Compound Microscope

It is used for microscopic examination of the samples in the laboratory.

i) Construction: A compound microscope consists of 3 major parts i.e. body, lens system and illumination system

a) The body of a microscope consists of the base, arm, body tube, stage, iris diaphragm and fine/coarse adjustment knobs etc. The base provides firm support for the rest of the microscope. Arm is used for carrying the microscope and supporting the optical/lens system.

Coarse adjustment knob moves the body tube and objectives to bring the specimen roughly into focus. The fine adjustment knob brings the specimen into exact focus. Upper body tube encloses light path between objective and ocular. Lower body tube provides movable section for focusing objectives. Revolving nosepiece holds the objectives, which can be rotated into light path. The stage provides a platform with an opening in the centre on which a slide is placed which contains the specimen to be observed. The clamps or a movable slide holder is used to hold the slide in place. Condenser collects the light rays and focuses them on the specimen. Iris diaphragm is an assembly of thin metal leaves controllable by a lever to produce variable sized openings.

b) The lens system consists of eye piece, objectives and condenser

Eyepiece or the ocular of the compound microscope is composed of 2 or more lenses; the upper compartment or the eye lens is the magnifier and the lower compartment is called the field lens. The commonly used eyepieces are available with magnifications like 1X, 2X, 5X and 10X etc.

Objectives are considered to be the most important part of a compound microscope since they affect the quality of image formation. Most compound microscopes are equipped with 3 objectives having different magnifying powers. They are low power (10X), high power (40X) and oil-immersion (100X) objectives. Low power objective is the shortest and oil-immersion is the longest.

The primary function of a condenser is to supply sufficient cone of light to fill the objective aperture for getting maximum resolving power. Generally, condensers also incorporate iris diaphragm and filter holder. Iris diaphragm is used to control the light intensity.

c) Proper illumination is essential for the efficient utilization of the magnification and resolution of a microscope. The readily available source of illumination is ordinary daylight.

As the intensity of daylight varies greatly, artificial light sources (generally a tungsten lamp) are more often used. The most precise of such light sources control the intensity, colour and size of the light beam. The size of the light cone differs with each objective. As the

magnification of objective lens increases, the working distance decreases, and the angle of aperture of the objective increases. Therefore, with increasing magnification a larger cone of light must enter the objective.

ii) Principle of working: A compound microscope works on the basic principle of magnification, resolving power and illumination.

Magnification is obtained by a series of 2 lens system, the lens system nearest the specimen called objective, magnifies the specimen and produces a real image. The ocular or eye lens system magnifies the real image, yielding a virtual image that is seen by the eye. The total magnification is equal to product of the ocular magnification and the objective magnification.

Resolving power of a lens is its ability to show two closely adjacent points on the object as distinct and separate. This characteristic of a microscope is a function of the wave length of the light used and a characteristic of the lens system known as its numerical aperture:

Wave length

Resolving power = Diameter of the smallest structure visible = Numerical aperture

The above relationship between the wavelength of light used and numerical aperture in determining resolving power holds good only for parallel light rays. When the specimen is illuminated with oblique rays in addition to direct light rays, the relationship becomes:

Wavelength

Resolving power = 2 x Numerical aperture

Use of compound microscope

 Place a slide on the stage with specimen side up, and centre the section to be examined as accurately as possible over the hole in the centre of the stage.

 Adjust the light source until it passes the maximum amount of light through the specimen. With low power objective in position, lower the body tube by means of the coarse adjustment until the objective is about 5-6 mm from the slide.

 Look through the eyepiece and slowly raise the objective with the coarse adjustment until the specimen is in approximate focus. Never focus downward while looking through the eyepiece. Bring the specimen into sharp focus with the fine adjustment.

Adjust the iris diaphragm and sub-stage condenser until the light intensity is optimum, being neither glaring nor dull.

 After examining the specimen with the low-power objective, shift to the high power dry objective by rotating the nosepiece until the objective clicks into place, first making certain that the portion of the specimen you wish to view is exactly centered in the field of the low power objective.

 Look through the eyepiece and slowly raise the body tube with the coarse adjustment until the specimen comes into approximate focus. Then bring the image into final accurate focus by using the fine adjustment.

 Focusing of the oil-immersion objective requires more care than that of the other objectives, but the procedure is essentially the same. First use the low power objective to locate the portion of the specimen to be examined. Raise the body tube and then rotate the nosepiece until the immersion oil such as clove oil or cedar oil on the portion of the slide directly under the objective. Watching the objective form the side, carefully lower it into the oil. Do not allow the objective to touch the slide. Look through the ocular and slowly focus upward with the fine adjustment until the image appears.

Precautions

 Never touch the lenses. If the lenses become dirty, wipe them gently with lens paper.

 Never leave a slide on the microscope when it is not in use.

 Always remove oil from the oil immersion objective after its use. If by accident oil gets on either of the lower power objectives, wipe it off immediately with lens paper.

 Keep the stage of the microscope clean and dry.

 Do not tilt the microscope when working with the oil-immersion system.

 When the microscope is not in use, keep it covered and in the microscope compartment.

 Never allow an objective lens to touch the cover glass or the slide.

 Never force the microscope. All adjustments should work freely and easily.

2. Autoclave

An autoclave is used for sterilization of media.

Principle: The principle of autoclave is that the water boils at about 100oC, depending upon the vapour pressure of the atmosphere. If the vapour pressure is increased, the temperature will be increased.

Construction: An autoclave is basically a double walled metallic vessel made of thick stainless steel or copper, one end of which has an opening fitted with a tightly closed lid. The lid is provided with pressure gauze to measure the steam pressure and a safety valve. There is also an exhaust valve below to let the steam escape from the bottom of the inner chamber.

Autoclave may be jacketed or non-jacketed types. In Jacketed types, the duration of heating is less than in non-jacketed types, however, in non-jacketed ones, water does not condense on objects and the steam is dry, i.e. it does not contain particulate water.

Working: For most purposes, sterilization in autoclave is done for 15 minutes at 121oC temperature which is achieved at 1.05 kg/cm2 pressure.

Precautions

 Autoclave should not be overloaded.

 All the air must be removed from within the autoclave before closing the exhaust valve by keeping the outlet valve open until a jet of continuous air comes out of it.

 Sterilization time must be counted not from the time it is switched on but from the time the required pressure is built up.

 Ensure that there is sufficient water in the autoclave before switching on.

 At the end of the sterilization period, allow the steam pressure to drop to zero and only then open the lid.