Deinococcus radiodurans Titering: Colony Counting Assay

 

A Summary of Bacterial Titration

 October 23rd, 2021

Introduction

The radiation-resistant bacterium Deinococcus radiodurans (D. radiodurans) has many biological advantages that allow it to thievingly grow under oxidative stress, UV radiation, desiccation and other stressful conditions, and this has made this bacterium the interest of multiple scientists and the subject of investigation of many research projects (Gerber et al, 2015). It is known that D. radiodurans "utilizes the pentose phosphate pathway (PPP) to aid in its survival and repair during oxidative stress" (Shawn S). In addition, the PPP also helps generate NADPH, a molecule used in anabolic reactions because it acts as an antioxidant, and it fights the reactive oxygen species that are made when the bacterium is under stress.* 

For the purpose of this lab, hydrogen peroxide is used to induce oxidative stress to then measure bacterial growth patterns. In the past, experiments were conducted to test for the amount of hydrogen peroxide suitable for increasing bacterial growth without killing the cells; the amounts of 25mM, 50mM and 1M were used. In all cases, the control D. radiodurans cultures (the ones with only sterile water added) grew in larger amounts than the treated D. radiodurans cultures (the ones with added water). This means that the amounts used caused more apoptosis (cell death) than growth. Since the results were consistent for all three amounts, experiments had to be redone to achieve more concrete results. During last week's lab, the test was to monitor anew whether 50 mM of hydrogen peroxide was enough to induce optimal growth of the D. radiodurans bacterium on a growth dish without causing apoptosis, and this was done through the colony counting assay. Colony counting aids in the calculation of the D. radiodurans titer that is then used to conclude whether the amount of hydrogen peroxide used induced or inhibited bacterial growth. 

Discussion

D. radiodurans titering was done in two different ways, microplate reading assay and colony counting assay, the method that will be described is the colony counting assay. The growing of D. radiodurans started by culturing it in a broth medium of necessary ingredients called TGY media. To do this, a culture of concentrated D. radiodurans previously grown in lab was inoculated into a fresh broth medium in two separate tubes then grown over two nights. The growth was measured after 48 hours by checking for broth opaqueness. Also, the cell density was measures using a NanoDrop Spectrophotometer. The results confirmed that the culture was ready to be treated with hydrogen peroxide. The next step includes adding 51 microliters of a 50 mM concentration of hydrogen peroxide to the broth culture of D. radiodurans in one of the tubes and the same volume of water to the control tube. An hour of wait time followed to allow the hydrogen peroxide to function as oxidative stress factor. Then, a series of dilutions were performed to aid in getting separate cultures when plating the bacteria on Petri dishes. Consequently, the two D. radiodurans broth cultures were plated on two separate plates labeled T. D. rad for treatment and C. D. rad for control. The plating followed the lawn method, where 10 microliters of each broth culture was distributed evenly on the corresponding Petri dish for the treatment and control. The plates were then incubated at 28 degree Celsius for two nights. After about 48 hours, the growth colonies of the treatment and control D. radiodurans were viewed and counted for a titer.  

Results and Conclusion

The D. radiodurans control and treatment colonies looked like the following: 

As visible, the control D. radiodurans colonies which were grown from a broth culture with just water added look more numerous than the treatment D. radiodurans colonies. To illustrate, the colony counting assay demonstrated that about 127 colonies appeared in one quadrant of the control Petri dish while only about 25 colonies appeared in one quadrant of the treatment Petri dish. The colony-forming unit (CFU), which denotes the titer of the D. radiodurans cells, was then determined for each dish by dividing the number of colonies in a single quadrant by the amount of broth culture used in each dish and multiplying that by the dilution amount. Calculation are as follows:

CFU C. D. rad: (127/0.01 ml) × 40,000 = 5.08E8 CFU/ml
CFU T. D. rad:  (25/0.01 ml) × 40,000 = 1.01E8 CFU/ml

The results obtained by comparing the titers still indicated that the control D. radiodurans grew in larger amounts than the treatment D. radiodurans. This means that, if all went well, 50 mM of hydrogen peroxide induced enough stress to result in cell apoptosis rather than increased growth. This experiment's results are consistent with previous results obtained from different amounts of hydrogen peroxide, so further investigation needs to take place to determine the best concentration of hydrogen peroxide capable of promoting D. radiodurans growth without harming the internal cellular functions. Perhaps other methods for determining the titer, like the microplate reader assay, would yield better results as it might lower margins of error or other variables that might distort the results. D. radiodurans previous growth curves obtained from the microplate reader assay are shown below for reference.

*Basic knowledge about D. radiodurans was learned from my mentor Shawn Soares, and most information in the introduction are in his words; as well as, the growth curves were created by him. 

References

Gerber, E., Bernard, R., Castang, S. et al. (March 2015) Deinococcus as new chassis for industrial biotechnology: biology, physiology and tools. Journal of Applied Microbiology 119, 1-10.