Bacterial Colony Counting
Introduction
Colony counting is used to obtain a count of living cells to observe growth and survival between a control group and an experimental group. The experimental group in this series was treated with hydrogen peroxide to induce oxidative stress as a means to analyze the impact on the growth of Deinococcus radiodurans and Deinococcus aquaticus. Deinococcus radiodurans is expected to have more growth than Deinocococcus aquaticus due to D. radiodurans being an extremophile, which is considered more robust and more resistant to oxidative stress.
Materials & Procedures
There are several materials and procedures used in preparation
for counting colonies. The materials are
as follows: Two broths; one broth made of TGY (tryptone, yeast extract, and
glucose) that was cultured with Deinococcus radiodurans. The other broth was also made of TGY that was cultured with Deinococcus aquaticus. A centrifuge, to separate cells from the
broth Potassium Phosphate buffer to remove excess glucose from the cells as this
compound could impact the growth of the cells as a food source. Hydrogen peroxide,
to induce oxidative stress. Spinner tubes, to use in the centrifuge. Six Petri dishes, disposable inoculation
loops, a pipette, an agar plate, and a vortexer.
To prepare for the centrifuge process, take original tubes with
cultured cells that have been treated and untreated and place them in the labeled
spinner tubes. All tubes that have been
labeled with a 2 have been treated with hydrogen peroxide. For example, the contents in the tube labeled
A2 were treated with. All tubes labeled with
a one were not treated, and are considered the control of the experiment. The
centrifuge speeds are at about 6.5 thousand rpm. The tubes will spin for a total of 5 mins in intervals
of 90 seconds and sit on ice for 1 minute.
This interval is used to protect the proteins within the cells from
denaturing through heat exposure.
Once the cells have been separated from the TGY broth, there will be a small amount of cells at the bottom of each tube. Remove the cells from the broth and add 500 microliters of potassium phosphate buffer. This is used to clean the cells of excess glucose and other elements within the TGY broth. Using a pipette siphon the cells and the buffer in and out of the pipette tip to flush the potassium phosphate buffer through the cells.
Once the final tubes have been homogenized, use a pipette to add diluted solution to an agar plate. Pipette 10 microliters in each quadrant. Then use the inoculation loop to spread the solution within the quadrant, using only one inoculation loop per quadrant. Do not use the same loop for any other quadrants, this is to control the concentration within each quadrant to account for any error that may have occurred during the dilution process.
After the agar plates have been incubated, there is growth of colonies in each plate. Now, to get the colony forming units (CFU), the colonies that have been grown need to be counted. Using a pen and a counter, mark each colony counted on the dish and use the counter to keep track. Make a diagram to show how many colonies grew in each quadrant. Then, take the average of the colonies of each plate. CFU/mL is calculated by the average number of colonies multiplied by the final dilution factor all divided by the volume used to culture in milliliters.
Results
According to the CFU calculations the D. radiodurans that were untreated had the most growth. However, the D. radiodurans that was treated had the least amount of growth. The treated D. aquaticus had the second largest amount of growth. It is unclear in the results if D. radiodurans is maintaining its composition in the state of oxidative stress or whether D. aquaticus is growing due to radical behavior within the cell.
Nice work! I've also seen CFU counting apps to make life a little easier, but the accuracy always seemed a little questionable.
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