Project Galileo – Biology
The Effect of Sodium Chloride Concentration on the Growth Rate of Aliivibrio fischeri over 24 Hours
Toshniwal Remi Ameya Ritesh (25)
Groupmate: Tan Yan Zu Julian (24)
St Joseph’s Institution 2026, Class ML403
Word count: 1579
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ABSTRACT
This experiment aims to investigate how varying sodium chloride (NaCl) concentration affects the growth rate of Aliivibrio fischeri, a bioluminescent marine bacterium. A. fischeri naturally inhabits marine environments with fluctuating salinity levels, including the light organs of the Hawaiian bobtail squid (Euprymna scolopes), where it engages in quorum sensing, a cell to cell communication process through which bacteria regulate gene expression based on population density. As cell density directly influences quorum sensing and bioluminescence, understanding the effect of salinity on bacterial growth is ecologically significant. NaCl concentration will be varied from 0% to 5% w/v, and growth rate will be calculated from OD600 readings taken using a spectrophotometer at t \= 0 h and t \= 24 h.
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INTRODUCTION
2.1 LITERATURE REVIEW
Aliivibrio fischeri is a bioluminescent marine bacterium found in seawater and in the light organs of marine animals, most notably the Hawaiian bobtail squid (Euprymna scolopes) (Visick et al., 2021).
In its natural environment, A. fischeri does not experience constant salinity. Coastal and estuarine habitats where the bacterium is found are subject to salinity fluctuations driven by rainfall diluting surface seawater, and river runoff reducing coastal salt concentrations (Soto et al., 2009). The bacterium also transitions between seawater and the interior of its squid host (Visick et al., 2021), two environments with different ionic compositions. Salinity therefore represents a key abiotic factor shaping where and how well A. fischeri can survive and grow.
When salinity rises sharply, cells experience hyperosmotic stress. Water exits the cell by osmosis, causing the cytoplasm to shrink, directly limiting the bacterium's ability to grow and reproduce (Soto et al., 2009; Csonka, 1989). Conversely, very low salinity creates hypotonic conditions in which excess water enters the cell by osmosis, risking osmotic lysis (Csonka, 1989). Both extremes impair growth, making the tolerated salinity range an ecologically meaningful boundary.
Population density is the crux of quorum sensing, a form of bacterial communication. Once autoinducer concentration crosses a threshold, the lux operon is activated, switching on bioluminescence as a coordinated population response (Nealson et al., 1970). Since growth rate governs how quickly that density threshold is reached, salinity indirectly controls whether and when bioluminescence occurs.
Despite the link between salinity, growth rate, and bioluminescence, most laboratory studies culture A. fischeri in standard media containing a fixed sodium chloride concentration of approximately 2% w/v (Christensen & Visick, 2020), and the effect of systematically varying NaCl on growth rate has not been comprehensively studied across the range the bacterium encounters in nature. This investigation examines how NaCl concentrations of 0%, 1%, 2%, 3%, 4%, and 5% w/v affect the growth rate of A. fischeri over 24 hours, measured via optical density at 600 nm (OD600). The findings will establish the salinity conditions that support optimal bacterial growth and determine how salinity shapes the conditions under which quorum sensing and bioluminescence occur.
2.2 RESEARCH QUESTION
How does increasing sodium chloride concentration (0%, 1%, 2%, 3%, 4%, and 5% w/v) affect the growth rate of Aliivibrio fischeri when cultured over 24 hours?
2.3 HYPOTHESIS
The higher the NaCl concentration, the higher the growth rate of A. fischeri, up to an optimal concentration of approximately 2–3% w/v, beyond which growth rate decreases as NaCl concentration increases.
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METHODOLOGY
3.1 VARIABLES
3.1.1 Independent Variable
The independent variable is NaCl concentration, measured in % w/v. Percentage weight per volume (% w/v) expresses the mass of NaCl in grams dissolved per 100 mL of solution, calculated as
% w/v \= (mass of NaCl (g) / volume of solution (mL)) × 100
NaCl concentration is varied by preparing six batches of Lysogeny Broth (LB), a standard nutrient medium containing tryptone and yeast extract, without NaCl in the base. A calculated mass of NaCl is dissolved into each batch separately to reach the target concentration. The six concentrations used are 0%, 1%, 2%, 3%, 4%, and 5% w/v. This range spans from low salt conditions (0%) through typical seawater salinity (\~3.5% w/v) to above marine conditions (5%) (Webb, 2021). The 1% w/v interval produces six evenly distributed data points sufficient to resolve a non-linear growth rate curve. The mass of NaCl required for each batch is calculated as
mass of NaCl (g) \= (% w/v / 100) × volume of solution (mL)
3.1.2 Dependent Variable
The dependent variable is the mean growth rate of A. fischeri over 24 hours, denoted μ and expressed in h⁻¹. Growth rate represents how quickly cell density increases over time and is calculated as
μ \= ln(OD₂₄ / OD₀) / 24
where OD₀ is the optical density at 600 nm (OD600) measured immediately after adding the bacteria to each flask (t \= 0 h), OD₂₄ is OD600 measured after 24 hours of incubation (t \= 24 h), and 24 is the incubation duration in hours.
Before each reading, the spectrophotometer is zeroed using the medium with no bacteria at the same NaCl concentration to remove any light absorbance caused by NaCl. The experiment is repeated three times per NaCl concentration, giving 18 flasks in total.
If OD₂₄ exceeds 0.8, the sample is diluted with the medium of the same NaCl concentration before reading, and the measured OD600 is multiplied by the dilution factor to obtain the true value of OD₂₄.
The formula assumes net exponential growth over 24 hours and since only two readings are taken, it gives an average growth rate over 24 hours rather than the actual growth rate at any specific point in time. This is acknowledged as a limitation of this investigation.
3.1.3 Controlled Variables
| Controlled Variable | Method of Control | Reason for Control |
|---|---|---|
| Temperature | All flasks incubated at 30°C in a temperature-controlled incubator throughout the 24 hour period | If temperature varies between flasks, differences in OD₂₄ would reflect thermal effects on metabolic rate rather than the effect of NaCl concentration |
| Initial cell density | One starter culture is prepared and its OD600 measured on the day of the experiment. The volume added to each flask is calculated using V \= 2.5 / ODstarter to achieve OD₀ \= 0.05 across all 18 flasks. | Unequal starting densities mean flasks may be at different stages of growth at t \= 0 h, causing differences in OD₂₄ that reflect initial density rather than NaCl concentration |
| Bacterial strain | All cultures use A. fischeri strain ES114 throughout | Different strains may differ in salt tolerance |
| Culture volume | 50 mL dispensed into each 250 mL conical flask using a 50 mL measuring cylinder | Keeps the amount of air above the medium the same in each flask, ensuring equal oxygen availability across conditions |
| pH of medium | Measured with a calibrated pH meter and adjusted to 7.0 ± 0.1 using 0.1 M NaOH or 0.1 M HCl before the bacteria are added | pH affects enzyme function and membrane transport independently of NaCl concentration |
Table 1: Controlled Variables
One variable that cannot be fully controlled is dissolved oxygen. Since cultures are not physically shaken, oxygen enters the medium by diffusion from the air above only. In the setups where A. fischeri grows faster, the bacteria will consume oxygen more rapidly, potentially limiting growth earlier.
3.2 MATERIAL LIST
| Material / Equipment | Quantity | Notes |
|---|---|---|
| A. fischeri strain ES114 stock | 1 plate | Stored on marine agar at 4°C |
| Tryptone | 25 g | Component of LB medium |
| Yeast extract | 12.5 g | Component of LB medium |
| NaCl | 50 g | For preparing six NaCl concentrations |
| Distilled water | 2 L | For preparing all media and solutions |
| Marine agar plates | 2 | For maintaining ES114 stock between uses |
| 500 mL conical flasks | 6 | One per NaCl condition for batch preparation |
| 250 mL conical flasks | 19 | 18 for cultures (3 replicates × 6 conditions), 1 for starter culture |
| Mass balance | 1 | For weighing NaCl, tryptone, and yeast extract |
| Spectrophotometer | 1 | For OD600 readings |
| Cuvettes | 1 | Rinse thoroughly between readings |
| Temperature-controlled incubator | 1 | Set to 30°C |
| Autoclave | 1 | For sterilising medium at 121°C for 15 minutes |
| Calibrated pH meter | 1 | For measuring and adjusting medium pH |
| 50 mL measuring cylinder | 1 | For dispensing 45 mL into each culture flask |
| 25 mL burette | 1 | For adding starter culture |
| 1 mL pipette | 1 | For transferring samples |
| Inoculation loop | 1 | For transferring bacteria from stock plate to starter culture |
| 0.1 M NaOH | 100 mL | For raising pH if needed |
| 0.1 M HCl | 100 mL | For lowering pH if needed |
| Test tubes with caps | 6 | For storing reference medium set aside before bacteria are added |
| Marker pen and labelling tape | 1 each | For labelling flasks |
| Disposable gloves | 1 box | Personal protective equipment |
| Lab coat | 1 | Personal protective equipment |
| 70% Ethanol solution | 100 mL | For wiping surfaces |
| Paper towels | 1 roll | For wiping surfaces after applying ethanol |
Table 2: Materials List
3.3 PROCEDURE
- For each NaCl concentration, weigh out the corresponding mass of NaCl from Table 3 into a separate 500 mL conical flask. Add 2.5 g of tryptone and 1.25 g of yeast extract to the same flask.
| NaCl Concentration (% w/v) | Mass of NaCl per 250 mL batch (g) |
|---|---|
| 0 | 0 |
| 1 | 2.5 |
| 2 | 5.0 |
| 3 | 7.5 |
| 4 | 10.0 |
| 5 | 12.5 |
Table 3: Mass of NaCl required per 250 mL batch at each target concentration
- Add approximately 200 mL of distilled water to each flask and stir until all solids are fully dissolved.
- Top up each flask to 250 mL with distilled water.
- Measure the pH of each batch using a calibrated pH meter. Add 0.1 M NaOH drop by drop to raise pH or 0.1 M HCl drop by drop to lower it until each batch reads 7.0 ± 0.1.
- Stopper each flask loosely and autoclave all six at 121°C for 15 minutes.
- Allow all flasks to cool to room temperature.
- Once cooled, pour approximately 10 mL of each medium into a capped test tube labelled with its NaCl concentration. Set these aside as reference samples.
Carry out steps 8 to 12 near a flame to reduce the risk of contamination.
- Using a sterile inoculation loop, transfer A. fischeri strain ES114 from the stock plate into a 250 mL conical flask containing 150 mL of LB medium at 2% w/v NaCl.
- Incubate the starter culture overnight at 30°C. The following day, measure the OD600 of the starter culture using the spectrophotometer. Record this value as ODstarter.
- Label 18 conical flasks (250 mL) with their NaCl concentration and replicate number (R1, R2, R3).
- Using a 50 mL measuring cylinder, measure 45 mL of the corresponding medium and transfer it to each labelled flask.
- Calculate the volume of starter culture to add to each flask as
volume (mL) \= 2.5 / ODstarter
Using a 25 mL burette, add this volume to each labelled flask, then top up each flask to 50 mL with its corresponding medium and gently swirl to mix.
- Immediately after step 12, take an OD600 reading from each flask.
- a. Zero the spectrophotometer using the reference sample from the same NaCl condition.
- b. Transfer 1 mL from the flask into a clean cuvette using a 1 mL pipette.
- c. Record the OD600 as OD₀.
- Place all 18 flasks in the incubator set to 30°C and leave undisturbed for 24 hours.
- After 24 hours, repeat steps 13a to 13c for each flask and record the OD600 as OD₂₄.
- If any reading exceeds 0.8, dilute the sample with the reference medium of the same NaCl condition, record the dilution factor, take the reading, then multiply by the dilution factor to obtain the true OD₂₄.
- For each flask, calculate growth rate using μ \= ln(OD₂₄ / OD₀) / 24.
- Calculate the mean μ across the three replicates for each NaCl concentration.
- Autoclave all 18 culture flasks at 121°C for 15 minutes before disposal.
3.4 RISK ASSESSMENT
3.4.1 Safety Considerations
A. fischeri strain ES114 is not known to cause infection in healthy individuals (Christensen & Visick, 2020). All bacterial transfers are carried out near a flame using aseptic technique, and disposable gloves and a lab coat are worn throughout to prevent skin contact. All work surfaces are wiped with 70% ethanol solution before and after handling bacteria.
The autoclave operates at 121°C. 0.1 M NaOH and 0.1 M HCl are mildly corrosive and can cause skin and eye irritation. Gloves are worn during all pH adjustment steps, and any spills are immediately diluted with water and wiped clean.
3.4.2 Environmental Considerations
The primary environmental concern is improper disposal of live *A. fischeri*. If living bacteria are disposed of without prior deactivation, they could establish in water bodies and disrupt native ecosystems. To prevent this, all 18 culture flasks and the starter culture flask are autoclaved at 121°C for 15 minutes before disposal, killing the bacteria cells.
3.4.3 Ethical Considerations
This investigation does not involve human subjects or vertebrate animals and therefore does not have ethical concerns.
4. BIBLIOGRAPHY
Christensen, D. G., & Visick, K. L. (2020). Vibrio fischeri: Laboratory cultivation, storage, and common phenotypic assays. Current Protocols in Microbiology, 57(1), e103. https://pmc.ncbi.nlm.nih.gov/articles/PMC7337994/
Csonka, L. N. (1989). Physiological and genetic responses of bacteria to osmotic stress. Microbiological Reviews, 53(1), 121–147. https://journals.asm.org/doi/10.1128/mr.53.1.121-147.1989
Nealson, K. H., Platt, T., & Hastings, J. W. (1970). Cellular control of the synthesis and activity of the bacterial luminescent system. Journal of Bacteriology, 104(1), 313–322. https://pmc.ncbi.nlm.nih.gov/articles/PMC248216/
Soto, W., Gutierrez, J., Remmenga, M. D., & Nishiguchi, M. K. (2009). Salinity and temperature effects on physiological responses of Vibrio fischeri from diverse ecological niches. Microbial Ecology, 57(1), 140–150. https://pmc.ncbi.nlm.nih.gov/articles/PMC2703662/
Visick, K. L., Stabb, E. V., & Ruby, E. G. (2021). A lasting symbiosis: How Vibrio fischeri finds a squid partner and persists within its natural host. Nature Reviews Microbiology, 19(10), 654–665.
https://pmc.ncbi.nlm.nih.gov/articles/PMC8529645/
Webb, P. (2021). Salinity patterns. In Introduction to oceanography. Roger Williams University.
https://rwu.pressbooks.pub/webboceanography/chapter/5-3-salinity-patterns/