Obtaining a Pure Culture from Isolated Colonies: Part 2

By TT -

Introduction

Previously, a 15mL tube with 5mL of TGY broth with live cultures of D. radiodurans was incubated for 48 hours. When the incubation process was stopped, there was little to no growth resulting in an OD600 reading of -0.06 nm.  There was a small orange-colored pellet at the bottom of the tube and the media was clear with a slight orangish tint. 

This led to a hypothesis that the lack of growth could have been from the lack of oxygen allowed to flow into the tube, hindering the growth process. So, if more oxygen was allowed to flow into the tube then there will be more growth of the cells. 

Materials & Procedure

Materials that were used are as follows: A cultured TGY broth with D. radiodurans cells, from part 1 of this experiment in a15 mL centrifuge tube, a large tub, balance tubes, and an orbital floor shaker.  A vortexer, P20 pipette, kimwipes, deionized (D.I.) water, uncultured TGY broth, and a nanodrop (nanometer) were also used in the results stage of this experiment. 

Since the cap on the centrifuge tube was closed during the previous incubation period, the cap, in the second incubation phase, was sat on the top of the tub and taped down so that the opening of the tube was covered but loose enough to allow gas exchange.  The tube was then placed into a large tube and balance tubes were used to hold the centrifuge tube in place so that there was security during the agitation stage while incubating the cells. The test tube was then put into the orbital floor shaker for another 48-hour incubation period. 

After the incubation period, the vortexer was used to resuspend the cells into the liquid as preparation to measure the cell concentration with the nanometer.  The nanodrop was cleaned with a kimwipe and deionized water.  Then, 2 µL of uncultured TGY broth was used to blank the nanometer, using the P20 pipette.  This step is done to rule out the liquid measurement of the broth that the D. radiodurans cells were suspended in. 

After, the machine was blanked, it was cleaned with the kimwipes and D.I. water.  Then, 2 µL of the cultured broth was placed on the nanometer using the P20 pipette. 

Results

After a second 48-hour incubation period, there was a larger orange-colored pellet at the bottom of the centrifuge tube. The liquid was turbid, these observations indicated more cell growth.  The results from the nanometer OD600 reading was 2.26 nm.  So, the cells were able to grow once more oxygen was allowed into the tube during the incubation period.  This cultured broth was used on a TGY agar plate for colony growth. 

Comments

  1. EricaMarch 30, 2022 at 4:59 PM

    Nice summary, but I would like to mention that the black/white font set up is super hard to read.

    ReplyDelete

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