Post date: Aug 1, 2013 12:16:33 AM
Previously I discussed the prep leading up to the experiment which ended in the creation of multiple serum bottles each filled with a different strain of denitrifying bacteria. Two days before starting the time series experiment, each serum bottle was injected with a solution full of nitrate, which is a vital nutrient for the denitrifying bacteria. Two days after this spike we took a small sample from each serum bottle to see if there was any nitrate that had not been consumed by the bacteria already. In the nitrogen cycle, nitrate is converted to nitrite and then converted into varying forms of nitrogen gas. Mixing our samples with two solutions SAN and NED we were able to determine that the bacteria had fully consumed the nitrate because of our mixtures remaining yellow tint. If there was nitrate still in the serum bottles, the mixture of SAN and NED with the sample would create an obvious pink solution.
Hungry for more nitrogen the bacteria were ready for their next spike: nitrite. The time series begins when the nitrite is first introduced to the first serum bottle. Over the course of two hours we would take a 10 mL sample from each serum bottle every 20 minutes. After taking a sample from a serum bottle using a syringe the contents are then passed through a special filter. These filters remove the bacteria from the sample and thus provide an accurate measurement of how much nitrite has been consumed by the bacteria at that moment. A very important factor of the experiment is the time that the exact time the sample was taken during the time series. The time of when the sample is taken is recorded the moment the sample has passed through the filter. So we would expect that the first sample would have a high concentration of nitrite while the last sample would have very low concentrations.
After each sample is taken it is then brought across the room to the NOx box. With walking approximately 54 samples across the lab to the NOx box and carrying each individual serum bottle to and from the shaker table, this process can get slightly chaotic. Before samples can be inserted, the NOx box needs to reach a baseline, which can sometimes take up to two hours. The NOx box is a very old machine that measures purely the intensity of a sample. Using a sodium iodide and acetic acid reagent, the NOx box is able to measure the intensity of the nitrite that has been mixed with vinegar and thus turned into a gaseous form. After the sample is injected into the NOx box a sharp peak will rise and then fall back down to baseline. The area of the peak determines the intensity of nitrite of a solution. We will repeat this process of growing bacteria, transferring the bacteria to a liquid form, feeding the bacteria nitrogen, taking samples, and measuring the nitrite content several times in hopes of reaching more accurate data through several trials.
Aside from the time series we ran a density test in hopes of determining an approximate number of bacteria within each serum bottle. To do this a very small sample was taken from each serum bottle. These samples were taken before the time series and after testing the bottles for nitrate. The sample was diluted and diluted and further diluted with the very pure MilliQ water to a very low known concentration of the original sample. And again a very small sample of our very dilute solution was placed onto a petri dish. Next using a turn table and a handmade glass instrument which was created with its elbow bend shape specifically for this purpose, the sample was evenly spread across the petri dish with the combined spinning motion of the table and the smearing of the elbow shaped instrument. This even distribution of a dilute bacteria sample is necessary so that a few days later obvious colonies would be visible and spread out enough to count. This count could then provide us with an idea regarding how much bacterial growth is occurring in each individual bottle.
The next step in our experiment would be to transfer our samples across the Stanford campus to the nitrogen isotope lab. The mass spectrometer, a multimillion dollar piece of equipment, can use a huge magnet to distinguish between a 15N and a 14N. The ratio of 15N to 14N will provide more information about the isotope effect of microbial denitrification.
work station where I spend many hours
The magic NOx box
Data output - Used to calculate how much nitrite is in a sample