University of Rochester, Materials science program
Surface near sinks in our home might be the paradise of microbes. Due to some design defects, water spattered out stagnates there. Giving it time, dirty stains is growing on the surface.
For growing, I am not talking about "accumulation". These stains are alive. According to my observation, stains radiate from several centers expanding their areas. Some stains are brown and some are almost black. When the brown expands, the black retreat; when the black spread out, the brown shrink. They seem fighting for territories and I do not know what move the balance or to which side. Therefore, I suspect those stains might be microbe colonies.
To determine what exactly they are, I collected sample and used microscope to observe them carefully. Seven techniques were utilized, which were HMDS drying, coating, secondary electron imaging, backscatter electron imaging, energy-dispersive x-ray spectroscopy, and colorization. All results show as following.
2. Sample Preparation
This step was conducted under guidance from Brian McIntyre. Considering the sample may consist of lots of microbes, it was regarded as biologic materials. Glutaraldehyde was firstly used to fix the morphology of cells and kill them to prevent potential biological hazard. Then, we used water to wash the glutaraldehyde away and leave an amount of water in the bottle. About 1 ml of pure ethanol was added to bottle, rested for a while and then another 1 ml pure ethanol. Repeated this step three times so the concentration of ethanol rose gradually from 0 to about 90%. Poured away liquid and add pure ethanol this time, rested, and poured away. Finally, immersed the sample in HMDS and put a strip of filter paper in it to let the liquid climb and evaporate. This was done in a fume cupboard. HMDS is toxic.
After we successfully obtained dried sample, spread it on a drop of carbon adhesive on carbon tape stuck on a stub. Then put the stub into gold sputter machine. Thin film of gold coating is necessary to make our sample conductive. The difficulty was the sample formed as aggregated powders which were very hard to give it a conformal coat. And gold tended to deposit on carbon adhesive around the sample. After twice coating, the sample became conductive enough to get a relative decent SEM image free of charging.
3. Light Microscope Imaging
Figure 1 is an image of the sample put on stub surround by carbon adhesive. The carbon adhesive glitter with gold. In contrast, the sample is grey and dark, which illustrate that most gold atoms flied to surroundings not the sample during the coating. The poor depth of field of a light microscope just shows the rough topography of the surface of the sample, which is another reason for its hard to coating. But I do not get enough information to determine what the sample is made.
4. Secondary Electrons Imaging
Figure 2 to 9 were taken by a scanning electron microscope (SEM) with a secondary electron (SE) detector. Under the high magnification of SEM. We finally revealed the mystery of what grows in the stains. Countless microbes are living in there. There are at least 4 types of microbes. The spheres and rods that you can find in most image might be cocci and bacilli. Figure 2 is a family portrait. You can find cocci, bacilli, strip-like structures, pasta-like structures, fine fibers, big shrunk cells.
Strip like structure is clear seen on figure 4a, as the red arrow shows. The twisted texture may result from cell shrinkage after drying. Figure 4b is an SE image of mold I took before. Same strip-like structure of mold is observed. So, the strip-like structure in figure 3a is highly suspected to be mold, as well. Orange arrows point to fine fibers with diameter about nanometers. It might be secretions of microbes. It could be protein. Lots of cocci also can be observed in this image.
Figure 5 contains two interesting structure. Pointed by red arrow is a very long bacillus. It has 8 microns long while other bacilli are about 2 microns. But its diameter is basically as same as others. How does it happen? The other interesting structure is pointed out by blue arrow. There is a pasta-like matter covering that area. Maybe, a cell exploded and spread all inside matters out. If it come from secretion, then I think it should cover everywhere not just there.
There are two big shrunk cells in figure 6 as pointed by red arrow. They could be protozoans or cells from human tissue. They are too big to be bacteria. Figure 7 is a colony mostly formed by bacilli. A dividing one is in the center of the image. figure 8 is a fine hair very likely from human body. Some bacteria grow on it.
5. Backscatter Electron Imaging
Figure 9 is a backscatter electron (BSE) image. Contrast come from topography not composition. There is no significant difference in terms of atomic weights of elements. The dominant elements are carbon and oxygen. Both of them have no capability to produce strong backscatter effect.
There is something amazing in this picture shown by the arrow. There is a hole on that cell (maybe) and its diameter is just consistent with that of the bacillus putting its "head" on it. It looks like that the bacillus is drilling in.
6. Energy-dispersive X-ray Spectroscopy
To characterize the element composition of the sample, I use energy-dispersive x-ray spectroscopy (EDX or EDS) collect the signal of characteristic x-rays from different areas of the sample. Figure 10 is an EDX element map. Figure 11 is spectra from a bacillus and a coccus, respectively. As expected, carbon and oxygen are dominant. Gold is from coating. However, there is plenty of calcium, silicon and aluminum. Ceramics happen to be very rich of these three elements. It is reasonable to conjecture that they come from ceramic materials composing the surface near sink. There is no obvious distribution pattern of these elements among the whole area. When we collect EDS data from a single point on a bacillus and coccus, respectively, a difference reveal. Compare two spectra in Figure 11, Ca peak is stronger in the coccus while Al is stronger in the bacillus. Does it show different preference of element intake? The data is too rough to be examined quantitatively. The difference may or may not exist. Therefore, it is evident to get any conclusion in that.
At the end of this project, I present a beautiful colorized picture here, figure 12, which was collected by SE detector and processed by Adobe Photoshop Elements. Enjoy these cute bacteria!
Now we know what is growing on the surface near the sink. Those stains are made by microbes and other dirt like hairs. Among these microbes, there are bacteria, mold, perhaps protozoans. So, keep the surface near a sink clean and dry, otherwise it become a paradise of microbes.
Thank you to Brian McIntyre for giving knowledge to me without reservation and answering my endless questions!
Thank you to Caleb Whittier for help me through all labs!
And I would also thank Kristen Frantz for always answering me when I need help!