Nematode Pests of Horticultural Crops and their Management (1+1)

Practical - 5

Exercise: Handling and Use of Microscopes.
Definition: A microscope may be defined as an optical instrument consisting of a lens or combination of lenses for making magnified image of minute objects.

Simple microscope: A simple microscope consists of a single lens held in an adjustable frame provided with a stand for conveniently holding the object to be viewed and a mirror for reflecting the light.

Compound microscope:

• A compound microscope has two sets of lenses, one known as objective and other as eye piece (ocular) mounted in a holder commonly known as Body tube. The objective is nearest to the specimen and magnifies the specimen a definite number of times. The second lens system i.e. ocular further magnifies the image formed by the objective. The image seen by the eye has a magnification equal to the product of the magnification of two systems.
• Accurate focusing is attained by a special screw appliance known as fine adjustment.

Objective: Handling of microscopes to view nematodes and their body parts.

• Since plant parasitic nematodes are miniature in form, they can be viewed only with the help of microscopes, the preliminary information regarding the microscopes used in Nematology and their handling is necessary. Generally two types of microscopes are used for Nematological investigations: Stereoscopic microscope and Compound microscope.

1. STEREOSCOPIC MICROSCOPE (Fig. 9):
Purpose: This microscopes is used to fulfill the following objectives:

• When nematode specimens are comparatively large.
• Very high magnification is not required.
• Maneuvering with live specimens e.g. counting and picking.
• Nematode dissection
• Slide preparation etc.
• Three dimensional and erect image of the specimen is desired.

Parts of the microscope

• Basewith a transparent glass object stage provided with clips for holding the slides and reflector/illumination source of light (transmitted/incident light).
• Objectives of 1X, 2X or 4X. In case of stereoscopic zoom microscope, magnification can be continuously changed by moving the magnification changer labeled as 1 X to 4X.
• Focusing knobs.
• Binocular ocular tube
• Eye pieces of 10X or 15X. Lever for setting the inter-pupillary distance.

Handling

• Place the object on stage.
• Adjust the reflector so that light beam is transmitted on to the object.
• Move thefocusing knob to focus the object.
• For viewing under high magnification either move the objective or shift the magnification changer.

Fig. 9. Stereoscopic microscope

This picture has incident light, please prefer the one with transmitted light for nematology

2. COMPOUND MICROSCOPE (Fig. 10)

Purpose: Compound microscope is used to fulfill following purposes

• To study various morphological structures of nematodes in detail at higher magnification (40X to 1,000X). Generally temporary and permanent slides are studied under compound microscopes.
• Measurements of nematodes can be taken under compound microscope by attaching camera lucida apparatus to it.

Parts of compound microscope:

• Base: with a hollow rounded structure for the attachment of a lamp and/or a reflector mirror.
• Condenser: with condenser lens, an aperture diaphragm, filter holder and a knob for its movement.
• Rectangular object stage with clips for holding slide and coaxial knobs for the movement of the slide.
• Coarse and fine focusing knobs.
• Nose piece: Revolving nose piece with objective of 4X,10X,40X and 100X. 100X objective lens is also known as oil immersion lens because when we use this lens, immersion oil (u=1.515) is placed over the cover slip.
• Neck of the microscope.
• Binocular ocular tube with eye pieces of 10X or 15X. Lever for setting inter-pupillary distance.

Handling:

• Place the slide on the stage and secure it with clips.
• Focus under 4X objective with a coarse focussing knob. Then use fine adjustment knobs for exact focussing.
• Move the condenser lens and adjust diaphragm to control contrast.
• For viewing under immersion oil lens first focus under 4X/10X lens. Move the nose piece. Put a drop of immersion oil on the cover slip and then focus under 100X lens, using a fine focussing knob.

Fig. 10. Compound research microscope

Precautions while handling microscope:

• While using binocular microscopes, eye piece should be adjusted according to individual requirement and specimen should be viewed with both the eyes open.
• Light focusing should be appropriate.
• Only fine focussing knob should be adjusted with precaution while viewing the specimen at high magnification with oil immersion to prevent breakage of cover slip and crushing ofspecimen.

Differences between stereoscopic and compound microscope

Stereoscopic microscope	Compound microscope
Low magnification	High magnification
(Usually less than 70X)	(Usually 40-1000X)
Working distance is high.	Working distance is low.
Image formed is erect.	Image formed is inverted.
Three dimensional image is formed.	Three dimensional image is not formed