All Glass Nebulizer
EMS Catalog #70505
Negative Staining Using an All Glass Nebulizer
- 4% Phosphotungstic acid ( PTA ) - neutralize to pH 6.3-7.0 with potassium hydroxide pellets. Stir vigorously during neutralization to avoid localized alkaline conditions giving a precipitate. If the PTA clouds up, discard it. Neither the pH nor PTA concentration is critical. The K+ titration gives a salt of very high solubility (80%) and increased electron scattering power. It can yield a virtually grainless background if examined in moist films.
- 1.0% bovine serum albumin (Cohn's fraction V) in water-the protein concentration is not critical. It is best to use it freshly prepared. Add 40-60 mg of powdered BSA to 5 or 6ml of water in a screw-capped test tube. To avoid denaturing the BSA, do not shake the suspension. Lay it down on a bench top an roll it over to wet down the wall. Let it stand 15 to 20 minutes with an occasional roll to get the BSA into solution. Denaturing the BSA destroys its surface activity and its floccules can be seen in the PSA at high magnifications. Highly purified serum albumins, e.g., crystalline samples, do not work as well as the cruder Cohn Fraction V powder.
For a preliminary trial start with the following conditions:
- 20 drops - distilled water
- 4 drops - 4% PTA
- 1 drops - 1.0% BSA
- 1 drop - particle suspension in water or a swab sample.
Mix gently in a depression dish with a Pasteur pipette and apply to a carbon -coated colloidon-filmed 200-mesh grid with an all-glass nebulizer, preferably a Vaponefrin type. Examine immediately in the EM using double condenser illumination to reduce the beam spot size and contamination as well as improve the beam's coherency. Under these conditions, suspensions of viruses in crude cell culture fluids seem to come out okay. The sample dilution of 1 to 20 in the PTA and water usually dilutes the media salts to a level that does not interfere with spreading or contrast. After a quick examination, adjust the PTA , BSA or sample concentration to optimize the spreading and contrast for the particular sample. The best conditions are when the objects of interest are well separated in a thin film of the negative stain. This can be achieved very readily with the use of the nebulizer and appropriate sample dilutions.
Inorganic salts and proteins from culture media and body fluids cause two separate and objectionable conditions to be dealt with. Salts, especially from density gradient samples, tend to form "trees" of crystallites in the droplet centers or may encompass the entire spray droplet. This condition not only obscures the objects of interest but may lead to grossly misleading artifacts. Serum proteins and sucrose may act as surface active agents giving very poor contrast in the specimen because they cause uncontrollable spreading. Both factors may be minimized by use of one or more of the following techniques: centrifuging and resuspending the sample in water; gel filtration on 2 or 4% agarose columns using water as the eluting fluid; or dialysis. Gel filtration seems to be the best all around method.
Under the conditions given above, intact bacteria usually lie in deep puddles of the PTA and are overly contrasted. Adequate contrast is obtained by using only 1 or 2 drops of the 4% PTA with 20 drops of water. Flagella, phages and pili are best examined in the more contrasting medium but can usually be found sufficiently contrasted in one or more spray droplets.
Carbon coated colloidon filmed grids are generally used for two reasons. Under the focused beam, the colloidon sublimes leaving only a carbon film under the specimen area being examined. The carbon film seems to give a consistently hydrophobic surface so the BSA surface activity is under control by adjusting its concentration in the preparation. Formvar films are often hydrophilic giving too much spreading of stray droplets. They also seem to char or contaminate under the beam detracting from the resolution and contrast of finer details on virus particles.
The All-glass nebulizer should produce a fine mist at 2 to 3 mm from its muzzle. Its glass bead just in front of the jet helps to break up large droplets, which should not reach the muzzle. THE best size of droplets is one that gives spread areas somewhat less the size of the opening of a 200 mesh grid. With droplets of this size it is possible to obtain random samples of the original specimen. This makes possible quantitative measurements on the number of particles, e.g., by adding a known volume of some reference suspension, which can serve as an internal standard against which other objects are counted.
For a good general discussion of negative staining and the use of an all glass nebulizer see R.W. Horn's article, "Electron Microscopy of Isolated Virus Particles and Their Components" that appears as chapter 11 (Vol. III ) of Methods in Virology, edited by K. Marmorosch and H. Koprowski, Academic press, New York 1967, pp 521-574.
Care and Cleaning Of All-Glass Nebulizer:
Solutions should not be allowed to evaporate to dryness in the nebulizer. After use, plug up the openings with corks.
To clean, pour out any remaining sample solution and aspirate with the rubber bulb to completely empty the capillary feed tube. Rinse out the unit thoroughly with hot water ( or a detergent solution) and aspirate again to empty the capillary feed tube. Repeat as often as necessary to insure complete flushing with clean water. Usually three or four times is adequate. After the final aspiration, dry the nebulizer upside down so that water will not siphon up into the capillary feed tube.
With infectious samples the nebulizer may be soaked in a strong disinfectant (amphyl or Cidex, etc.) before flushing and rinsing as described above.