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In this article we will discuss about:- 1. Essential Parts of Compound Microscope 2. Magnification of the Image of the Object by Compound Microscope 3. Resolution Power 4. Method for Studying Microbes 5. Measurement of the Size of Objects.
Essential Parts of Compound Microscope:
The essential parts of usually used monocular compound microscope (Fig. 15.1) are the following:
(i) Lenses:
The eyepiece with different magnification (5-20 times). It has field-lens towards the object and eye-lens close to the observer’s eye. The objectives are generally with three different magnifications viz., low power (10X), high power (40-45X) and oil-immersion (90-100X).
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The focal length of these are 16 mm, 4 mm, and 1.8-2.0 mm respectively. These objectives are mounted on a revolving nosepiece for convenience. The eyepiece and objectives are fitted at the two ends of a hollow tube called the ‘body tube’.
(ii) Adjustment of Objective Lens:
In some microscopes coarse and fine focussing adjustment knobs are provided in order to lower or raise the body tube with lenses for rendering image clear. This is done by rotation of the knobs. The coarse adjustment is meant to bring to object into vision whereas the fine adjustment is used for focussing finer details.
(iii) Stage:
The object to be observed is kept on a glass slide and placed on the stage. It may have clips to keep the slide in desired position or a mechanical stage for horizontal movement of the object. In some microscopes the stage may be raised or lowered with coarse and fine adjustment for focussing the object.
(iv) Mirror:
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The mirror reflects light which is transmitted through the object for observing it. The mirror has two planes, one concave and the other plane.
When natural light is available the plane mirror may be used for reflection of light because concave mirror would form window images. However, with artificial illumination, the concave mirror is necessary for higher magnifications whereas for lower, the plane mirror may be used.
(v) Sub-Stage Diaphragm:
This is meant to control the amount of light transmitted through the object.
(vi) Sub-Stage Condenser:
The sub stage condenser consists of convex lenses which concentrate and intensify the light reflected by the mirror. With objectives of magnifications exceeding 10X, the use of condenser becomes necessary for narrowing the core of transmitted light which would fill the smaller aperture of the objective. The condensers usually employed are called ‘Abbe’ condensers and these are used with plane mirrors.
Magnification of the Image of the Object by Compound Microscope:
A bright-field or compound microscope is primarily used to enlarge or magnify the image of the object that is being viewed, which can not otherwise be seen by the naked eye. Magnification may be defined as the degree of enlargement of the image of an object provided by the microscope.
Magnification by a microscope is the product of the individual magnifying ability of the oculars and the objectives. For example if the ocular is 10X, and objective is 40X, the specimen is magnified 400 times. If an oil immersion objective (100X) is used along with 10X ocular, the specimen is magnified 1000 times.
The following factors play an important role in magnification:
(i) Optical tube length.
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(ii) Focal length of the objective lens.
(iii) Magnifying ability of the ocular.
The total magnification of the object-image can be calculated using the following equation:
Total magnification = Length of the optical tube/Focal length of the objective x Magnification of ocular.
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Theoretically, if the magnifying power of the ocular and objectives of a compound microscope are increased, it should be possible to get higher and higher magnifications.
A magnification up to 3000 can be obtained by using high powered lenses, but the image will be blurred and details will not be clear. This is due to the fact that in a microscope not only the lenses, but the wave length of the light is also important and this decides the resolving power of the microscope.
Resolution Power (Resolving Power) of Compound Microscope:
Resolution power (resolving power) of a bright-field or compound microscope is defined as its ability to distinguish between two particles situated very close. In a magnified image, the object should not only larger but the details should also be clear.
This is possible when a microscope has the ability to see two points situated very close as two distinct entities. In other words, resolution power may be said to be the minimum distance at which two structural entities of an object can be seen as discrete individual structures even in the magnified image.
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This explanation can be understood clearly with a comparison with the human eye. Human eye functions on the same principal as that of a bright-field or light microscope, i.e., one can see objects because of the light reflected by them.
The human eye has a resolving power about 0.25 mm in the sense, two dots situated 0.25 mm (or more) apart can only be seen as two dots; anything closer than this distance will appear like a single dot.
Factors of Resolution Power:
The resolution power of a bright-field (light) microscope depends on two factors:
(a) Wave length of the light and
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(b) Numerical aperature (NA) of the objective.
(a) Wave length of the light:
In light (bright field) microscopes, the wave length of the light used for illumination falls in the visible range (400-750 nm). If light of shorter wave length is used within this range the resolution will be higher. For example, blue light has a shorter wavelength than red light. Greater resolution can be obtained by using a blue light as a source of illumination than a red light.
(b) Numerical aperture (NA) of the Objective:
Numerical aperture (NA) is defined as the property of a lens that determines the quantity of light that can enter into it. It depends on two factors.
(i) Refractive index of the medium that fills the space between the specimen and the front of the objective lens, and
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(ii) The angular aperture, i.e., the angle between the most divergent rays passing through the lens and optical axis of the lens. (The more divergent or oblique rays that an objective can admit, greater is the resolution power).
Numerical aperture (NA) can be mathematically calculated with the help of following formula.
NA = n sin f
Where, n = refractive index of the medium
f = angular aperature
Calculation of Resolution Power:
The resolution power of a bright-field microscope can be calculated using the following formula:
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Resolution (resolving) power (RP) = Wave length of light used for illumination/2 x Numerical aperture (NA)
For convenience, if yellow light of wave length of 580 nm with numerical aperture (NA) of 1.0 is used in the microscope, the resolution power (RP) of the microscope will be:
Resolution power (RP) = 580/2 x 1 = 290 nm
Method for Studying Microbes with Compound Microscope:
Two methods are generally used, ‘wet method’ and ‘dry’ and fix method’.
A. Wet Method:
There are two primary methods generally used for studying microorganisms in wet conditions:
(a) Wet mount method, and
(b) Hanging drop method.
(a) Wet mounts method:
It is the most widely used method (Fig. 15.2). A drop of fluid containing microorganisms to be examined is put on a glass-slide and a coverslip made of thin glass is placed on it. The fluid spreads out in a thin layer between coverslip and slide. The mount is now examined under the microscope. For higher magnifications (e.g. with 100 X objective) the oil-immersion technique is employed.
A drop of immersion oil is put between the objective lens and cover slip before the microorganisms are examined under the microscope. The immersion oil fills the space between the specimen and the objective lens and thus replaces the air present between the specimen and the objective lens. The result is that the numerical aperture (NA) is improved and the level of magnification is increased.
(b) Hanging drop method:
It is used to observe the motility, germination, or fission of microorganisms. In this method (Fig. 15.3) a cavity slide which has a circular concavity in the centre is used.
The periphery of the concavity on the cavity-slide is smeared with vase-line. A drop of liquid microbial culture is placed in the centre of the cover glass if it is a liquid culture. If the culture is solid, it is mixed with a drop of distilled water before placing on the cover glass.
The cover glass is inverted over the concavity so that the drop hangs freely and the edge of cover glass adheres tightly to the vase-line coated periphery of the concavity. The microorganisms present in the hanging drop are now observed under the microscope.
(ii) Dry and Fix Method:
Microorganisms, particularly bacteria, being too small need their permanent preparation be made by drying and fixing them on clean slide with or without staining. For preparing a dry mount, a drop of distilled water with a small amount of culture is spread as a thin smear on a clean slide.
The smear is allowed to dry and it is then ‘fixed’ by passing it through a flame two to three times with the smeared slide away from the flame. If desired, this dried and fixed amount may be stained and the preparation dried again for observation under the microscope.
Measurement of the Size of Objects by Compound Microscope:
The size of objects viewed under the compound microscope can be accurately determined using a micrometer. The latter consists of two scales, the eyepiece scale, (also called ‘graticule’ or ‘ocular’) and the stage micrometer scale. The eyepiece scale is calibrated with the help of stage micrometer and the former is then used for measurements.
The eyepiece scale is placed inside the microscope eyepiece, and the stage micrometer on the microscope stage. The scale on the latter is exactly 1 mm long and divided into 100 divisions, so that each division is 10 µm. As stated earlier, the stage micrometer is used to calibrate the eyepiece scale.
(i) Calibration (Fig. 15.4):
1. It is noted first that which objective lens is in use on the microscope.
2. Stage micrometer is positioned in such a way that it is in the field of view.
3. The eyepiece is rotated so that the two scales, the eyepiece or ocular scale and the stage micrometer scale, are parallel.
4. The stage micrometer is now moved so that the first division marks of the two scales are in line.
One can now see how many divisions on the eyepiece scale as well as on the stage micrometer scale correspond to each other. Since 1 division on the stage micrometer equals 10 µm, one can find the value of one division of the eyepiece scale.
For instance, in illustration ‘iii’ of Fig. 15.4, four divisions on the eyepiece scale equal 10 divisions (i.e., 100 µm) of the stage micrometer scale; 1 division on the eyepiece scale = 25 nm for the particular objective lens used in this case.
Above positions are repeated using objective lenses and following information’s are recorded on an adhesive label. Information recorded on adhesive label is stuck to the base of the microscope for future reference.
(ii) Use:
Having calibrated the eyepiece scale for all the objective lenses on the microscope, one can use it to measure the dimensions of cellular and sub-cellular structures, e.g., bacterial cells, fungal spores onion epidermal cells etc.
