How Important are Pellets in a Laboratory?

How Are Bacterial Pellets Made?

               On Friday, for the first time I learned how to grow bacterial pellets. The entire process was pretty repetitive and time-consuming, but overall the whole agenda was interesting. First off, I want to clarify what is a bacterial pellet and why we, researchers at the Biodesign Institute, need to grow them.

                A bacterial pellet is a clump of dense bacterial cells that grow together in a laboratory bottle due to a centripetal force. What produces the centripetal force, you may ask? Well, in the laboratory we use a centrifuge to produce this force as we spin down two or more balanced bacterial media bottles; in order to separate out the bacteria cells from the liquid media. The most important rule I learned about using a centrifuge is to make sure the lab bottles are balanced. By balanced I mean that both bottles have the same mass amount by using a common mass scale. In my case, whenever I needed to add more media solution into one of the bottles; the trick is to just add water into the less massed bottle, until the mass of the bottle is about the same as the other bottle. The reason why the bottles have to be balanced is so that the rotor in the centrifuge doesn't get damaged by spinning so fast where the unbalanced side of the cup holders doesn't teeter and totter, causing the rotor to break. In our case, we spun the bacteria at around 5000 rpm, which is very fast if any you get the chance to see it. It took around 10 to 15 minutes for us to spin the bottles down, but all the work was worth it since the bacterial pellet grew. See picture:

The brown patch in the picture is the bacterial pellet.

Notice how compact the pellet appears, where this tells me how the bacterial cells are clumping together due to centripetal force.

So far I have only talked about how the bacterial pellets grew, but now I still need to answer one of my questions about why we need to use them? When we grow bacteria particularly the Agrobacterium strain, we are focusing on bunching up all of the bacterial cells together so that we can have a huge concentration of these cells to infiltrate. Hence, the bacterial cells will clump together to form the pellet, as shown in the picture. The more concentrated bacterial cells we have then the more concentrated media solution we can produce to which the gene of interest, the gene that codes for the antibodies, can be encoded in the plants' tissue. In order to grow the media solution, I had to put the bacterial cells in a MES buffer solution, so that the bacteria can grow in the solution and be stimulated to transfer genetic material to each other. As the bacteria are growing in the solution, I have to then pour a little sample of the solution into a cuvette, which is a small container that fits into a spectrometer, so that I can place the cuvette into the spectrometer to measure out the optical density of the bacteria cells in the sample.  What the spectrometer does in order to give me the optical density of the sample is by passing a 600 nm wavelength light through the cuvette, where any light that was blocked by the cells themselves would be determined from the amount of light that was able to pass through the cuvette, which is measured out by the machine. The density value will then be used to calculate out an estimate of how much bacterial cells there could be in these large 500mL flasks, that contain the bacterial cell "waste" solution left out from the pellet.

Here is a picture of a lab bottle filled with concentrated bacterial cells after using the vortex

Whenever I use the centrifuge, the liquid that is still in the lab bottles where the pellet forms at the bottom is disposed into labeled bacterial strain flasks. The liquid may still has some bacterial cells in the solution, but most often then not it will be the MES buffer solution that I mentioned. I keep pouring in more solution into each specific lab bottle, until the pellet has formed completely. After that I can use a vortex spin machine to grind out the pellets to dilute the bacterial cells into a clear white liquid. That white liquid will be added to another solution of MES where then it can be used for infiltration. Other than that that is pretty much all the skills you will all need to know in order to make a bacterial pellet.

Here is a picture of a vortex machine.