I am involved in materials science and not biology. However, such a fundamental question should probably be addressed in a different forum.

There is undoubtedly much written about preparing bacterial samples for EM. Please check the literature. Also, vendors in the field will often publish whitepapers on such subjects showing how their wares prove helpful. To do so, they often include plenty of details.

You might have specifically indicated bacteria in your title and TEM (or SEM, but the recommendation would have been the same.

Sample Preparation for Electron Microscopy Imaging Preparing samples for electron microscopy (EM) is a critical step to ensure clear, high-resolution imaging of both biological and material specimens. The preparation method depends on the type of EM technique being used—Transmission Electron Microscopy (TEM) or Scanning Electron Microscopy (SEM)—as well as the nature of the sample. The goal is to preserve structural integrity, prevent artifacts, and enhance contrast for effective visualization under the electron beam.

For Transmission Electron Microscopy (TEM), the sample needs to be extremely thin, typically around 50 to 100 nanometers, to allow electrons to transmit through it. The process begins with chemical fixation using agents like glutaraldehyde to preserve protein structures, followed by osmium tetroxide to stabilize lipid components and enhance contrast. After fixation, the sample undergoes a dehydration process through a graded ethanol or acetone series to remove water, which is incompatible with the vacuum inside the microscope. The dehydrated sample is then embedded in a resin to provide support for ultrathin sectioning. Using an ultramicrotome, sections are cut and placed onto copper grids. These sections are then stained with heavy metals such as uranyl acetate and lead citrate to improve electron density and contrast. Once stained, the sample is ready for imaging under TEM.

In the case of Scanning Electron Microscopy (SEM), the emphasis is on examining the surface morphology of the sample rather than internal structures. The sample is first fixed with glutaraldehyde, and for biological specimens, post-fixation with osmium tetroxide is also performed. Dehydration is carried out using an ethanol or acetone series, similar to TEM preparation. To prevent structural collapse during drying, critical point drying (CPD) is often used, especially for delicate biological specimens. The dried sample is mounted onto a metal stub using conductive adhesives like carbon tape. To prevent charging under the electron beam, a thin conductive coating of gold, platinum, or carbon is applied via sputter coating. The coated sample can then be imaged under SEM in a vacuum environment.

Additionally, for highly sensitive or hydrated samples, specialized techniques like Cryo-EM are employed. In this method, samples are rapidly frozen to preserve their native state without the need for staining or dehydration. For materials science applications, techniques like ion milling or focused ion beam (FIB) preparation may be used to create thin sections suitable for TEM analysis. Overall, meticulous sample preparation is essential to obtain artifact-free, high-contrast images in electron microscopy, tailored to the specific requirements of the specimen and the imaging technique used.