For this week, I will guide you through the basics of the reproduction and storage of bacteria. Plus, I will show you an insight into the difficulties of working sterile on a bench. For scientists working with a certain species of microbe, it is crucial to keep their ‘pets’ in an always available condition. So in any case of new experiments they can reach back for a pool which is exactly the same, therefore representative in a forthcoming study. The three most common ways of doing this are lyophilization, storage in liquid nitrogen or passaging. During this week’s lab session we gained practice in the last one, so I just quickly summarize the first two.
|Some fellows in the sun.|
Lyophilization is also called freeze-drying. It starts with cooling down our sample to a very low temperature,
than applying vacuum on it. Using this method, we can get rid of all the frozen water in the material, therefore it is easily preserved. In case of microbes, even after many years you could make your creatures come alive by simply dropping that lyophilized bacteria strain to a bottle of culturing medium. Moreover, lyophilization is not a secret tool used only by microbiologists. I’m sure you drink instant coffee sometimes. Well, those little crunchy powder-like particles you pour water on are indeed lyophilized.
I should confess that liquid nitrogen is real fun! Imagine a liquid with a temperature of −196 °C; −321 °F. It was not at our seminar, but fortunately sometimes I had the chance to ‘play with’ this material. It looks like as the clearest water, but immediately creating a cloud of steam as you pour it to a container. Slowly that cloud reduces itself to a thing layer of mist swirling over the surface of the pool you created. As you drop some tubes of cells into it, that seemingly calm ocean starts on hissing and creating more and more mist, while your tube is happily cavorting around. Personally, I always think of it as some kind of futuristic material. If you don’t have the chance to meet this substance, search for some youtube videos, surely you will find some literally cool. Back to our topic, at this extremely low temperature bacteria can be stored safely, and brought alive much later just as in the case of lyophilization.
Making the 'indented agar tubes'. The culturing medium mixed with agar
is heated up, poured into the test-tubes, then it is left to cool down and
solidify on this indented rack.
The descendant story, I stroke up before, should be explained. Otherwise, there might be too much unanswered questions left in you. Bacteria reproduce themselves usually by binary fission, this term simply means that one cell doubles its DNA and divides into two daughter cells. These daughters are absolutely identical to each other in their genome (all hereditary information of an organism), and also have no difference to their preexistent ‘mum’. Therefore, they are called clones. That is why I’m going to be able to nurse Elsa for this whole semester. However, it should be made clear that if I would keep Elsa at conditions which are the best for her to proliferate, then I would end up having a half room of Elsas. Not being my intention, I should turn to some other practice. Yes, I could grow a certain size of population, dry-freeze them in smaller portions or store them in liquid nitrogen. Than every week pick a little tube of Elsa, ‘melt it’ do my tests on her, next week a new tube, and on and on… However we are to approach this problem from another angle. We are continuously going to passage a little pinch of her every week to a new, empty agar surface. Storing this little group of clones on
28 °C will render us only a plate full of Elsa for next week, which can be used for experiments and for passaging a crumb of Elsa for next weeks observations.
|If you enlarge this pic, you can see the sticks end smoldering.|
This passaging process is relatively easy, however requires a little bit of practice. Sterile work is critical; otherwise, you might contaminate your plate with other species and cannot proceed with your experiment. We used a Bunsen burner as the primary sterilizing tool. In approximately the
15 centimeter radius of the flame the air can be announced bacteria free, because of the heat. Our chosen bacteria were kept on agar, but in a test tube. (The pictures tell more about the creation of such storing equipments.) During the whole passaging process we tried to keep everything close to the flame. As an initial step we heated up a little metal stick so it is completely sterile. For handling the tubes, the tube’s caps and the stick with two hands, we were taught a practical hold. I won’t spend many lines detailing this, rather I decided to shoot a short footage of that process next class, so you can get more close to the work done in lab. I’ll only give a basic explanation. We touched the mouth of the open test-tubes into the flame, so they are sterile, and just after this did we plug our stick into the tube to pick out a little amount of our creatures. After that we turned to a new microbe-free agar test-tube, spread the clump of microbes from our stick to the surface of it. This resulted in a new agar surface with much less cells on it, so we can let them thrive there for a week, without overgrowth.