Oct 21, 2021
Growth Curves of S. elongatus under salt stress and high carbon
- Akashdutta 1
- 1IISER Pune
- iGEM IISER Pune India 2021
Protocol Citation: Akashdutta 2021. Growth Curves of S. elongatus under salt stress and high carbon. protocols.io https://dx.doi.org/10.17504/protocols.io.bzc2p2ye
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: October 21, 2021
Last Modified: October 21, 2021
Protocol Integer ID: 54394
Keywords: distinct phases of growth, typical bacterial growth curve, growth curve, such growth curve, growth curves for certain freshwater, reduced growth rate, growth rate, slow growth, optimal growth rate, initial low cell density, growth, cells over time, sucrose production in the particular strain, exponential phase, net loss of viable cell, cell population, death of cell, death phase, cell division, mean generation time, dilution of exoenzyme, trends in growth, stationary phase, sucrose production, viable cell, bacteria, lack of growth, lag phase, cell, death rate, number of cell, strain, dilution, changes in the optical density, physiological adaptation of cell
Abstract
When liquid media is inoculated with bacteria and the cell population is counted at intervals, it is possible to plot a typical bacterial growth curve that shows the increase in the number of cells over time. Such growth curves show four distinct phases of growth:1
Lag phase: There is slow growth or lack of growth due to the physiological adaptation of cells to culture conditions or the dilution of exoenzymes (due to initial low cell densities).
Log or exponential phase: Optimal growth rates are seen in this phase. Cell numbers double at discrete time intervals known as the mean generation time.
Stationary phase: During this phase, the growth (cell division) and death of cells occur at the same rate, resulting in the number of cells being constant. The reduced growth rate is usually due to a lack of nutrients and/or a buildup of toxic waste constituents.
Decline or death phase: Here, the death rate exceeds the growth rate, resulting in a net loss of viable cells.
This is one of the simplest methods used to analyze trends in growth because it uses a spectrophotometer to track changes in the optical density (OD) over time. In other words, as the number of cells in a sample increases, the transmission of light through the sample will decrease.2
Growth curves for certain freshwater cyanobacterial species are carried out under salt stress to account for sucrose production in the particular strain. This is because sucrose is naturally produced intracellularly in these strains to balance the osmotic pressure of a saline environment.3
This experiment was carried out in two iterations: one at 0.04% of carbon dioxide - i.e, ambient carbon dioxide from the atmosphere - and the other with bicarbonate added to be equivalent to 0.6% of carbon dioxide. Find our results for these iterations here.
Troubleshooting
Grow 100 mL of culture in a 250 mL flask in CoBG-11 medium.
To this add bicarbonate equivalent to 0.6% carbon dioxide., or 2 g/L of sodium bicarbonate in CoBG-11
12 hours later, take 3 1 mL samples from each flask in 2 microcentrifuge tubes
Use one set of samples to take the OD measurements
Take the other set of samples and centrifuge at 4000 rpm, 25°C, 00:07:00
7m
Take the supernatant in fresh microcentrifuge tubes and store it at -20 °C , for later analysis. Discard the pellets.
Take the readings every twelve hours until the OD730 value reaches 1.0
Add the bicarbonate at regular intervals of initially once every 24 hours, increasing the frequency if the culture turns a dull yellow.