Part IV.Appendices

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Laboratory equipment needed for selected diagnostic procedures
Glossary

 

Laboratory equipment needed for selected diagnostic procedures

Richard F. Lee
Citrus Research and Education Center
University of Florida (Lake Alfred)
United States of America

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 problem 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) will be given, with the current price range in the United States of America.

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 worldwide 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 ending US$25 to International Scientific Communications Inc., 30 Controls Dr., PO Box 870, Shelton, CT 06484-0870, United States of America (Telephone (203)926-9300, Telex 964292, FAX (203)926-9310).In addition to the more specialized equipment needed for some of the diagnostic procedures, there are a few basic requirements for any laboratory.

Size

A minimum area of about 55 m 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

There needs to be a stable source of electrical power. If the power source is subject to interruptions, or may be not working for long periods, a generator should be provided to power important equipment and to permit work to continue. Most equipment manufactured in the United States of America for use here 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 susceptible 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, the availability of 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

It should be 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 handheld unit and rise depending on the features selected.

Refrigerator

Needed for storage of reagents, chemicals, and seed at 4C, the refrigerator 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 -2C and to produce ice needed for use in ice-baths. Select a freezer which 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

Its sensitivity should be at least 0.01 g. A top-loading electronic balance with dual range sensitivity of 0.01 to 120 g and 0.1 to 1 200 g ranges 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 it should be a refrigerated model with a rotor capable of holding 50-ml centrifuge tubes and adapters to accommodate smaller volume tubes. Prices of such a centrifuge 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 the reduced g force as specified in the protocol.

Magnetic stirrer and hot plate

They 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 NEEDED 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, UV-visible spectrophotometer and simple chromatography are needed if IgG and conjugates are to be prepared. Other equipment needed for ELISA is:

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 $125-250). It is highly desirable to have a set of three adjustable pipettes (0-20 ml,20-200 ml, and 200- 1 000 ml) at a cost of about $ 125-250 each. If much ELISA is to be performed, a multichannel adjustable (50-250 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 0-20 ml 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, VirTis, or Tissu-Tearor in a price range of $7002 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 adsorbance read in a regular spectrophotometer. Manually operated ELISA plate readers, available from about $4 000 upwards, 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 NEEDED 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. There are currently many models of both vertical slab and tube gel apparatus sold commercially, with prices starting at 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, also 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 power supply is essential for electrophoresis. For versatility, the power supply should be capable of running at constant volts (0-500 V range or greater) or at constant current (0-400 mA range or greater). Cost begins at about $900 and increases depending on the model and features desired. Again, be aware of the cycles per second (Hz) on purchased equipment and be sure it will be compatible with the cycles per second in your country.

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 vacuum after being collected as ethanal-precipitated 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 upwards.

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 straining 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 handheld 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 or more sensitive than ethidium bromide staining and eliminates the need fore 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. AUV- 1 filter between the camera and the UV light source is commonly used. Polaroid cameras are routinely used. Instant cameras, with a hood which 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, a 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 1520 min 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 procaryotes such as citrus greening.

Autoclave

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

Transfer hood

It is desirable to have a transfer hood which is 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 and used for culture work. This will increase the number of contaminations occurring, and will not be satisfactory at all in hot, humid climates.

Incubators

These need to be available. Shaking capability can be easily 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 procaryotes from culture. Costs for a suitable microscope begin at about $2 000 and increase as quality increases. A stereoscope is also needed as an aid to culture work and also for general use and shoot-tip grafting. Costs for a stereoscope begin at about $1 200.

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