Manual on general laboratory handling techniques in plant pathology

TABLE OF CONTENTS

CONTENT

1. GENERAL REQUIREMENTS AND FACILITIES FOR A PLANT QUARANTINE LABORATORY

2. KNOWING LABORATORY EQUIPMENTS, APPLIANCES, GLASSWARE AND CHEMICALS WITH THEIR SAFE HANDLING TECHNIQUES [PLANT PATHOLOGY AND ENTOMOLOGY]

3. KILLING, PRESERVATION AND TRANSPORTATION TECHNIQUES FOR INSECT PESTS

4. STERILIZATION TECHNIQUES IN LABORATORY

5. PREPARATION OF CULTURE MEDIA FOR FUNGI AND BACTERIA

6. ISOLATION OF PLANT PATHOGENIC FUNGI FROM DISEASED PLANT MATERIAL

7. ISOLATION OF PHYTO-PATHOGENIC BACTERIA FROM DISEASED PLANTS

8. ISOLATION OR EXTRACTION METHODS FOR NEMATODES FROM PLANT AND SOIL SAMPLE

9. MAINTENANCE AND STORAGE OF VIRUS ISOLATES

10. PRESERVATION OF DISEASE SAMPLES

11. PURE CULTURE MAINTENANCE OF PLANT PATHOGENS

1.GENERAL REQUIREMENTS AND FACILITIES FOR A PLANT QUARANTINE LABORATORY

1. Infrastructure & Utilities

Laboratory space with furnitures and immovable fittings for example: Working tables, laboratory chairs with adjustable height, book case, chemical rack, cupboards to store apparatus and supplies, specimen storing rack, microscopes etc

Water (sink/basin with hot and cold water taps with mixing facilities with plastic tubing, good drainage system leading to concrete tank outside the laboratory)

Electricity with several electrical wire outlets

Information facilities (telephone, internet, email etc)

Wash room and lavatories

Guide keys, reference publications, databases

Communication systems

Transport (Standby Vehicle used for laboratory works)

Storage/preservation facilities – Herbaria/culture collections

2. Human resources

Trained diagnosticians/taxonomists

Technical support staff

Non-technical staff

Service managers/co-ordinators

Outreach/Public liaison staff

3. Location of the laboratory

The laboratory should be located in accessible area nearby the office. It is better to have laboratory in ground floor. The specimens collected should easily be carried in to laboratory.

4. Laboratory layout

When designing a laboratory there are many aspects to consider. It is important that work should be carried out in a logical order and, that particular parts of the diagnostic protocol are separated from one another. Figure 1 is the layout of a general diagnostic laboratory mainly of plant pathogens. General plant protection laboratory may have the following different rooms and chamber as appropriate.

Preparation room

The preparation room is used for preparing media, including sterilizing items in the autoclave, sterilizing petri dishes in an oven, washing glassware and storing glassware, chemicals and other basic items. This room should have an exhaust fan to remove hot air produced by the autoclave and the oven.

Clean room

The clean room is used for isolating fungi and bacteria from cleaned subsamples of diseased plant tissue into pure cultures. It is also used for growing cultures under clean conditions. The microscopes are located in this room for examining cultures and fungal structures.

This room should be air-conditioned, if possible, to protect equipment and cultures. It should also be kept free from dust and insects. If, do not have an airtight clean room or humidity will be too high and fungus (mould) will develop on walls and equipment. A dehumidifier is useful in this room. No soil is allowed in the clean room as soil is a source of fungus-eating mites that can contaminate cultures.

Specific chambers within the rooms (for specific diagnostic laboratory): If there is single room for a laboratory , it should have following specific chambers.

Media preparation chamber: It is mainly to prepare media and should have a continuous and sufficient supply of single and double distilled water. It should also have diststill water for storage, a double distillation unit, pressure cookers of ultra-dent type (30 cm *30 cm) to steam the media are provided in this chamber. Various instruments including pH meters, balances, centrifuge, refrigerators, deep freeze can be adjusted in this chamber. Vaccum lines and compressed air facility are necessary for filter sterilizing the solutions.

Inoculation Chamber: This chamber should harbor inoculation cabined/ laminar flow cabinet (convenient for two persons to sit and work at the same time), the floor should be covered with linoleum or tiles to facilitate proper cleaning. The atmosphere in this chamber should be free from all types of contamination. This chamber should preferably be air conditioned for good air circulation and to maintain a constant temperature. A power back up system is required to protect incubated cultures

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Laminar flow cabinet

Incubation chamber: Incubation of plant parts and tissues at a specified temperature (around 25 degree celcius) after the aseptic transfers needs a chamber. This has to be maintained with an air cooler or by window air conditioners. Controlled heating system may be needed in some check posts in the high mountains.

Washing and drying chamber: A sink has to be fitted in one corner of the chamber. racks to train and dry should be placed adjacent to the sink to facilitate the drying of cleaned glasswares.

Plugging Chamber: This chamber should have tables of sitting and standing height. Space to store the cleaned glassware to be provided. Glassware to be plugged with nonabsorbent cotton.

Media storage chamber: Storing of the media before its actual use is done in this chamber.

Sterilization chamber: Horizontal autoclaves of several dimensions may be kept in this chamber. An electric oven for drying the glassware may be accommodated. In one corner a sink may be fitted. Exhaust fans for proper ventilation should be provided.

Shaker chamber: It should be accommodate rotary shakers with variable speed controls, illuminated with fluorescent tube lights (40 w) may be fitted 60 cm above the shaker so that they can provide an intensity of 2000 lux to the cultures. A lux meter can be used to measure the light intensity in the inoculation and shaker chambers.

Other Chambers: Office, meeting hall and data collection/management chamber with communication facility as listed above should also be provided. An acclimatization chamber fitted with humidifier and temperature and air conditioning system should also be managed. The acclimatization chamber should also have the facility similar to the illuminated incubation chambers to regulate the variable light intensity.

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Figure: Typical arrangement of equipment in a diagnostic laboratory: a) and (b) two views of clean room, (c) and (d) two views of preparation room.

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Figure: Typical floor plan and the layout of general plant quarantine/protection diagnostic laboratory [some modification can be made with floor availability and type of laboratory]

2. KNOWING LABORATORY EQUIPMENTS, APPLIANCES, GLASSWARE AND CHEMICALS WITH THEIR SAFE HANDLING TECHNIQUES

A LABORATORY EQUIPMENTS

1. Analytical Balances

An instrument used for accurate measurement /determination of weight of a sample in various operations.

1.1 Conventional type : Free swinging balance, Equal arm balance, Two pan balances

1.2 Electric balance- no mechanical failure and greatly reduced sensitivity to vibration with it

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Care and maintenance of analytical balance

Never exceed the stated maximum load of the balance

The balance must be kept clean

Objects to be weighed should never be handled with the fingers

Objects to weighed should be allowed to attain the temperature of balance

No objects which might injure the balance pan should ever be placed directly on it. Always use a watch glass or weighing bottle for weighing substance

Nothing must be left on the pan when the weighing has been completed

Exposure of the balance to corrosive atmosphere must be avoided

2. Heating

Apparatus Ovens

Ovens are the boxes designed to maintain a constant internal temperature; they are electrically heated and thermostatically controlled for drying the glass wares and other solids with very high melting points. Ovens are designed to mentain the differential internal temperature ranging from room temperature to about 250 - 300˚ C ± 1 – 2 ˚C. They are also useful for determining the moisture content of materials

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Hot air oven Micro-wave oven

Incubators They are the boxes designed to maintain a constant internal temperature by the use of thermostat; and are used for rearing insects and prematurely born infants. An incubator comprises a transparent chamber and the equipment that regulates its temperature, humidity and ventilation and; is used in biological experiments to allow growth of organisms in optimal conditions of temperature and humidity.

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General Incubator Biological Oxygen Demand

Distillation Plant

It is an instrument used to purify the water through distillation. The working mechanism of the distillatioln plant allows the water to vapourized and condensed to form the distilled water.

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Hot Plates

The electrically heated hot plate, is with three controls - low – medium – high

The heating elements and the internal wiring are protected from fumes or spilled liquids

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Autoclave

A thick walled vessel with a tightly fitting lid, in which substances may be heated under pressure.

It is used for sterilizing the media.

Heating Mantles

An equipment with a flexible knotted fiber glass sheath in it which fits snugly around a flask and contains an electrical heating element which operates at back heat

It is particularly designed for the heating flasks

It has wide application in distillation operations

Crucibles and Beaker Tongs

Apparatus such as crucibles evaporating basins and beakers which have been heated need to be handled with suitable tongs

Berlese Funnel

An apparatus comprising of funnel, lamp and collecting jar.

An apparatus for collecting and extracting small invertebrate animals from grains soil and decomposing litter.

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Autoclave

Berlese funnel extraction works in the following way:

A 60-watt light bulb, housed in a reflective shade, is suspended above a funnel containing approximately 1,000 grams of suspect grain.

A collecting jar is placed under the funnel.

Water in the jar prevents insects from escaping.

Over several hours, light and heat generated from the bulb and reflector cause stored product insect pests to move through the grain into the collecting jar where they can be identified

Cooling Apparatus

Refrigerators

Refrigeration is generally the cooling of a body by the transfer of a portion of its heat away from it

A refrigerator (often shortened to fridge) is an electrical or gas appliance that uses refrigeration to help preserve materials

They may consist of either a cooling compartment only (a larger refrigerator) or a freezing compartment only (a freezer) or contain both

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Fridge Deep Freeze

4. Desiccators

A desiccators is a covered glass containers designed for the storage of objects in a dry atmospheres

It is charged with some drying agent such as silica gel, anhydrous calcium chloride

The exhausted silica gel (spent material) can be regenerated by heating in an electric oven at 150 180 ˚c

5. Magnifying Instruments

i. Hand lens
ii. Pocket lens
iii. Microscopes

Microscope is an important instrument, which enlarge the image of an object with the help of lenses so as to facilitate the morphological, anatomical and other characteristics of an object. It also facilitates measuring sizes of smaller objects.

Dissecting microscope, binocular microscope and electric microscope are common types of microscopes being used in laboratory. The smaller objects are measured by ocular and stage micrometers.

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Dissecting Microscope Compound Microscopes

iv. Micrometer

An instrument for the accurate measurement of small distances or angles

Stage micrometer is a glass slide in which one millimeter scale is divided into 100 equal divisions and each division measures 0.01 mm (10 microns).

Ocular micrometer is a small glass disc which can be fitted into an eyepiece of microscope. It has a scale of one centimeter long, divided into 100 equal parts and each division measures 0.1 mm (100 microns).

Care and maintenance of microscope

i. Carry the microscope in an upright position, by putting one hand on the handle and supporting the base with other hand.
ii. Avoid tarring suddenly
iii. Keep microscope free from dirt and dust
iv. Clean lenses with lens paper only
v. Always use clean glass slides with specimen on it. vi. Reflect the mirror towards the light source
vii. Always start focusing with low power to bring the object in the objective field. Use higher magnification gradually.
viii. Clean the microscope after finishing your work.
ix Store the microscope in a cabinet with a cover over it

6. Rotary evaporator: concentration and purification

7. Suction pump: useful to filter mixture with suction mechanisms through any filter media

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