Agenda 6 : Stain Synechocystis cells with Nile Red for visualization of PHB

Materials Needed

1. Synechocystis sp. culture
2. Nile Red stain (Sigma-Aldrich, N3013)
3. Dimethyl sulfoxide (DMSO) or ethanol (for Nile Red stock solution)
4. Phosphate-buffered saline (PBS)
5. Glass slides
6. Coverslips
7. Fluorescent microscope
8. Pipettes and tips
9. Microcentrifuge tubes
10. Vortex mixer

Procedure

1. Preparation of Nile Red Stock Solution
   1. Dissolve Nile Red in DMSO or ethanol to prepare a 1 mg/mL stock solution.
   2. Store the stock solution at room temperature in a dark container to protect it from light.
2. Culture Preparation
   1. Grow Synechocystis cells in a modified BG11 growth medium until they reach the desired density (typically in the exponential growth phase).
   2. Harvest the cells by centrifugation at 5000 x g for 5 minutes.
   3. Discard the supernatant and resuspend the cell pellet in PBS to wash the cells.
   4. Centrifuge again at 5000 x g for 5 minutes and discard the supernatant.
   5. Resuspend the washed cell pellet in PBS to the desired concentration (OD750 of 0.5).
   
   [Figure 19: Kor-HS Team Member Adding a Plate into the Centrifuge]
3. Staining Procedure
   1. Take 1 mL of the resuspended Synechocystis cells in a microcentrifuge tube.
   2. Add 5 µL of Nile Red stock solution to the cell suspension (final concentration of 5 µg/mL).
   3. Vortex briefly to mix.
   4. Incubate the mixture in the dark at room temperature for 10-15 minutes.
   
   [Figure 20: Kor-HS Team Member Inserting Nile Red]
4. Washing
   1. After incubation, centrifuge the stained cells at 5000 x g for 5 minutes.
   2. Discard the supernatant and wash the cell pellet with 1 mL of PBS to remove excess stain.
   3. Centrifuge again at 5000 x g for 5 minutes and discard the supernatant.
   4. Resuspend the cell pellet in 100 µL of PBS for microscopy.
5. Microscopy
   1. Place a small drop (10 µL) of the stained cell suspension onto a clean glass slide.
   2. Gently place a cover slip over the drop.
   3. Observe the stained cells under a fluorescent microscope using appropriate filter settings (excitation at 488 nm and emission at 600 nm).
   4. Capture images.
   
   [Figure 21: Kor-HS Team Member Preparing for Microscopy]
   
   [Figure 22: Kor-HS Team Member Looking into the Stained PHB]

Protocol for Quantifying Synechocystis Cell Number Using a Microplate Reader

Objective : To quantify the number of Synechocystis cells using a microplate reader by measuring optical density (OD) at 750 nm.

Materials Needed

1. Synechocystis sp. culture
2. Phosphate-buffered saline (PBS)
3. 96-well microplate (clear, flat-bottom)
4. Pipettes and tips
5. Microplate reader capable of measuring absorbance at 750 nm
6. Sterile cuvettes (for calibration curve preparation)
7. Spectrophotometer (for calibration curve preparation)

Procedure

1. Preparation of Synechocystis Culture
   1. Grow Synechocystis cells in BG11 growth medium until they reach the desired density (typically in the exponential growth phase).
2. Preparation of Serial Dilutions
   1. Prepare a series of serial dilutions of the Synechocystis culture to create a calibration curve. Typically, prepare dilutions such as 1:2, 1:4, 1:8, 1:16, and so on, using PBS.
   2. Transfer 1 mL of each dilution into sterile cuvettes.
3. Measurement of Optical Density for Calibration Curve
   1. Measure the OD at 750 nm of each dilution using a spectrophotometer.
   2. Record the OD values and corresponding cell concentrations (cells/mL) for each dilution.
   3. Plot the OD values against cell concentrations to create a standard calibration curve.
4. Sample Preparation
   1. Harvest the Synechocystis cells by centrifugation at 5000 x g for 5 minutes.
   2. Discard the supernatant and resuspend the cell pellet in PBS.
   3. Adjust the cell suspension to an appropriate concentration range based on the calibration curve.
5. Loading the Microplate
   1. Transfer 100 µL of each sample and standard (serial dilutions) into the wells of a 96-well microplate. Ensure to include PBS as a blank.
   2. It is recommended to perform each measurement in triplicate for accuracy.
6. Measurement Using Microplate Reader
   1. Measure the absorbance of each well at 750 nm using the microplate reader.
   2. Ensure the microplate reader is set to the correct wavelength (750 nm).
   
   [Figure 23: Placing a Plate into the Quantification Machine]
7. Data Analysis
   1. Subtract the blank (PBS) absorbance value from all sample absorbance values to correct for background absorbance.
   2. Use the calibration curve to convert the corrected OD750 values of the samples to cell concentrations (cells/mL).
8. Calculation
   1. If needed, calculate the total number of cells in your original sample by multiplying the concentration (cells/mL) by the total volume of your culture.

Experiments Day 5

Agenda 7 : PHB Extraction from Synechocystis

Experimental Principle

• The primary objective was to extract PHB from the Synechocystis bacteria.
  • Utilize Synechocystis cells that reached their stationary phase to maximize PHB accumulation
  • Use sodium nitrite to lyse cells and release PHB granules
  • Chloroform was used to dissolve the PHB, allowing it to be extracted.
  • The final product was stored and purified for further use.

Materials Needed

1. Synechocystis culture
2. Chloroform
3. Sodium Nitrate (NaNO2)
4. Ethanol (4 degrees Celcius)
5. Centrifuge
6. Water bath or shaker
7. Glass vials or tubes
8. Micropipettes
9. Micropipette tips
10. Analytical Balance
11. Drying oven or desiccator
12. Protective glass screen

Procedure

1. Cell Preparation
   1. Grow Synechocystis cells under suitable conditions until they reach the stationary phase for higher PHB accumulation.
   2. Harvest the cells by centrifuging the culture at 5000 x g for 10 minutes.
   
   [Figure 24: Korea-HS Team Member Centrifuging the Culture]
2. Cell Lysis
   1. Resuspend the cell pellet in a suitable volume of distilled water.
   2. Add 1% (w/v) sodium nitrite (NaNO₂) to the cell suspension.
   3. Incubate the mixture at 36.5°C for 1-2 hours to facilitate cell lysis and release of PHB granules. Must work quickly to transfer samples into tubes to prevent evaporation.
3. PHB Extraction
   1. After cell lysis, add chloroform to the suspension in a 1:1 ratio (v/v).
      
      [Figure 25: Korea-HS Team Member Adding Chloroform to the Suspension]
   2. Vigorously mix or shake the solution for 30 minutes to allow PHB to dissolve in the chloroform phase. Use a micropipette set to 300 microL to inject chloroform, replacing the tip after each use to avoid damage.
4. Phase Separation
   1. Centrifuge the mixture at 5000 x g for 10 minutes to separate the chloroform layer containing PHB from the aqueous phase.
   2. Carefully collect the bottom chloroform phase using a micropipette. Pull down slowly to avoid disturbing the layers.
      
      [Figure 26: Korea-HS Team Member Removing the Bottom Layer of the Chloroform Phase using a Micropipette]
   3. Remove the top layer of the solution as well.
5. Precipitation of PHB
   1. To precipitate PHB, add an excess of cold ethanol (4°C) to the chloroform extract.
   2. Let the mixture stand at low temperature for several hours
   
   [Figure 27: Kor-HS Team Member Precipitating Ethanol]
   
   [Figure 28: Images of samples after precipitation]
6. Collection and Purification
   1. Centrifuge the mixture again to collect the PHB precipitate.
   2. Wash the precipitate with ethanol or water to remove impurities.
   3. Dry the PHB in a drying oven at 50-60°C or under a vacuum in a desiccator.
7. Quantification
   1. Weigh the dried PHB to determine the yield.
8. Storage
   1. Store the purified PHB in a cool, dry place until further use.

Agenda 8 : Analyzing the Cytotoxicity of PHB on Human Skin Cells

Experimental Principle

• The aim is to assess the cytotoxic effects of extracted PHB on human skin cells.
• PHB samples are placed in a non-toxic solvent to ensure safe exposure to cells.
• The PrestoBlue™Cell Viability Assay is used to measure metabolic activity to show cell heath.
• A dose-response curve is created to find the relationship between PHB concentration and cell viability.

Materials Needed

1. PHB samples
2. Human skin cells
3. Dulbecco’s Modified Eagle Medium (DMEM)
4. Fetal bovine serum (FBS)
5. Penicillin-streptomycin solution
6. Phosphate-buffered saline (PBS)
7. PrestoBlue™ Cell Viability Reagent
8. Trypsin-EDTA solution
9. Incubator (37°C, 5% CO₂)
10. Centrifuge
11. Microplate reader

Procedure

1. Preparation of PHB Samples
   1. Dissolve the PHB in chloroform or DMSO and ensure that the solvent concentration is non-toxic.
   2. Prepare the PHB concentrations by serial dilution in the culture medium and sterilize by filtering through a 0.22 µm filter.
2. Cell Culture
   1. Cultivate human skin cells in DMEM with 10% FBS and 1% penicillin-streptomycin.
   2. Incubate cells at 37 degrees Celcius in a humidified atmosphere with 5% CO₂.
   3. When cells reach 70-80% confluence, detach them using trypsin-EDTA.
   4. Add trypsin using a micropipette to degrade the protein membrane from the cells.
   5. Incubate the cell culture with trypsin for 5 minutes in the CO₂ incubator at 37 degrees Celsius.
   6. Centrifuge the detached cells at 300 x g for 5 minutes.
   7. Resuspend the cell pellet in a fresh culture medium.
   
   [Figure 29: Centrifuging Process of Detached Cells]
3. Seeding Cells in 96-Well Plates
   1. Use a micropipette to see the cells into 96-well plates at a density of 5,000-10,000 cells per well in 100µL of complete medium.
   2. Allow the cells to adhere and grow for 24 hours before treatment.
   
   [Figure 30: Kor-HS Team Member Adding Skin Cells into a 96 Well Plate]
4. Treatment with PHB
   1. Replace the culture medium with 100 µL of fresh medium containing various PHB concentrations.
   2. Include negative control (cells with culture medium only) and positive control (known cytotoxic agent).
   3. Incubate cells with PHB for 24 to 72 hours.
5. PrestoBlue™ Assay for Cytotoxicity
   1. Add 10 µL of PrestoBlue™ reagent to each well.
   2. Gently mix the plate for even distribution.
   3. Incubate at 37°C for 1 to 2 hours (1 hour is typically sufficient).
   
   [Figure 31: Kor-HS Member Adding PrestoBlue into a 96 Well Plate]
6. Measurement and Data Analysis
   1. Measure fluorescence (excitation 560 nm, emission 590 nm) or absorbance (570 nm).
   2. Normalize readings by subtracting background.
   3. Calculate cell viability percentage relative to control.
   4. Plot the dose-response curve and determine IC50 if applicable.
7. Statistical Analysis
   1. Perform ANOVA followed by post hoc tests (e.g., Tukey's test) for multiple comparisons.

Experiments Day 6

Agenda 9 : PHB Cytotoxicity Test Under Modified Lab Conditions

Experimental Principle

• To quantify and analyze the extent of PHB cytotoxicity and synthesis under various conditions fostered through previous labs.
  • Evolutionary selection to identify strains with enhanced PHB production (reverse approach) differentiates the results extracted from conventional methods.
  • PrestoBlue™ Assay was conducted with a spectrophotometer to determine the cytotoxicity.
  • ANOVA test was performed as a statistical test to assess the significance of the results.

Materials Needed

1. Centrifuge
2. Oven
3. Test tubes
4. Electronic balance
5. Incubator
6. Distilled Water
7. PrestoBlue
8. PHB culture medium
9. Micropipettes
10. Micropipettes tips

Procedure

1. Wash each test tube 3 times using 500 microliters of distilled water.
2. Vortex each test tube after each wash to remove impurities.
3. Wash PHB with distilled water.
   
   [Figure 32: Kor-HS Team Member Adding Water to Wash PHB]
4. Centrifuge the PHB in test tubes at maximum speed in order to collect PHB.
5. Measure the weight of the PHB produced from the cells.
   
   [Figure 33: Weighing the Centrifuged Test Tube]
   1. The test tubes were placed in a dry oven to evaporate all water, ensuring accurate weight measurements.
   2. To determine the weight of the PHB, the weight of 4 empty test tubes was first measured and recorded.
   3. Then, PHB was added to each of the test tubes, and the weight of the tubes with PHB was recorded.
   4. The weight of the PHB was obtained by subtracting the weight of the empty tube from the weight of the tube with PHB.
6. Take the image and compare the weight of PHB produced from different conditions.
7. After 24 hours, replace the culture medium with 100 µL of fresh medium containing the various concentrations of PHB.
   1. Include a negative control (cells with culture medium only) and a positive control (cells treated with a known cytotoxic agent).
      1. The negative control provides a baseline measurement for cell viability and growth in the absence of PHB.
      2. The positive control ensures that the assay can detect cytotoxic effects by using a known cytotoxic agent; it validates the sensitivity and reliability of the assay.
8. Incubate the cells with PHB for 24 to 72 hours, depending on the desired exposure time.
9. After the treatment period, add 10 µL of PrestoBlue™ reagent directly to each well containing 100 µL of cell culture medium.
   
   [Figure 34: Kor-HS Team Member Inserting PrestoBlue into a 96 Well Plate]
   1. When adding the reagent, a new micropipette tip was used for each addition to avoid cross-contamination.
   2. The reagent was pipetted gently to the side rather than to the center of the well as it was difficult to sense the depth of the well and therefore minimize the impact of punctures.
   3. The solution (PrestoBlue) is a membrane-permeable chemical. Therefore, when it penetrates the skin cells it creates a reducing condition that turns the blue color into pink.
   4. If the blue color remains, this signifies that there are not a lot of cells (measures for the cell viability) as cells have intact membranes so permeation of the membranes will cause a color change.
   5. Negative control wells (cells with culture medium only) exhibited no color change.
   
   [Figure 35: A Full Image of the 96 Well Plate after having PrestoBlue Assay]
10. Gently mix the plate to ensure even distribution of the reagent.
11. Incubate the plate at 37°C for 30 minutes.
    1. During incubation, the metabolically active cells reduce the PrestoBlue™ reagent, causing a color change.
12. Measure the absorbance between 570 nm and 600 nm (using a spectrophotometer, the wavelengths used were 570 nm and 600 nm)
    
    [Figure 36: 570nm Absorbance Test Results]
    
    [Figure 37: 600nm Absorbance Test Results]
13. Normalize the readings by subtracting the background (e.g., a well with culture medium and PrestoBlue™ but no cells).
    1. Background absorbance (culture medium + reagent without cells) was subtracted from sample readings.
14. Calculate the percentage of cell viability relative to the control (untreated cells) using the following formula:
    1. Cell viability (%) = [(absorbance of sample - background) / [absorbance of untreated control - background)] * 100
15. Plot the cell viability against the concentration of PHB to generate a dose-response curve. If applicable, determine the IC50 value (the concentration of PHB that reduces cell viability by 50%).
16. Perform statistical analysis to assess the significance of the results, typically using ANOVA followed by post hoc tests (e.g., Tukey's test) for multiple comparisons.