Wednesday, March 30, 2011

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.

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