Replicas, Shadowing, and Negative 271 CHAPTER 8 TECHNIQUES

Vacuum Evaporation

Vacuum evaporators are used to coat substrates or specimens with carbon and/or metals and to pro• duce pure carbon substrates for specimen support. The duration of evaporation, temperature of electrodes during evaporation (amperage used), angle of evaporation, vacuum at the time of evaporation, material being evaporated, and the temperature of the stage holding the specimen or support being coated are all factors for the end-product desired. As stated earlier, the shortest duration that delivers a usable coat, the lowest source (electrode) temperature that will cause evaporation, the largest angle between the source and substrate, the highest vacuum, the metal with the smallest grain structure, and a cooled stage will work together to produce the finest grain structure on the coated substrate. However, since other factors must be considered, all of these conditions cannot always be met. When shadowing a substrate with topography (e.g., a freeze-fracture specimen), contrast is developed by the dep• osition of metal/carbon on one side of a particulate specimen, with a clear area behind the specimen that is not coated. This area is known as the shadow. Taller objects (like protozoan cells) are typically shadowed from one direction with metal at an angle of 45°, followed by a stabilizing carbon coat from sources directly overhead. Alternatively, carbon and metal are deposited simultaneously at a 45" angle. Specimens with little relief (nucleic acids, bacterial pili, ) are shadowed at a lower (10°) angle (Fig. 165A). If specimens are supported on a stage that is rotated rapidly during evaporation, the specimen is coated from all sides, producing very little shadow, but giving overall bulk to the sample (Fig. 1658). Carbon is evaporated onto most metal coatings to provide further mechanical stability to the coat. It is also evaporated onto plastic film-coated grids to provide mechanical stability and electrical conductivity to decrease thermal drift when the electron beam interacts with the coated grid. The barest perceptible car• bon coat is usually adequate for this purpose. Finally, as already described in the section on film-coating grids, thicker carbon coats can be evapo• rated onto mica, which are then stripped from the mica substrate. The carbon film is then used to coat grids, ultimately serving as a support film for specimens. A variety of metals requiring different evaporative temperatures can be used in shadowing. Those requiring higher evaporative temperatures have a finer grain structure. Using metal alloys or evaporating a metal and carbon at the same time produces finer-grain coatings, because nucleation of single metals takes place more rapidly than mixtures, resulting in larger deposited grains. For freeze-fracturing and similar applications, the objective is to produce an electron-transparent replica of the specimen surface. The specimen is digested with acids or bases so that only the replicated sur• face of the specimen is examined with the electron . In the past, vacuum evaporators were used to coat SEM samples by attaching the samples to rotating, tilting stages during the coating process in an attempt to coat all the hills and valleys in typical SEM speci• mens with lots of topographic detail. With the advent of sputter coaters that have nondirectional metal deposition on sample surfaces, the vacuum evaporator has been retired from most SEM specimen preparations.

Shadow Casting 1. Applications and Objectives Shadow casting metals onto specimens at high or low angles from a fixed direction is used to develop contrast in a specimen (tall or short, respectively) as a result of topographic differences in specimen details. Shadow casting of specimens on a rotating stage confers bulk to a sample with little topography. It is also possible to calculate the height of specimen structures by measuring shadow length and relating it to the angle of shadow.

M. J. Dykstra et al., Biological Electron © Michael J. Dykstra 2003 272 Chapter 8

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Figure 165. Escherichia coli coated with platinum-palladium. (A) Low-angle. fixed-direction coating. 51.474 x. (B) Low-angle, rotary coating. 65,337X.

2. Materials Needed

• Vacuum evaporator • Carbon electrodes • Metal electrodes of choice • Specimen (dried or, in the case of freeze-fracture, frozen) • Grids • Pieces of 3 X 5 in. (7.6 X 12.7 cm) card stock • Carbon electrode sharpener • 400-grit emery paper Replicas, Shadowing, and Negative Staining 273

3. Procedure

Carbon Electrode Preparation 1. Polish the ends of both carbon electrodes by first sanding them on a piece of 400-grit emery paper on a counter top and then polishing the sanded surface on a piece of cardboard or card stock until the sanded surface is shiny. 2. Use the electrode sharpener to sharpen the polished end of one electrode so that the final electrode has a reduced-diameter end about 5 mm long (Fig. 160). 3. Install the carbon rods with the sharpened rod in the driven electrode and the other attached to the fixed electrode. The rods should meet midway between the two electrodes, which are spread as far apart as possible. After the rods are installed, make sure that the driven electrode is free to travel when the carbon rods are evaporated.

Wire Evaporation (Platinum-Palladium)

1. Ball up 1.5 cm of platinum-palladium wire with a pair of coarse-tipped forceps. 2. Place the wire into a tungsten basket attached to a set of electrodes. 3. Load the specimen and place a small piece of 3 X 5 in. (7.6 X 12.7 cm) card stock for monitoring coating thickness near the specimen. The card should have been dried previously in a resin poly• merizing oven so that no residual atomospheric moisture remains, which would slow down the pumping speed of the vacuum evaporator. Pump down the vacuum system as specified in the vacuum evaporator instructions. 4. Once the vacuum evaporator has reached high vacuum (1.3 X 10-2 Pa or better), slowly tum up the electrode current with the electrode rheostat until the wire in the basket melts. It is important to do this slowly enough so that the wire fuses with the tungsten basket. If the current is raised too quickly without careful visual monitoring, the noble metal wire can simply be cut by the heated tungsten and fall in large lumps to the vacuum chamber floor, producing no coating on the specimen. Caution: Wear welder's goggles to prevent eye injury from the bright electrodes.

Firing the Wire Electrodes 1. For a fixed-angle bum, once the platinum-palladium is welded to the tungsten basket, continue rais• ing the electrode current slowly until the card stock begins to darken. When this is noted, do not increase the current any further. Observe the lumps of platinum-palladium welded to the basket. After they have evaporated, return the electrode rheostat to the off position. 2. For a rotary bum, the procedure is identical except that the rotary specimen holder should be turned on prior to the bum. The specimen should rotate at about 100 rpm.

Firing the Carbon Electrodes 1. Carbon rods are fired alone to coat polymer film-coated grids or to produce pure carbon films as specimen supports. They are also fired after the platinum-palladium has been evaporated onto a specimen surface to add electron-transparent physical support to the metal coat. To fire the electrodes, switch the electrode control to the set bearing the carbon rods, and slowly tum up the electrode rheostat until the pointed carbon rod begins to evaporate. Some sparking during this process is acceptable, but excessive sparks indicate that large chunks of the carbon rod are being discharged, which can result in debris on the specimen. 2. When coating a plastic support film or evaporating carbon onto a metal layer previously evaporated onto the specimen surface, a very thin carbon coat (light brown) is adequate. If coating a mica sub• strate to produce a carbon support film, a thicker (black) film is needed, so bum the electrodes until the sharpened tip has been consumed. 274 Chapter 8

4. Results Expected

The evaporated metal coat from a fixed-direction shadowing run will have a fine grain and will produce a shadow where no metal has been deposited behind a specimen area with topography. A rotary coating run will produce a specimen with increased bulk but without much evident shadow. A carbon coating will not be noticeable under the electron beam, but will have contributed structural stability and electrical conductivity to the specimen.

5. Cautionary Statements Always protect your eyes with welder's goggles during electrode firing, as the high-intensity light from the electrodes can damage the retina. The use of card stock to monitor coat thickness works well in the absence of an expensive crystal film monitor that can actually record the number of nanometers of film being deposited during electrode firing. Some workers use immersion oil or diffusion oil drops on a piece of broken white porcelain crucible lid to monitor film thickness. The vacuum may cause vaporization of the oil, however, coating surfaces within the vacuum chamber, including the specimen, and leading to problems for developing a good vacuum over time and to poor coating of the specimen surface. If oil is used, diffusion oil is preferred, because it is already part of the pumping system and has a lower vapor pressure than immer• sion oil.

DNA (Plasmid) Preparation for TEM' 1. Application and Objectives

This is a quick method for the preparation of purified short segments of nucleic acids (e.g., plasmids) for visualization in a transmission . The technique confers sufficient bulk and contrast to isolated short pieces of nucleic acids for the purpose of measuring their length or assessing whether they are single-stranded or double-stranded utilizing a transmission electron microscope.

2. Materials Needed • I mg/ml of cytochrome C (Sigma 7752, Type VI) in distilled water • 0.25 M ammonium acetate in distilled water, pH 7.5 • Distilled water • 50% ethanol • 0.5-5.0 fLg/ml of nucleic acid in distilled water • Parafilm ™ • Petri dishes • Micropipets (2-10 fLm and 10-100 fLm adjustable) and tips • 4% uranyl acetate stock (alcoholic or aqueous) • 200-mesh Formvar-coated grids • Vacuum evaporator with flat rotary stage • Double-stick tape • 1.5-cm platinum-palladium wire • Microfuge tubes • Shell vial or scintillation tube for mixing uranyl acetate • Forceps • Whatman™ No.1 filter paper (9-cm disks)

I Special thanks are due to Chris Dykstra, College of Veterinary Medicine, Auburn University, for introducing us to this technique. Replicas, Shadowing, and Negative Staining 275

3. Procedure

I. Mix the following in a microfuge tube: .40 ILl of 0.25 M ammonium acetate, pH 7.5 • 10 ILl of 0.5-5.0 ILg/ml of DNA in distilled water 2. Let the mixture sit for I min in the microfuge tube. 3. To the microfuge tube, add 1.0 ILl of 1 mg/ml of cytochrome C (Sigma 7752, Type VI) in water. Invert the capped tube several times. 4. Place 51 ILl of solution from microfuge tube onto Parafilm™ in a Petri dish, cover the dish, and wait for 1 min. 5. Touch the Formvar-coated side of a grid to the drop. More than one grid can be touched to the same drop. 6. Immediately stain in dilute uranyl acetate in 50% ethanol prepared by adding one drop of normal poststain stock to 20 ml of ethanol in a shell vial. Dip the grid into the shell vial of uranyl acetate in ethanol for 5 sec. 7. To destain the stained grid, dip the stained grid into 50% ethanol for 5 sec immediately after it is removed from the staining mixture. 8. Dry the destained grid quickly and completely with a piece of filter paper after destaining (see section in Chapter 4 on grid drying procedure). 9. After 15 min, stick the edge of the grid specimen-side up to a piece of double-stick tape previously applied to the surface of the horizontally rotating specimen support in the vacuum evaporator. Put the grid near the center of the rotating stage, and situate the center of the stage 1.5 cm below and 8 cm horizontally away from the platinum-palladium source. This gives a 10° shadowing angle. 10. Pump the vacuum evaporator chamber down to 1.33 X 10-3 Pa, and then fuse the platinum• palladium wire with the tungsten basket, as described in the instructions for shadowing. II. Tum on the rotary stage and allow to rotate at 100 rpm. 12. Fire the platinum-palladium-coated tungsten basket, as described under shadowing, and remove the grid from the vacuum chamber. 13. Examine the coated nucleic acid with a TEM. The nucleic acids should be easily seen at a TEM screen magnification of 12,000X or above.

4. Results Expected The nucleic acids should be well spread out on the surface of the coated grid and should have ade• quate bulk to be seen easily (Fig. 166). The metal coating should not be objectionably grainy.

5. Cautionary Statements All cytochrome C is not the same. The cytochrome C is used to spread the nucleic acid over the drop to which the coated grid is touched. If the nucleic acid is not evenly spread over the grid surface, try another pro• duction batch of cytochrome C. Once a satisfactory stock of cytochrome C is demonstrated, it is good practice to make up aliquots of appropriate quantity in microfuge tubes and to store them frozen at - 20°C until needed. Before proceeding with this method, review the instructions for shadowing and the specific instruc• tions for your vacuum evaporator. Also, review the cautionary statements about uranyl acetate provided in the section on post staining in Chapter 4.

Negative Staining

Before negative staining, particulate samples must generally be concentrated (see the sections on viral preparation and agar embedment of particulates for methods). The actual final concentration of specimens prepared for negative staining is often empirical, but some starting points are available. With , a faintly turbid suspension is desired. Harris and Home (1991) suggest that 106 to 109 virions/ml will provide 276 Chapter 8

Figure 166. Plasmid DNA stained with uranyl acetate and shadowed with platinum-palladium. 68,654X. a good preparation. while solutions should contain 0.l-0.2 mg/ml, suspensions should be con• centrated to 0.1-0.5 mg/ml, and cell membrane suspensions should contain 0.5-1.0 mg/ml. Materials can be suspended in 5-10 mM saline or buffer, but should not be suspended in sucrose from sucrose gradients because it will interfere with negative staining.

Negative Staining with 1. Applications and Objectives Negative staining with slightly alkaline PTA is commonly used for many types of particulate samples. Nermut (1982) reports that this stain is most appropriate for the proteinaceous capsomeres of viruses, while uranyl acetate at acidic pH is superior for the membranous components and glycoprotein spikes associated with the surfaces of enveloped viruses. The objective is to produce a finely electron-dense coating over the entire surface of a film-coated grid bearing particulate materials, with only slight stain pooling around the particulates, allowing easy visualization of surface structures of the particulate samples (viral capsomeres, bacterial pili or flagella, protozoan cell surface scales, etc.).

2. Materials Needed

• Sodium phosphotungstate • INNaOH • 100-ml stock bottle • Distilled water • Pasteur pipets • pH meter • Forrnvar-coated grids • Concentrated particulate sample suspended in a small volume of fluid • Forceps • Whatman™ No. I filter paper (9-cm disks) Replicas, Shadowing, and Negative Staining 277

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Figure 167. Negative stain procedure. (Al Forceps lying on a Petri dish lid with the grid held in jaws kept closed with forceps locking ring.

3. Procedure Stain Preparation. Add 1 g of sodium phosphotungstate powder to 50 ml of distilled water in a 100-ml stock bottle. Check the PTA solution pH with the pH meter, and adjust the pH to 7.2-7.4 with the 0.1 N NaOH.

Staining Method

1. Pick up a Formvar-coated grid with forceps and push the forceps locking ring down so that the grid is held firmly. Lay the forceps down on the Petri dish lid with the tips extending over the edge with the grid held coated-side up (Fig. 167 A). 2. Add one drop of concentrated particulate suspension to the grid with a Pasteur pipet (Fig. l67B). See the section on preparing viral samples for negative staining for preparation suggestions. 3. After 3-5 min, remove the suspension by touching the ragged, tom edge of filter paper to the edge of the forceps jaws where they contact the grid (Fig. l67C) until the grid surface is nearly dry. Never let the grid surface totally dry out because it will produce a coating of culture or sample materials and yield an excessively dirty grid. 4. Add one drop of 2% PTA to the grid. 5. After I min, dry the grid as before with the ragged tom edge of filter paper, except, this time, dry the grid quickly and completely. Immediately touch the sample side of the grid to a clean piece of filter paper in the bottom of a Petri dish. Slide a fresh piece of filter paper down between the forceps jaws to push the dried grid out of the forceps tips and onto a clean, dry part of the filter paper in the Petri dish. 6. Let the grid dry for 15 min in the Petri dish (covered) and then examine with the TEM.

4. Results Expected

The grid will have a finely granular electron density over the entire surface, with slightly darker regions surrounding the particulate sample, but the stain will be generally excluded from intact particulate materials (Fig. 168). 278 Chapter 8

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Figure 167. (B) Addition of one drop of particulate suspension to film·coated grid with Pasteur pipet. (C) Wicking grid almost to dryness by means of a piece of Whatman ™ No. 1 filter paper.

5. Cautionary Statements Negative stains are all and thus should be handled as the toxic and hazardous materials that they are. Dried droplets of stain should be handled with care to avoid inhalation. All wastes should be disposed of according to the institutional guidelines for hazardous wastes. If working with pathogenic viruses or bacteria. it should be noted that negative staining does not kill all pathogens. Thus, the infectious agent should be inactivated prior to negative staining, or the grid should be sterilized by gas or ultraviolet radiation prior to being handled or put into an electron microscope. If the viability of a pathogen is in question, proper safety precautions should be employed, and, following negative staining, the grid should be tested for pathogen viability before determining the necessary safety procedures to be employed in future studies of the agent. Replicas, Shadowing, and Negative Staining 279

Figure 168. Adenovirus particles negatively stained with 2% PTA. 81,731 x.

Some older papers and procedure manuals suggest the use of nebulizers to produce a fine mist of viral suspension that is directed toward a coated grid. This procedure should not be used under any circumstances, since atomizing a suspension of viral particles does not conform to safe laboratory practices. A PTA solution will keep at 4°C for at least 3 years.

Negative Staining with Uranyl Acetate

1. Applications and Objectives

Uranyl acetate used as a negative stain at an acidic pH is superior for the demonstration of glycopro• tein spikes and knobs associated with enveloped viruses, and develops a greater contrast than ammonium molybdate and PTA. The grain size of the uranyl acetate stain is greater than that of these other two negative stains (Nermut, 1982). Uranyl acetate negative staining is also successful for staining ultrathin frozen sections produced by the Tokuyasu technique (see Chapter 2). Proper staining with uranyl acetate will produce a faint electron density over the entire Formvar-coated grid surface, with minimal pooling around the particulate sample and minimal evidence of uranyl acetate crystals.

2. Materials Needed The same materials are needed as listed for the PTA staining method, except that a 0.2-4.0% aque• ous solution of uranyl acetate with a pH of 4-5 is used in place of PTA.

3. Procedure

The procedure is identical to that used for PTA negative staining.

4. Results Expected

The grid surface will be slightly electron-dense overall. The particulate sample will be revealed by negative contrast where the uranyl acetate is excluded. Some of the particulate sample will also exhibit positive staining from the uranyl acetate. 280 Chapter 8

5. Cautionary Statements

A 4% aqueous solution of uranyl acetate is saturated, so large crystals of insoluble uranyl acetate will be found on the bottom of the storage bottle. For this reason, it is important not to pipet the stain from the bottom of the container to avoid the presence of numerous needle-like crystals of uranyl acetate on the surface of the negatively stained grid. A solution of uranyl acetate will keep for weeks at room temperature in a dark bottle. If the solution appears turbid, do not use it. Hayat and Miller (1990) state that uranyl acetate stains are not stable above pH 6.0 and that a I % aqueous solution is good for visualizing macromolecules. Uranyl acetate is a toxic heavy metal and should be used and disposed of accordingly.

Negative Staining with Ammonium Molybdate

1. Applications and Objectives

Ammonium molybdate is an ideal fine-grain negative stain that will confer electron density and con• trast to ultrathin frozen sections.

2. Materials Needed

This technique requires the same materials as called for with PTA negative staining, except that instead of PTA, a 3% aqueous solution of ammonium molybdate is used. This solution will have a pH of 6.5 without any adjustment.

3. Procedure

The staining procedure is identical to that described for PTA.

4. Results Expected

Sections stained with ammonium molybdate will have a reasonable contrast and a fine grain structure, revealing details of the frozen tissue/cell structures as a negative image.

5. Cautionary Statements

As with all the negative stains, this heavy metal should be handled and disposed of with methods appropriate to hazardous materials. The effect of this metal on the viability of negatively stained samples is not thoroughly documented, so it should be assumed that pathogens are still viable after staining unless they have been specifically tested.

Wetting Agents Used for Negative Staining

1. Applications and Objectives

If negatively stained preparations exhibit uneven spreading of the stain or sample, or have excessive pooling of the negative stain around the perimeter of the sample particulates, or if large structures such as protozoan wall plates (scales) roll up rather than remaining flat (Dykstra, 1976), wetting agents may help. The most commonly utilized wetting agents are the antibiotic bacitracin (Gregory and Pirie, 1973) or BSA. Both of these substances are and are, in themselves, visible after negative staining. These agents cause the sample and/or the negative stain itself to spread evenly on the film-coated grid surface. Replicas, Shadowing, and Negative Staining 281

2. Materials Needed

• 500 j..lg/ml of bacitracin in distilled water or 0.01--0.02% BSA in distilled water • Formvar-coated grids • Forceps • Pasteur pipets • Parafilm™ • Whatman™ No.1 filter paper (9-cm disks) • Petri dishes (lO-cm)

3. Procedure For Spreading the Negative Stain

I. Place a suspension of the particulates on the surface of the filmed grid for 3-5 min and then wick almost to dryness. 2. Immediately replace with a drop of the chosen negative stain mixed 1: 1 with either of the wetting agents (bacitracin, BSA) just before use. 3. After 30-60 sec, wick the grid quickly and completely to dryness, as described for the PTA nega• tive stain procedure.

For "Relaxing" Large Specimens I. Mix one drop of bacitracin solution with one drop of concentrated particulate suspension on a piece of Parafilm TM. 2. Place one drop of mixture on a Formvar-coated grid for 3-5 min. 3. Wick almost to dryness, add the negative stain of choice, and wick to total dryness after 30-60 s, as previously described.

4. Results Expected The negative stain should be evenly spread on the Formvar-coated grid surface, with minimal pool• ing around the particulate sample. Any large structures in the particulate sample should be well relaxed and spread out (Fig. 169).

5. Cautionary Statements All normal precautions for negative stain procedures listed under negative stains should be observed. Both bacitracin and BSA will be seen after negative staining as small white dots excluding the negative stain. Bacitracin is a smaller molecule than BSA, so the dots will be smaller with the former wetting agent.

Preparation of Samples for Transmission Electron Microscopy

The preparation of viral samples for negative staining and examination with the transmission electron microscope may be accomplished by the application of several different techniques. If large amounts of virus can be harvested and an ultracentrifuge is available, ultracentrifugation techniques work quite well. If only small amounts of virus are available from the sample being examined, immune electron microscopy tech• niques may be the best approach. Viruses that cannot be grown in tissue culture can be negatively stained after isolation from the host tissues or body fluids. Viruses also can be passed through cell cultures and then negatively stained. 282 Chapter 8

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Figure 169. Cell wall scales of the protozoan Sorodiplophrys stercorea. (A) Negatively stained scales before using a wetting agent. 37,340X. (8) Scales after being "relaxed" by exposure to bacitracin. 28,938X.

Ultracentrifugation Technique for Viral Sample Preparation

1. Applications and Objectives This technique is utilized to concentrate viral particles from infected cell cultures, tissues, or body fluids. For this technique to be highly successful, the number of viral particles needs to be fairly high. Replicas, Shadowing, and Negative Staining 283

2. Materials Needed

• Pasteur pipets • IS-ml clinical centrifuge tubes • Ultracentrifuge tubes • Clinical centrifuge • Ultracentrifuge • Distilled water

3. Procedure

1. Collect the sample, dilute about 1: to with PBS, and store frozen if necessary. Do not sonicate cell samples or scrape mucosal linings to release viral particles from cells. Sometimes, it is useful to freeze (at -20°C) and thaw infected cell cultures several times to release viral particles. 2. Centrifuge the sample at approximately 1,000 rcf for S min or until clarified on a clinical centrifuge. 3. Centrifuge the supernate from step 2 at lO,OOOrcf for 30 min. 4. Centrifuge the supernate from step 3 at 40,000 rcf for 60 min. S. Discard the supernate from step 4, and resuspend the remaining pellet in about 0.1 ml of distilled water or PBS. If a pellet can be seen after the final centrifugation, remove all but about 0.1 ml of the supernate very gently with a Pasteur pipet. 6. Negative stain the resuspended pellet material (or O.I-ml droplet) according to the instructions for the negative stain of your choice.

4. Results Expected

Viral particles will be visible after negative staining procedures.

5. Cautionary Statements

All normal precautions for dealing with potential viral pathogens should be observed, and all cautions for handling and disposal of the heavy metal negative stains should be followed. Sonication is not recommended at any time for cells containing viral particles, since membranes tend to become disorganized, and many will reanneal with each other to produce extremely small vesicles that can be confused with some viral particles. Freeze-thawing will release virions from cells without producing vast numbers of small vesicles.

Immune Electron Microscopy for Concentrating Viruses (King and Lecce, 1980)

1. Applications and Objectives If an antibody has been raised against a virus, it can be used to concentrate and identify the virus prior to negative staining. In addition to providing a technique to specifically identify the viral isolate involved, the technique can be used to concentrate relatively small amounts of virus for negative staining.

2. Materials Needed

• Antibody against virus (IgG fraction against rotavirus, for example) • Formvar-coated grids • Negative stain of choice • Forceps • Whatman™ No.1 filter paper (9-cm disks) • Pasteur pipets • Petri dishes (lO-cm) 284 Chapter 8

3. Procedure 1. Prepare antibody to the virus. 2. Pipet a drop of antibody suspension onto a Formvar-coated grid, let sit for 5 min, dip briefly in dis• tilled water, and then wick to dryness with a piece of filter paper. 3. Pipet a drop of fluid suspected of containing virus onto the antibody-coated grid. After 3-5 min, dip briefly in distilled water and wick almost to dryness with filter paper. 4. Negative-stain as described for PTA (or stain of your choice) for 30-60 sec and dry.

4. Results Expected

If the virus is recognized by the antibody, the virions will be bound to the coated grid and will be vis• ible after negative staining (Fig. 170).

Figure 170. Immunoelectron microscopy of virus showing a coronavirus and a smaller helper virus coaggregated with specific antibody raised against the viruses. Negatively stained with PTA. 48,894 X.

5. Cautionary Statements Handle the viral suspension and the heavy metal stain with all the necessary precautions called for with potential pathogens and heavy metals. If the grid is not dipped briefly after the antibody is bound to the Formvar coat, excessive negative stain may be bound to nonspecific serum proteins dried to the Formvar surface. An alternative method involves mixing the antibody and viral suspension prior to placing it on a Formvar-coated grid. This would reveal clumps of antibody/virus after negative staining.

Viral Concentration with the Beckman Airfuge2

1. Applications and Objectives This is a quick method to prepare viral samples from fecal samples or fractionated cell cultures for negative staining and transmission electron microscopy.

2 Special thanks are due to Dr. William Steffens, College of Veterinary Medicine, University of Georgia, for introducing us to this technique. Replicas, Shadowing, and Negative Staining 285

2. Materials Needed

• Beckman Airfuge with 4 Position rotor • 1.5-ml polycarbonate tubes (S/16X 1%,0.8 X 3.5 em) • 15-ml conical centrifuge tubes • Clinical centrifuge • DPBS or appropriate buffer • Vortexing device • Pasteur pipets • Forceps • Whatman™ No. I filter paper (9-cm disks) • Petri dishes (IO-cm) • Negative stain

3. Procedure

1. If working with cells, freeze-thaw them several times (alternating -20°C and room temperature) to fractionate the cells. 2. Place the fractionated cells or fecal sample (the latter diluted approximately 1:15 with DPBS) in a 15-ml conical centrifuge tube. 3. Centrifuge the samples at top speed on a clinical centrifuge for 5 min (or approximately 1,000 ref). 4. Remove the supernate with a Pasteur pipet, and centrifuge for 2 min at half speed on a Beckman Airfuge in polycarbonate tubes. 5. Remove the supernate with a Pasteur pipet, and centrifuge for 2 min at full speed on the Beckman Airfuge. 6. Remove all but one drop of supernate with a Pasteur pipet. 7. Resuspend the "pellet" in the drop of supernate remaining by vortexing the tube. 8. Remove a single drop and place this on a Formvar-coated grid, leave for 4-5 min, wick almost to dryness, and negative-stain with the appropriate heavy metal, as described under the section on negative staining.

4. Results Expected The virions will be sufficiently concentrated so that negative staining will reveal them on the Formvar-coated grids.

5. Cautionary Statements All the normal precautions for handling potential pathogens and heavy metals should be observed.

References

Dykstra, MJ. 1976. Wall and membrane biogenesis in the unusuallabyrinthulid-like organism Sorodiplophrys stercorea. Protoplasma 87: 329. Gregory, D.W. , and Pirie, J.S. 1973. Wetting agents for biological electron microscopy: l. General considerations and negative staining. 1. Microsc. 99: 261. Harris, R., and Home, R. 1991. Negative staining. In: J.R. Harris (ed.), Electron microscopy in biology. A practical approach (pp. 203-228). Oxford University Press, New York. Hayat, M.A., and Miller, S.E. 1990. Negative staining. McGraw-Hill, New York. King, M.W., and Lecce, J.G. 1980. Immune affinity electron microscopy: A rapid method for the detection of rotavirus in fecal suspensions. Proc. Southeast Electron. Microsc. Soc. 3: 28. Nermut, M.V. 1982. Advanced methods in electron microscopy of viruses. In: C.R. Howard (ed.), New developments in practical virology. Alan R. Liss, New York.