growing bacteria

Post date: Jul 16, 2013 1:02:06 AM

My search to expand my knowledge on the different fields of science has landed me in the middle of biochemistry and oceanographic applications and implications. While spending my summer in a lab in the basement of Stanford’s Y2E2 I will be researching the nitrogen cycle, specifically where nitrite is converted to gaseous forms. Using denitrifying bacteria, nitrogen isotopes 14 and 15, mass-spectrometers, NOx boxes, and many other interesting machines we will hopefully reach a conclusion regarding the varying isotopic signatures of nitrogen being released and consumed throughout the environment.

A few weeks prior to data collection we must begin to prep our experiment, which means growing our live bacteria. From Stanford's treasure trove of frozen bacteria samples kept tight in a -80 ˚C freezer we chose four different strains of denitrifying bacteria. We next carefully thawed the bacteria placed a drop of them upon petri dishes with a thick, solid layer of tryptic soy broth and agar. Using sterile technique, we spread the bacteria evenly out across the plates. Then using special parafilm tape we sealed closed the dishes to create an environment for the bacteria that would not come into contact with the outside world. And four days later out bacteria were looking beautiful as you can see here.

The next step was to create a liquid media solution for the bacteria to grow in. We created a very simple solution using extremely purified Milli-Q water and Luria broth. We then filled approximately 16 serum bottles with 120 mL of the dilute Luria broth. After clamping the serum bottles we put them in the autoclave, which cooks the bottles at extremely high temperatures to kill anything that could possibly be living within the bottles. The serum bottles are an important element of our experiment providing an environment which can be manipulated while also being free of contamination. We also took a sample of media from a serum bottle to test it's pH; the pH reading of 6.586 will prevent equilibrium isotope fractionation.

The next step is to create a nitrate solution to add to the media where the bacteria will grow. Due to the fact that the bacteria are denitrifying, they need ample amounts of nitrate as food to thrive. After several hours of challenging stoichiometry and using a very sensitive balance, we were able to create our 100 µM solution of KNO3. We then carefully injected 1 mL of this solution into eight of our serum bottles. We used a special filter that filtered any possible biological contamination out of the solution, thus ensuring a sterile environment for our bacteria. Next we used a similar needle and injected the bacteria into the serum bottles, but without a filter. These bottles were placed on the shaker table to circulate the bacteria and nutrients. Hopefully in a few days our bacteria will have fully consumed the NO3- so we can begin our nitrite spike experiment.