The resolution of the microscope depends on the wavelength of the light used, the way the electron beam acts on the sample and the imaging principle is different, the scope of application is also different, at present the most commonly used is the transmission electron microscope and scanning electron microscope. This experiment focuses on the preparation of scanning electron microscope samples. Scanning electron microscope total magnification can be varied between 20 ~ 300000 times, observation requires the sample must be dry. The sample must be dry and the surface must be electrically conductive. Therefore, the preparation of biological samples for scanning electron microscopy generally requires the use of fixed dehydration, drying and surface gold plating and other processing steps. Content from Microbiology Laboratory (3rd Edition)
Operation method
General approach
Principle
1. The fine structure of biological samples is easily damaged. Therefore, fixation is necessary before sample preparation and electron microscopic observation. In order to maximize the retention of its life form. The use of water-soluble, low surface tension organic solutions such as ethanol gradient dehydration of the sample, but also in order to minimize the drying of samples to minimize the surface tension caused by the changes in the natural form of the sample. 2. Currently the most widely used and most effective method is the critical point drying method. The principle is that in a closed container with a solution, as the temperature rises, the evaporation rate accelerates, the density of the gas phase increases. Liquid phase density decreases. When the temperature is adjusted to a certain value, gas, liquid two-phase density is equal, the interface disappears, surface tension does not exist. The temperature and pressure at this time is called the critical point. The biological samples can be dried by replacing the internal dehydrating agent with a substance with a lower critical point, which can completely eliminate the damage of surface tension on the structure of the samples. The most commonly used replacement agent is carbon dioxide. Since carbon dioxide is not very miscible with ethanol, the samples are dehydrated in ethanol grades and then the ethanol is replaced with amyl acetate, which is a "mordant" that is miscible with both substances.
Materials and Instruments
E. coli slant/suspension Move 1. Fixation and dehydration 1.1 Cut the clean coverslips into 4-6 mm2 pieces. 1.2 The thicker E. coli suspension to be examined will be added to it dropwise, or the bacterial moss directly coated, can also be used to cover-slip small pieces of paste colonies on the surface of the natural drying of the optical microscope after microscopy, to the bacterial body is denser, but not piled up together as appropriate; 1.3 Mark the side of the coverslip with the sample; 1.4 Place the above samples in 1%~2% glutaraldehyde phosphate buffer (pH7.2), and fix them in the refrigerator at 4℃ overnight. 1.5 On the following day, the samples were rinsed with 0.15% of the same buffer solution and dehydrated with 40%, 70%, 90% and 100% ethanol for 15 min each time. 1.6 After dehydration, replace the ethanol with amyl acetate. 2. Drying The samples prepared above are placed in a critical point dryer, immersed in liquid carbon dioxide, and heated above the critical point temperature (31.4 °C, 72.8 atmospheres) to vaporize and dry. 3. Spray coating and observation The sample is placed in a vacuum coater, the gold is sprayed onto the surface of the sample, and the sample is removed for observation in a scanning electron microscope. Caveat After the sample has been dehydrated, the organic solvents crowd out the water and take over where the water used to be. The water is removed, but the sample is still impregnated with solvents, which must be "invited" out with as little surface tension as possible so that the sample is truly dry. Common Problems Another similar method of sample preparation is the use of centrifugal washing to sequentially immobilize and dehydrate the organisms and finally apply them to a slide. The advantages are: 1. contact between the organisms and air can be completely avoided during the fixation and dehydration process, thus minimizing the deformation of the organisms caused by natural drying. 2. it ensures that there is sufficient concentration of bacteria in the final slant sample, as the bacteria coated on the slide may sometimes be dislodged from the slide during the fixation and drying process. 3. Ensure that the side of the slide with the sample on it is not misplaced. For more product details, please visit Aladdin Scientific website.
Liquid carbon dioxide 1% to 2% glutaraldehyde phosphate buffer pentyl acetate concentrated sulfuric acid anhydrous ethanol sterile water
General optical microscope Sterile dropper Sterile tweezers Large head pin Slide Coverslip Vacuum coater Critical point dryer