Elza wearing makeup - Stained to death

This week I was able to have a closer look at Elsa. Although she is quite attractive in the rays of the setting sun as the white layer of her swirls into a piece of agar, an entirely new dimension has opened up in the last class. We practiced some dying procedures and used microscopes to observe our samples.

Elza stained with Safranin. These pics were also taken by me
it was quite fun, using a microscope and a phone.. :)

I guess, the tininess of microbes hasn’t been made clear yet. To start with, the word ‘micro’ meaning small, does have a precise definition in connection with measurement. In everyday life we don’t need to consider smaller distances than millimeters, or smaller weights than a gramm. However, the scale doesn’t end here! One micrometer is exactly the one millionth of a meter, a microgram is a millionth of a gram. It might turn out to be even more astonishing, if you fetch a piece of paper and write down: zero, point, five zeros, one. In meters this is the number for one micrometer! Showing allegiance to their name, most microbes fall between 0.5-5 micrometers. Using a 100x magnification objectives I was able to see individual cells of Elsa and try to deduce some information on their morphology.

The proof that Elza is Gram positive. If you enlarge,
you can see tiny white balls inside some cells,
these are the endospores.

Over the past almost three years it has occurred to me that in biology the funniest and also most beautiful names are of dyes. There is a wide range of them used for staining animal tissues, plant tissues, microbes, certain cell components, etc. Here are some names I especially adore: Malachite green, Azocarmine blue, Soudan black, Nile blue, Hematoxylin-Eosin, Xylene Cyanol, Ponceau, and this list goes on and on. I aim to show you only one staining process, probably the most famous one in microbiology, which was invented in 1884 by a Danish scientist. Using this so-called Gram staining enables us to distinguish between two main categories of bacteria. Actually, these categories were created on the basis of this staining… J

This method exploits the fact that there are two most prevalent types of bacterial cell walls. Due to their different nature of molecular components some of them are negative, some are positive to Gram staining.

Stained slides. We put a drop of immersion
oil onto them, then used the 100x objectives
on our light microscopes.

Here is an abridged protocol we used. First, we eliminated all fats and dirt from our glass slides, which we use as a storing surface for our samples. We did this by dipping them into 96% alcohol, than burning it off with the Bunsen-burner. Then we applied a drip of bacteria suspension and spread it, so almost the whole slide was covered with microbes. After letting them dry onto the slide (which process was accelerated by carefully using the heat of the burner) we applied the first dye, the Crystal Violet. We let them alone for one minute, than washed the stain off with water and applied the second color that contains iodine. The iodide ions are able to form a complex with the Crystal Violet. Until now, all kinds of bacteria behaved the same way, having a deep purple color. The distinctive part came when we used absolute alcohol to remove this Iodine-Violet complex from the cell wall. Those who were submissive enough to obey and let their colorful dress go, are called the Gram negative boys. As a final step, we applied a dye called Safranin which was able to give color to only the Gram negative cells, as the place reserved for stains had already been occupied in the Gram positives by the Iodine-Crystal Violet complex. We were able to conclude that all pinkish-reddish cells gained their color due to the Safranin, so they bear the name Gram negative. Those rocking with the deep purple are the positives. It shouldn't be missed that there is a quite unfortunate 'side-effect' of these staining processes. Our samples need to sacrifice themselves for the sake of science... But don't worry, I have passaged plenty of Elzas to a new agar medium, so we will be able to be mesmerized by her beauty next week.

A, is the process of Gram staining and results on both negative and positive microbes.
B, the most typical microbe shapes. Source.

As I have already informed you, our eventual task by the end of the semester will be to precisely characterize our chosen species, in my case Elsa. I compared her to all other samples made by the class and she resembles much the Bacillus subtilis… She is Gram positive, and has endospores. However, this is far from being enough for characterization, it does pretty much restrict the choices available.

Hold your breath!

This gorgeous creature is a Clostridium species. There are about 100 types of them.
Some living freely, some being pathogenic. This one in the picture is the Clostridium difficile,
which starts thriving in your gut after you used antibiotics to kill all the others. Then this guy
realizes that the place is free, then swings into motion, which you usually realize
by having antibiotic-associated diarrhea. Source.

This post aspires to give a brief overview on my first encounter with anaerobe microbes. Basically any organism, which doesn’t require oxygen for living is called anaerobic. There are certain categories, for example obligate and facultative anaerobes. Apparently, the obligate guys made up stricter rules for themselves. They love being without oxygen so much, that if they come across any amount of oxygen, they stop growing and finally give up living. The facultative ones are cleverer, (this is of course an exaggeration, there are instances when being obligate is more adaptive for one) they are happy growing without oxygen but don’t mind its prevalence in their neighborhood (for a certain level).

Schematic structure of an endospore. These
several layers are responsible for
saving the DNA from UV light and high
temperatures, for instance. Source.

The subject of our experiment was a genus (this is just a taxonomic category, which contains very similar species) called Clostridium sp. These microbes fall into the obligate category, also having a special feature: they produce endospores. This characteristic was the one we needed for selection, I’ll explain you why. Endospores are very inventive tools for surviving harsh environmental conditions, lack of food, draught, chemicals, UV light, extreme temperatures, etc. The endosporal life stage might be compared to the hibernation of let’s say grizzly bears. The metabolic activity (all biochemical reactions that function to produce energy from food and to use energy for any kind of processes, for instance motion) of bears changes and slows down, in contrast the these endosporic microbes go completely dormant, by arresting all metabolic pathways. Sporulation occurs relatively quickly, it involves secretion and formation of several layers, coats around the cell plus the inner structural changes, for instance DNA condensation (it gets more dense, packed into a smaller volume). Eight hours are sufficient for a bacterial cell to develop an endospore. Due to this completely dormant, inactive phase it is not only one winter, as in the case of bears, that such kind of organisms can survive.

This is the hot bath, where we
eliminated everybody
except for Clostridiums.

Now, a little history: in 1947 Clostridium endospores were put into a sterile medium and weren’t opened until 1981. It didn’t take more than 12 hours for the bacteria to reactivate themselves and start cheerfully thriving, 34 years later! If that won’t be enough for you, there are further, much bolder claims. In the 1990s a 25-40 million years old bee, preserved in amber was found. In itself that wouldn’t be exceptional at all, but some scientists isolated endospores from this oldish bee’s gut. Believe it or not, they were able to bring those bacteria back to life, by simply putting them into appropriate culturing medium!

This is and anaerobic 'booth'. This elephant trunk like thing
is where you put your hands and start operating inside that
box. It keeps oxygen out, so is perfect for experiments
done with anaerobic bacteria.

Our task in class was to selectively grow this famous Clostridium. Its name has a Greek origin, it refers to its rod like shape. First, we needed to eliminate all other species from our bacteria culture. We did it by placing our Clostridium suspensions into a hot bath, they stayed there for 10 minutes at 80 Celsius degrees. That process yielded us a suspension full of dead microbes, except for those who bore endospores. (Endospores can survive even 150 Celsius degrees). After that we used a syringe to place a little amount of this suspension to the bottom of an empty Petri dish (no agar!). We melted some agar/culturing medium solution and poured it over our samples. After mixing them, we placed the top of the Petri onto the still warm and soluble agar upside down, so it stuck to it. This tightly fitting construction, called the ‘Brewer plate’ was able to expel ‘all’ oxygen particles, letting our dear little Clostridiums to start reactivating. I can’t wait for Tuesday to see what they ended up with! 

As I promised, here is my little Elsa. You can observe her better if you enlarge
the picture. She is that white cream like guy on the surface of the yellow agar.
If you are careful enough you can notice how she started going deeper into the agar,
it is like little roots going down from the indented surface.

Meet Elsa!

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.

This week, all of us were given an unknown (at least by us, students) species of microbe. One of our tasks during the semester is to characterize and eventually determine our new friend. For this purpose we should keep our fellow alive, so every week we can pursue new tests on her, better said on her descendants. (I named my microbe Elsa, so that is why I’m arbitrarily gonna refer to her as a she. Before anyone wants to condemn me sexist, I declare that my decision to call her a she was purely based on my very first impression. She is grayish but rather white, seems to be pretty fragile and ethereal. I think she is beautiful in a sense.) Unfortunately, I can’t show a picture of her now, but I’ll upload one with the next post, I promise. (Yes, the title of this post was kind of a bait, I'm sorry. However, here is you reward: I appreciate you didn't give up reading! :)

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.

I end this post with a picture I like a lot. Our seminar is on every Tuesday from 14.00 to 19.00,
so around the end the lights of the sunset pierce in through the window. All these equipment,
our teacher writing on the board, don't tell me it is not nice..