Ch18

R.F. Lee

The importance of the plant laboratory cannot be overemphasized. The ability to grow indicator plants that can be effectively used to detect the presence of viruses is fundamental to the success of a clean stock programme. The availability of a laboratory and basic laboratory equipment for certain diagnostic procedures is also important. When a virus-detection laboratory is being established, the question soon arises as to what equipment is needed, where it can be bought and the cost. In this section the basic laboratory equipment and the specialized equipment needed for three commonly used diagnostic procedures (ELISA, electrophoresis and culturing) are given, with the recent price range in the United States.

Much of the following equipment may be obtained from local suppliers. As trade names may vary, the basic requirements are given rather than specific brands and model numbers. Each year the world-wide Laboratory buyer s guide is published for purchasers of laboratory equipment, chemicals and reagents. It includes product listings, a manufacturers' directory and a list of laboratory dealers around the world. The Laboratory buyer s guide may be obtained by sending US$25 to International Scientific Communications Inc., 30 Controls Dr., PO Box 870, Shelton, CT 06484-0870, USA [Telephone (203)926-9300, Telex 964292, Fax (203)9269310].

While more specialized equipment is needed for some of the diagnostic procedures, there are a few basic requirements for any laboratory.

Size

A minimum area of about 55 m² is needed for a diagnostic laboratory. Thought needs to be given to designing effective work areas so that equipment and items needed for a particular procedure are conveniently located. With a proper arrangement of work areas, up to four persons can work effectively in a laboratory of this size. There should be a fume hood available, plus a sink for dishwashing and cabinets for storage of glassware, equipment and reagents.

Electrical power

A source of stable electrical power is necessary. If the power source is subject to interruptions or may be down for long periods, a generator should be available to power important equipment and to permit work to continue. Most electrical equipment manufactured in the United States for domestic use is 60 Hz (cycles per second). It is important that power sources and equipment be able to handle the cycle sequence in the country of use.

Air conditioning

Electronic equipment and instruments depending upon optical filters and diffraction gratings for operation, such as spectrophotometers and ELISA plate readers, are sensitive to high temperatures and high humidity. Fungi can ruin filters and gratings essential to the operation of spectrophotometers, and high humidity will cause corrosion of even solid-state electronic circuitry.

Deionized water supply

Costs for this vary depending on local water quality; usually a mixed bed resin filter with a resistance meter is adequate. Culturing of microorganisms requires very high-quality water and a still may be needed.

Glassware

Although a single piece of glassware represents a small part of the total cost of equipping a laboratory, sufficient glassware is fundamental for effective operation of a diagnostic laboratory. A complete set of volumetric flasks and graduated cylinders needs to be available, as well as flasks, beakers, Petri dishes, test-tubes, centrifuge tubes, test-tube racks, ice containers, spatulas and containers for buffers and solutions. If 500 samples are to be run for ELISA, there need to be 500 tubes with racks to hold the samples. Allow US$2 000 to $3 000 for glassware to equip a diagnostic laboratory.

pH meter

The laboratory must have a pH meter capable of reading to 0.1 pH unit, with reference standards so that the instrument can be calibrated and tested. Select an electrode that will measure pH of Tris buffers. Prices start at about $100 for a hand-held unit and rise depending on the features selected.

Refrigerator

A refrigerator is needed for storage of reagents, chemicals and seed at 4°C. It should be large enough to hold racks of ELISA tubes until the ELISA test is complete. A refrigerator with sliding glass doors is desirable. If large enough, it can be used to run native gels for PAGE with the power supply located outside. Similarly, small centrifuges can be run inside a larger refrigerator and observed through the glass doors.

Freezer

A freezer is needed for storage of reagents, chemicals and samples at -2°C and to produce ice needed for use in ice-baths. Select a freezer that does not have an automatic defrost cycle; the heating cycles of an automatic defrost unit will cause a more rapid breakdown of reagents and nucleic acid preparations stored in the unit.

Balance

A balance is needed with sensitivity of at least 0.01 g. A top-loading electronic balance with duel-range sensitivity of 0.01 to 120 g and 0.1 to 1 200 g is ideal. Prices start at about $1 400.

Means of sterilizing equipment

An autoclave is useful for sterilizing equipment, glassware and reagents used for many diagnostic procedures, and it is absolutely essential for culturing. A large pressure-cooker will serve this purpose and is relatively inexpensive, while a small automated autoclave (prices begin at about $3 000) is convenient if much culturing is to be done. For sterilization of glassware, a glassware oven will be suitable.

Centrifuge

Ideally the centrifuge should be a refrigerated model with a rotor capable of holding 50-ml centrifuge tubes and adapters to accommodate smaller tubes. Prices of such centrifuges start at about $10 000. At the simplest, a clinical centrifuge with interchangeable rotors to accommodate different sizes of centrifuge tubes will suffice in many procedures (at a cost beginning at about $1 000). If the centrifuge is small enough, it can be operated in the bottom of a refrigerator for cooling. Often changes must be made to established protocols because of limitations in the ability to carry out the centrifugation steps. In some instances the sample must be divided into two tubes because the total volume specified will not fit into one tube, or the centrifuge time must be increased to account for a centrifugal force lower than that specified in the protocol.

Magnetic stirrer and hot plate

These can be purchased as separate units at costs starting at about $ 125 each or as a combined unit at costs starting at about $300.

SPECIALIZED EQUIPMENT FOR ELISA

Antisera and/or conjugates needed for ELISA are commonly available from type-culture collections, commercial sources or fellow research scientists. A low-speed centrifuge, UVvisible spectrophotometer and simple chromatography are needed if IgG and conjugates are to be prepared. Other equipment needed for ELISA is the following.

Repeating pipette

A repeating pipette which allows multiple pipettings with good accuracy is essential. At the minimum, a fixed volume (200 ml) pipette is required (cost about $125 to $ 250). It is highly desirable to have a set of three adjustable pipettes (0 to 20 ml, 20 to 200 ml and 200 to 1 000 ml) at a cost of about $125 to $250 each. If much ELISA is to be performed, a multichannel adjustable (50 to 250 ml) pipette should be considered at costs beginning at about $500. Microcapillary tubes or Drummond pipettes can be used to measure small volumes of IgG and conjugate if the 0 to 20 Ill pipette is not available.

Grinding equipment

Although samples may be homogenized with pestle and mortar, a mechanical device is desirable if large numbers of samples are to be assayed. Dispersion homogenizers with a generator shaft of 15 to 25 mm diameter are commonly used, e.g. Polytron, Tissumizer, Vir-Tis or Tissu-Tearor in a price range of $700 to $2 500. Rollers and stomacher devices work for some applications.

Evaluation of results

ELISA results can be estimated visually, but it is difficult to determine weak reactions, especially when there is a background. Photometric measurements must be made to obtain quantitative data. Aliquots of the reaction can be diluted in water and the absorbance read in a regular spectrophotometer. Manually operated ELISA plate readers, available from about $4 000 upward, can quickly and accurately read an ELISA plate. If ELISA is to be performed on a sizeable scale, consideration should be given to purchasing an automated ELISA plate reader with an RS 232 port linked to a personal computer. Costs for such a system begin at about $20 000.

SPECIALIZED EQUIPMENT FOR ELECTROPHORESIS

Electrophoresis apparatus

Electrophoresis is often used to diagnose viroids or for the analysis of dsRNAs to detect the presence of viruses. Electrophoresis for these procedures is most commonly performed on vertical slab gels, although tube gel Electrophoresis can also be used. Many models of both vertical slab and tube gel apparatus are sold commercially, with prices starting from about $225 for a small apparatus to $1 500 for larger units.

When the apparatus is being selected, its potential use should be taken into account. The same electrophoresis apparatus can also be used for SDS polyacrylamide gel electrophoresis for protein analyses and for non-denaturing gels for isozyme analysis. The larger units will cost more initially, will need more reagents because of the larger volume and will require longer run times, but they offer better resolution. The small units offer speed and lower cost, but often lack a high degree of resolution. For most diagnostic applications, the small unit is satisfactory. An alternative to purchasing a commercial unit is to custom-make a unit from Perspex. The most expensive component is the platinum wire needed for the electrode. This can be bought from an electron microscopy supply catalogue or an electronics shop.

Power supply

A stable power supply is essential for electrophoresis. For versatility, the power supply should be capable of running at constant voltage (0 to 500 V range or greater) or at constant current (0 to 400 mA range or greater). Cost of equipment that provides this range of control over the power supply begins at about $900 and increases depending on the model and features desired. Care must be taken to match the frequency (Hz) of equipment to that of the main electricity supply.

Vacuum pump and dessicator

These are often used to de-gas acrylamide solutions before pouring gels. In addition, nucleic acid preparations are usually dried in a vacuum after being collected as ethanalprecipitated pellets before being used for electrophoresis. A dessicator costs from about $50 depending on size. Hand vacuum pumps are available at costs beginning at about $30; water aspirators can also be used. An electric vacuum pump, which offers more versatility, costs from about $250 upward.

Visualization of samples on gels

The usual method used to detect nucleic acids (such as viroids and/or dsRNAs) on gels after electrophoresis is by staining with ethidium bromide, then viewing over an ultraviolet (UV) transilluminator. The nucleic acids fluoresce, and the resultant diagnostic bands on the gel can be visualized and photographed. Visualization by this method allows subsequent manipulations of the nucleic acids, such as infectivity assays, electrophoresis on denaturing gels and preparation of probes (Part III). A UV transilluminator with 302 nm wavelength is recommended for use with ethidium bromide staining, as hand-held short-wave UV lights do not have enough light intensity to visualize any but the strongest of gel bands. Prices for a UV transilluminator start at about $1 100 and increase in price as the filter size increases.

An alternative to viewing nucleic acids by fluorescence over UV light is to silver-stain the gels (Part III). Silver-staining is as sensitive as or more sensitive than ethidium bromide staining and eliminates the need for a UV transilluminator. However, silver-staining inactivates the nucleic acid and immobilizes it in the gel, which does not permit subsequent manipulation of the nucleic acid.

Documentation of the gel requires a means of photographing the gel. A UV- 1 filter between the camera and the UV light source is commonly used. Polaroid cameras are routinely used. Instant cameras with a hood that fits over the gel on the UV transilluminator and a fixed focal length are now available with prices beginning at about $400. More elaborate Polaroid set-ups begin at about $4 000. To minimize investment and film costs, an SLR 35-mm camera with macro-focus lens and auto exposure can be used with black-and-white film such as Kodak Contrast Process Pan or equivalent. After the gel has been photographed, the film can be processed immediately before moving the gel (usually 15 to 20 minutes of darkroom time) to verify that the gel bands have photographed well and that the focus is satisfactory.

SPECIALIZED EQUIPMENT FOR CULTURING

Culturing is often used to verify the presence of citrus stubborn and other disorders caused by harmful prokaryotes such as citrus greening and Pierce's disease of grapevine.

Autoclave

An autoclave is essential for culture work. This can be as simple as a large pressure-cooker, but if a lot of culture work is to be done the additional cost of an automated autoclave may be justified.

Transfer hood

It is desirable to have a transfer hood equipped with filtered air and a flame to sterilize transfer loops. In more arid climates, an open work space in a room with no air movement can be lined with wet paper towels and the surface sterilized with 70 percent alcohol for culture work. More contaminations will occur than when a hood is used, however, and this method will not be satisfactory at all in hot, humid climates.

Incubators

These need to be available. Shaking capability can easily be obtained by placing small shakers in incubator cabinets. Costs of small shakers start at about $300.

Microscopes

A light microscope with phase contrast and good optics is needed to verify the presence of spiroplasmas and harmful prokaryotes from culture. Costs for a suitable microscope begin at about $2 000 and increase as quality increases. A stereoscope is needed as well, as an aid to culture work and also for general use and shoottip grafting. Costs for a stereoscope begin at about $1 200.

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