On Monday we did microscopy with Mrs. Cindy. Strains 14, 17, 37, 40, 66, and 75 were selected for microscopy. Cindy first showed us how to make slides for microscopy. Cells were scraped off of the plates and innoculated into 2ul of water in a tube. The risk of the water is that it could cells to lyse; therefore, cells would not be visualized during the microscopy. The slides are covered with collodian which helps the cells to stick to the plates. 0.5 ul of the cell solution was pipetted onto a coverslip and the glass slide was placed on top. DAPi was added to the slides as well. DAPi is a fluorescent stain that allows you to see DNA because it binds in between bases in the DNA which causes the DAPi to change conformation. The location of DNA can be visualized using DAPi because it glows blue when viewed with UV light.
The results of our light microscopy were not phenomenol. However we are able to obtain five images of our strains. No image was obtained for strain #14 using light microscopy.
IMAGE 1: Strain 17
· Little and large round cells
· Pairs and tetrads
· Cells congregated at the edge of the water
IMAGE 2: Strain 37
· Cells clumped together
· These cells were larger than strain 17
IMAGE 3: Strain 40
· Possible motility
· Oval shape
· Variety of sizes and shapes
· Some looked like spirochetes
IMAGE 4: Strain 66
· Various shapes and sizes
· Irregular shape
· Big clumps
· Sort sort of dark metal present in cell solution
IMAGE 5: Strain 75
· Motile
· Rods
· Vibrio
The DAPI stain was unsuccessful using the light microscope. This may be due to the fact that DAPI could not penetrate the membranes of the cells. This is probably due to the fact that the cells are hydrobic because they have an extracellular polysaccharide layer. Treating the cells with a chemical such as toluene that would dissolve this polysaccharide layer could make cells permeable to DAPI.
Next, we performed transmission electron microscopy on our select strains. The slide preparation for electron microscopy is very different from light microscopy. A copper grid is used which contains is coated with carbon on one side. The copper acts as a thin film for maintaining cells on the grid. I’m not exactly sure why the copper is used? Somebody might comment and let me know. Anyways, a small drop of cells is placed on the parafilm. The carbon side of the grid is placed on top of the drop. The grid is then removed from the drop using tweezers and dabbed on filter paper. Next the grid is placed on a drop of uranyl acetate and dabbed again on the filter paper. The uranyl acetate is used to visualize the cells. Electrons cannot penetrate through uranyl acetate so it makes everything around the cells dark. It is kind of like a negative stain. After the grid is prepped it is placed onto the electron microscopy. The resolution of the TEM is higher than light microscopy because the wavelength of electrons is smaller than that of the white light used in electron microscopy. Liquid nitrogen is placed inside the TEM to condense the vapor in the vaccume. The vaccume is used because of the small wavelengths of the electrons. A beam of electrons is show down onto the sample. The screen gives off green light when excited by electrons. I’m not sure of the images number for TEM.
37: No images obtained; cells may have exploded
17: tetrads and pairs
14: very small; looked like collapsed soccer balls; long crystals
66 big black blog (some sort of metal precipitate)
75: no images taken
40: oval with flagella; lots of flagella present; detached flagella present; cells are joined together
On Wednesday we read temperature results temperature results for 10C, 45C, and 50C. None of our strains grew at 50C and only three grew at 45C. Most of our 26 strains grew at 10C. Two of the strains that grew at 45C were cream and the other one was black. Distinct color changes were observed in some strains at various temperatures. I’m not exactly sure what may cause this color change.
We also tested xylan, granular cellulose and avicell hydrolysis. The xylan plates were inoculated with iodine. None of our strains hydrolyzed xylan. The granular cellulose and avicell plates were inoculated with congo red for 15 minutes and for a salt solution for 15 minutes. The purpose of the salt solution was to wash away the ubound dye. Our results seem somewhat ambiguous because it was unclear if some of our plates were positive or not. We recorded several strains as being weakly positive for hydrolysis of granular cellulose and avicell.
Sean Michael rain the gel of our PCR results using the Geo specific primer. Our results were not satisfactory. However, I did learn how to interpret a gel. We used latter three. All our 6 strains from Dr. Rainey ran, but only one of our 6 soil isolates ran on the gel. The results are found in the image below. If anybody sees that I labeled anything wrong please let me know because I’m still learning how to do this.
The results of our light microscopy were not phenomenol. However we are able to obtain five images of our strains. No image was obtained for strain #14 using light microscopy.
IMAGE 1: Strain 17
· Little and large round cells
· Pairs and tetrads
· Cells congregated at the edge of the water
IMAGE 2: Strain 37
· Cells clumped together
· These cells were larger than strain 17
IMAGE 3: Strain 40
· Possible motility
· Oval shape
· Variety of sizes and shapes
· Some looked like spirochetes
IMAGE 4: Strain 66
· Various shapes and sizes
· Irregular shape
· Big clumps
· Sort sort of dark metal present in cell solution
IMAGE 5: Strain 75
· Motile
· Rods
· Vibrio
The DAPI stain was unsuccessful using the light microscope. This may be due to the fact that DAPI could not penetrate the membranes of the cells. This is probably due to the fact that the cells are hydrobic because they have an extracellular polysaccharide layer. Treating the cells with a chemical such as toluene that would dissolve this polysaccharide layer could make cells permeable to DAPI.
Next, we performed transmission electron microscopy on our select strains. The slide preparation for electron microscopy is very different from light microscopy. A copper grid is used which contains is coated with carbon on one side. The copper acts as a thin film for maintaining cells on the grid. I’m not exactly sure why the copper is used? Somebody might comment and let me know. Anyways, a small drop of cells is placed on the parafilm. The carbon side of the grid is placed on top of the drop. The grid is then removed from the drop using tweezers and dabbed on filter paper. Next the grid is placed on a drop of uranyl acetate and dabbed again on the filter paper. The uranyl acetate is used to visualize the cells. Electrons cannot penetrate through uranyl acetate so it makes everything around the cells dark. It is kind of like a negative stain. After the grid is prepped it is placed onto the electron microscopy. The resolution of the TEM is higher than light microscopy because the wavelength of electrons is smaller than that of the white light used in electron microscopy. Liquid nitrogen is placed inside the TEM to condense the vapor in the vaccume. The vaccume is used because of the small wavelengths of the electrons. A beam of electrons is show down onto the sample. The screen gives off green light when excited by electrons. I’m not sure of the images number for TEM.
37: No images obtained; cells may have exploded
17: tetrads and pairs
14: very small; looked like collapsed soccer balls; long crystals
66 big black blog (some sort of metal precipitate)
75: no images taken
40: oval with flagella; lots of flagella present; detached flagella present; cells are joined together
On Wednesday we read temperature results temperature results for 10C, 45C, and 50C. None of our strains grew at 50C and only three grew at 45C. Most of our 26 strains grew at 10C. Two of the strains that grew at 45C were cream and the other one was black. Distinct color changes were observed in some strains at various temperatures. I’m not exactly sure what may cause this color change.
We also tested xylan, granular cellulose and avicell hydrolysis. The xylan plates were inoculated with iodine. None of our strains hydrolyzed xylan. The granular cellulose and avicell plates were inoculated with congo red for 15 minutes and for a salt solution for 15 minutes. The purpose of the salt solution was to wash away the ubound dye. Our results seem somewhat ambiguous because it was unclear if some of our plates were positive or not. We recorded several strains as being weakly positive for hydrolysis of granular cellulose and avicell.
Sean Michael rain the gel of our PCR results using the Geo specific primer. Our results were not satisfactory. However, I did learn how to interpret a gel. We used latter three. All our 6 strains from Dr. Rainey ran, but only one of our 6 soil isolates ran on the gel. The results are found in the image below. If anybody sees that I labeled anything wrong please let me know because I’m still learning how to do this.
i swear it took forever to label my gel in paint
ReplyDeleteoh and i think the carbon was used so that the organisms don't just fall through the holes in the grid.... wait did you ask what the carbon or copper was for? :)
ReplyDeletethe copper is the grid...right? and then the carbon is the film on top to keep the cells from falling through the grid?
ReplyDeleteIs the copper just to have an orientation when looking at the slides or does it have some other purpose? I didn't know if maybe some charged molescules on or in the cell were attracted to the copper to make it stick to the grid or what.
ReplyDeleteGrid is a structure on which to place your cells. It is fairly stable to bombardment with electrons...
ReplyDeleteOk thanks!
ReplyDelete