Sunday, August 29, 2010

Microbe Monday: A sterile stick and other tools

My previous advisor used to joke that all you need to do microbiology is a sterile wooden stick. You can use this simple tool too isolate colonies, pick colonies, inoculate media and isolate DNA*. If you were to visit my bench, you would find a bucket of sterile sticks as I use them almost every day. However, I need a lot more than a wooden stick to get my research done. The machines I use most often are the thermocycler, FPLC, biacore and any kind of spectrophotometer. Unfortunately, my favorite tool, the microscope, is one I rarely use.
One of the major reasons I love this instrument because it enables me to actually see the microbes I am studying. Since my work typically involves using RNA, DNA or protein, I spend most of my time looking at tubes containing a clear solution, or a if I'm lucky, a solution containing a dye. If not a tube, then I'm probably viewing a stained gel (acrylamide or agarose). It's just not as visually stimulating as looking at something under the microscope.**
Another reason I like to use the microscope is because you can take pictures or video of what you observe. Pictures and videos typically go over really well in a seminar and when applicable, they are great in a paper. The presence of images or video in a paper was often a deciding factor when I was deciding on a paper for journal club. I think everyone agrees that it's nice to see something besides a graph or table every now and then.***
For researchers like me, there just isn't usually a reason to have photos or video from a microscope. However, when I can, I choose assays that utilize a microscope. For example, to assess translation, I'll use a reporter gene like GFP so that not only do I get to use the microscope, but I get to look at fluorescence and that is way cool. I've also used fluorescence to assess protein-protein interactions. In both of these cases I can use the microscope for qualitative "yes or no"-type of data and then if need be, I can use a fluorometer for more quantitative data.
To illustrate my point, here are images from wikimedia commons. I wanted to include videos, but blogger wasn't cooperating.****


Original figure legend: Multiple fluorescence 2PE imaging. 2PE multiple fluorescence image from a 16 μm cryostat section of mouse intestine stained with a combination of fluorescent stains (F-24631, Molecular Probes). Alexa Fluor 350 wheat germ agglutinin, a blue-fluorescent lectin, was used to stain the mucus of goblet cells. The filamentous actin prevalent in the brush border was stained with red-fluorescent Alexa Flu or 568 phalloidin. Finally, the nuclei were stained with SYTOX ® Green nucleic acid stain (2).


Polymicrobic biofilm grown on a stainless steel surface in a laboratory potable water biofilm reactor for 14 days, then stained with 4,6-diamidino-2-phenylindole (DAPI) and examined by epifluorescence microscopy (3).











*Once the DNA is precipitated, you can wind it around a sterile stick or toothpick in leu of centrifugation. This is not really effective if you aren't isolating a large quantity of DNA.
** Yes, you can visualize your data in ways that don't involve a microscope and as a general rule it's awesome to obtain data, be that data in the form of a band, a sensogram or absorbance reading, but in my opinion seeing your data in a living organism is way cool.
***Not that anything is wrong with a table or graph. Pretty much all my data is presented in this form.
****I know that there are many compelling images and videos utilizing fluorescence microscopy, but I am not sure what the rules are regarding image and video usage from scientific papers. If anyone would like to enlighten me, please feel free. If anyone knows of some better images, that I can post without committing any kind of offense or spending money, please let me know and I will be happy to post them.


(2) Multi-photon excitation microscopy. BioMedical Engineering OnLine, 2006, 5:36.DOI:10.1186/1475-925X-5-36.
(3) Centers for Disease Control and Prevention Rodney M. Donlan: "Biofilms: Microbial Life on Surfaces"

6 comments:

Girlpostdoc said...

I use a lot of sterile sticks, but like you my tools are diverse and none of them involve looking directly at the organism. In part, my little critter is difficult to see without a high powered scope. I first fell in love with science when my mom brought home a microscope and I could see blood cells up close and personal.

Micro Dr. O said...

I hadn't used a microscope for my research in ages until this past week. After relearning how to do a simple gram stain, I got to use old school microbiology to look at the effects of a drug on morphology. With all the molecular biology I'm used to doing, this simple experiment was incredibly exciting and fulfilling! :)

Thomas Joseph said...

When isolating a novel organism, we get to ship our samples off for electron microscopy. The image on the left side of my banner is one such example.

chall said...

oh sterile sticks! :)

The swirling the DNA is one of my fave things to do since I can at least SEE it. THe microscope is awesome, and I love seeing my bugs.

I do would like to take some of those "confocal" pics thuogh. Make beautifuk pictures for walls at home. I'm so in mood for some science art :)

Bori said...

Hmm, for some reasons I use sterile tips (the 10µL ones) for all my bacterial inoculation, plates streaking, colonies picking- just a variation on the topic I guess :-)
About the fluorescent images: isn't it ok to use open access articles for this- providing you cite them properly?

microbiologist xx said...

girlpostdoc - How funny. I think the first thing I ever looked at under a scope was blood cells. I loved it!!

Micro Dr. O - LOL! I would have to relearn how to Gram stain too. I haven't done one since undergrad.

TJ - I HATE you!! OK. I don't. It's just jealousy rearing its ugly little head. Really, that is awesome...I think I want to work where you work. :)

chall - SEEing things is sooo exciting. My last lab had a decon. microscope and it looked like you coulf perform brain surgery with that thing. It seemed like it took 15 button pushed just to get everything turned on, but boy, did it ever make nice images. I never got the chance to use it. :(

Bori - Whenever i use the sterile tips, I just end up gouging holes and trenches in my plate. You're right though, it's the same concept. :)
I'll have to check about the open access journals. It seems like it should be fine, seeing how the journal is open access and all. I wasn't sure, so I didn't want to take the chance. Thanks for the suggestion.