Pure Culturing of Deinococcus radiodurans in Liquid and Solid Media

A Summary of Bacterial Culturing

December 6th, 2021

Introduction

This week's laboratory procedure may be simple and well known to every laboratory scientist or scientist-to-be, but it is still the most commonly executed procedure in lab in addition to it being fairly underrated -- bacterial culturing. There are different types of bacterial cultures, some grown in solid media, others in semisolid while a good majority are grown in liquid media. When it comes to D. radiodurans, it is helpful to obtain both liquid cultures in broth and solid agar plate colonies. Broth cultures ensure larger quantity growth, but do not show the characteristics of the grown microorganism. On the other hand, growing microorganisms on agar plates shows diversity and certain characteristics, and it makes it easier to isolate pure cultures. As known, the starter medium for D. radiodurans is TGY, containing tryptone, glucose and yeast extract. This medium provides all the necessary nutrients in a controlled environment for D. radiodurans to have optimal growth, that is in 28 degree Celsius. Culturing D. radiodurans is the first building block to further experiments and applications, thus it is necessary to renew older cultures of bacteria by inoculating them in fresh media. 

Materials and Methods

For culturing D. radiodurans in liquid and solid media, the following materials are used:

• 50 ml tube for the new broth culture
• A 500 ml flask with newly made TGY medium
• Inoculation Loop
• Bunsen Burner
• Older agar plate with grown colonies
• Fresh agar plate containing TGY and agar
• Electronic Pipette

    On a sterile lab bench near the Bunsen Burner, the materials are brought closer for sterilization. To begin with, using the Aseptic Technique, the top of the medium flask is flamed over the Bunsen Burner for sterilization along with the aluminum foil that covers it. Using an electronic pipette, 20 ml of fresh TGY is transferred from the flask into a labeled 50 ml tube. Then, the Inoculation Loop is sterilized over the flame for the next step. After about 10-15 seconds of wait time to let the loop cool down, the loop is used to carry one bacterial colony from the older agar plate with grown colonies into the 50 ml tube with fresh TGY medium. Once the loop is inside the medium, a little stirring does the job of homogenizing the liquid with the D. radiodurans cells. The loop is then sterilized on the flame to be used again. This summarizes the procedure for culturing D. radiodurans in a liquid medium. The 50 ml tube with TGY medium and the bacteria is then incubated at 28 degree Celsius for 2-3 days.  

    Further, the culturing of D. radiodurans on a fresh agar plate is done using the Four-Way Streak Plate Method. The new agar plate is labeled with the medium type, bacterial type, initials and date and divided into four quadrants for this method. To start with, the Inoculation Loop is first flamed for sterilization. Next, a bacterial colony from the old agar plate is carried with the tip of the loop to the first quadrant on the agar plate held upside down where it is streaked back and forth until it covers all the space in that quadrant. The loop is flamed again, and it is passed over the first quadrant of the new agar plate to carry over some D. radiodurans cells to the second quadrant where it is streaked in a similar manner. This method of streaking and flaming in between is then repeated for the third and fourth quadrants. The loop is flamed one last time before being stored. Finally, the agar plate with the newly streaked bacterial cells is then taped and incubated at 28 degree Celsius for 2-3 days. 

Results and Conclusion

After the incubation period is over, the 50 ml tube should show a turbid broth culture indicating very large quantities of cells, and the agar plate should show circular, red colonies of bacteria in one or two quadrants and red streak lines in the other quadrants. The broth culture can then be diluted to create other broth cultures using serial dilutions and to be turned into pure colonies using the Spread Plate Method which can be used in the Colony Counting Assay. The applications of bacterial broth cultures are many, some of which include the 96-Well Assay, DNA Purification, protein extraction, and numerous D. radiodurans stress-induced growth experiments using UV radiation or chemicals compounds like hydrogen peroxide. Nevertheless, agar plate colonies are not in any way inferior to broth cultures as they can be used to start new pure cultures, to demonstrate certain phenotypes and patterns of growth, to test for growth and resistance in a medium with certain ingredients added or taken out, and in many many other applications. All in all, bacterial culturing is the first step to any and all types of bacterial experiments in the laboratory.