Negative staining solution preparation

Measurements show some variation depending upon the staining solution used and the method of application. In dried and fixed smears, the cell wall and slime layer do not stain with weakly staining dyes such as methylene blue but do stain with the intensely staining pararosaniline, new fuchsin, crystal violet, and methyl violet. The great majority of bacteria have been measured in fixed and stained preparations. In some instances dried, negatively stained smears have been used. Therefore, the method employed should be specified when measurements of bacteria are reported otherwise the results will be of doubtful v alue. 

TEM has been used to determine the shape and particle size of nanoparticles [27, 33]. Samples are prepared by placing a drop of preparation on copper grids, followed by negative staining with an aqueous solution of sodium phosphotungstate, phosphotungstic acid, or uranyl acetate [27, 163, 164]. Freeze fracturing with TEM has been... 

Prepare a positive control bytreatingthe tissue or cells with DNase I for 10 min at 25°C. For negative control, incubate with staining solution only without the enzyme. 

Fig. 7 TEM image of aggregates of PAELi-fc-PLPheg negatively stained with uranyl acetate specimen was prepared by deposition of a drop of a 0.2 wt % polymer solution on a carbon-coated copper grid, drawing-off the solution with filter paper, and subsequent drying in vacuo. Reprinted with permission from [42], copyright (1997) Hiithig Wepf...

Support Films. Since the specimens to be negative stained are generally in solution, some support medium is needed to mount the preparation on a grid for observation. While there are a number of choices in this regard, plastic and carbon are the two main compositional categories. 

Cryogenic transmission microscopy (Cryo-TEM) is an excellent method, by using which the objects in solution can be visualized without fixing and staining. As mentioned above, negative staining is a convenient method for the preparation of TEM specimen however, the object can be visualized only in dry state, which is sometimes much different from the structme in solution. In addition, using Cryo-TEM the structmal information inside the object can be obtained in contrast to freeze-fracture replica method. 

Shaffer et al [365] have continued to modify staining techniques for TEM of latex particles. Recent work on structured latex particles prepared by seeded emulsion polymerization focused on the effects of changes in polymerization variables, such as batch versus semicontinuous, core-shell ratio, shell thickness and shell composition. In this system the core was poly(n-butyl acrylate) and the shell was poly(benzyl methacrylate-styrene). A few drops of the latex was combined with a few drops of a 2% uranyl acetate solution which serves as a negative stain. A drop of that mixture was deposited on a stainless steel formvar-coated grid. After drying it was stained in ruthenium tetroxide vapor to differentiate the rubbery core, which is not... 

This test depends on attaching virus protein to a small laboratory dish. A serum sample is prepared from the blood of the individual to be tested, and it is placed in the dish containing bound HIV viral proteins. If HIV-specific antibodies are present in the serum, they will become tightly bound to the dish by way of the HIV proteins. The serum is then removed, and the dish is washed during this procedure, only antibodies specific for HIV will be retained. The dish is then reacted with a stain that will detect any human antibodies. Thus, dishes that were exposed to serum containing HIV-specific antibodies will be stained, while dishes from antibody-negative serum samples will be unstained. A modified ELISA test was developed in which the virus proteins are attached to small beads that can float in solution, instead of to the bottom of the dish. The test is carried out in a test tube and proceeds as before. 

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