E.coli Survival Test
Since we did not know if E.coli can survive in methanol solution, we decided to test the E.coli survival rate in different concentrations of methanol and ethanol using three different methods: Spectrophotometry, double staining fluorescence microscopy, and clonogenic assay.
Our first method, spectrophotometry, was designed based on the fact that when a bacterium dies, its membrane breaks and all the proteins are released. The spectrophotometer is a machine that can be used to detect the concentration of these proteins by measuring the light absorbance of a certain wavelength. By measuring the change in protein concentration after adding methanol, we sought to find the cell death rate according to the formula found in the literature, but when actually put into practice, the reading fluctuated considerably and so was unable to give us an accurate result, probably because of the protein settling and degradation in methanol.
Double Staining Fluorescence Microscopy
The second method we tried is double staining fluorescence microscopy. Two fluorescent dyes, propidium iodide and hoechst 33342, both stain bacterial DNA, but they differ in their permeability and in their fluorescence. Hoechst 33342 is a nucleic acid stain that emits blue fluorescence when bound to double stranded DNA. This dye is highly permeable and can enter the cell membranes of both dead and live E.coli. The other important fluorescent dye we used is propidium iodide, which emits red light when bound to DNA. Unlike hoechst, propidium iodide can only penetrate the cell membrane when E.coli is undergoing lysis, indicating that they are dead or starting to die, making it a very useful tool to test the survival rate. We had to play around with different conditions to see which ones will give us the best visual result. For example, we tested different dilutions for the bacterial suspension, different concentrations of the dyes, incubation time, dye ratio and amount. We also had difficulties at first finding our E.coli. There were a lot of residues on both side of the coverslips that we accidentally mistaken as E.coli. After deep cleaning our microscope slides and coverslips with the ultrasonic cleaner, we finally identified our E.coli under 400x magnification. After numerous testings, we found that 50% methanol is deadly to the E.coli, as indicated by all the red fluorescent signals. We overlapped the blue and red images to visualize how much E.coli has died. The purple denote bacteria which are dead, while the blue show those which are still alive. Additionally, we determined that ethanol had a more detrimental effect on the E.coli, with 30% being the fatal concentration.
In order to determine whether or not our fluorescence microscope results were reliable and accurate, we utilized another method called clonogenic assay to test the E.coli survival rate in ethanol/methanol solutions. This method involves applying a treatment to a sample of cells and then spreading these cells on agar plates to allow them to grow overnight. The next day, you can observe distinct colonies of bacteria on the agar plates. The number of colonies able to grow on the plate signifies the number of live bacteria originally deposited onto the plate. Comparing the number of colonies on the treated plate to an untreated plate gives a rough idea of the percentage of cells that survived the treatment. For our experiment, we first determined the ideal dilution of our bacterial suspension for a visible result. Without a high enough dilution, it will result in a bacterial lawn rather than in distinct colonies. After diluting our overnight bacterial suspension by 10,000 times, we were able to obtain fairly decent results. As for the treatment, we tested various concentrations of ethanol and methanol, ranging from 10% to 50%, with varying incubation times. After incubating the E.coli with the treatment for a certain period of time, we removed the methanol/ethanol solution, diluted the sample using LB nutrient broth, and spread 10uL of that sample on the agar plates. As we increase the concentration of ethanol/methanol, we observed a slight decrease in the number of colonies and the colony size. In the figure shown here of our methanol treated E. coli, the number of colonies significantly decrease in size at 40% and do not even grow at 50%. Similarly, in the ethanol treatment test, there appears to be very little change between the control and the 20% ethanol treatment, but no growth at all under 30% treatment. These results -- that no colonies form at 30% ethanol nor 50% methanol, are consistent with the results we found using the fluorescence microscope technique.