Bright Field Microscopy

Microscopy Methods in Microbiology
Microroscopy Methods in Microbiology Part 2
Microroscopy Methods in Microbiology - Introduction
Observation of Microbes in the Living State
Observation of Microbes in Smears
Dyes and Staining
Useful pH Range and Colour Change of Some Indicator Dyes
Factors Affecting Staining
Microscopy and Micrometry
Microscopy and Micrometry - Introduction
Behaviour of Light
Light Theories
Resolving Power
Refraction and Reflection
Bright Field Microscopy
Use of Bright Field Microscope

Bright Field Microscopy

	Bright Field Microscope Bright field microscope is absolutely indispensable to the biologists in general and to the microbiologists in particular. This instrument provides magnification f the objects which are often invisible to the naked eye (A) Lens Systems: Compound microscopes designed by different manufactures differ enormously in their outer appearance, but essentially they consists of three lens systems; (1) the objective, (2) the eye piece or ocular and (3) the condenser. Several of these microscope components
(1) Objectives: Objectives are considered to be the most important parts since, they affect the quality of image formation. According to their degree of correction for chromatic aberration, they are catergorized into three classes are (i) achromats, (ii) fluorites and (iii) apochromats. Achromatic objectives are corrected for coma and for primary and secondary spherical aberration in they yellow-green region, but over corrected for blue and under corrected for the red portion of the spectrum. They are less expensive and commonly used.
Other two are more expensive and are more corrected for aberration and thus, give better resolution and image definition Most compound microscopes are equipped with here objectives those have different magnifying powers. They are low power, high power and oil-immersion objectives. They can be easily recognised from their length and N.A. value engraved on the barrel. Low powder objective is the shortest and oil-immersion objective is the longest.
(a) Numerical aperture: We have already discussed the ability to see details of object and a limit of resolution by microscope. When we look through ocular, we are not observing the object, but the intermediate image formed by the objective. The eyes to not have capacity to resolve further this image and therefore, the resolving power of objective is of great value. The resolving power of the objective is proportional to the width of the pencil of light used for illumination. The largest cone of rays admitted by the objective lens depends upon the refractive index of the medium between object and objective. As shown in figure 2-5a, angle (θ), the measure of aperture of objective, is the half aperture angle. The magnitude of this angle is expressed as sin value
	The sin value of the halg aperture angle multiplied by the refractive index (n) of to medium between object and objective gives the value of numerical aperture Thus, N.A = n sin θ The working aperture depends upon the refractive index of medium in which the lens works. It is clear that the objective Used in air will never effective N.A greater than one, because the refractive index of air is one and sin θ cannot exceed one. Obviously, to increase N.A., objective should be immersed in a medium having refractive index similar to that of glass.

This will result into, large cone of light to enter the objective giving higher θ value. The resolving power obtained with the help of oil-immersion objective is nearly 0.25 μm. Refractive indices of some mounting media is listed in Table that of glass. This will result into, large cone of light to enter the objective giving higher a value. The resolving power obtained with the help of oil-immersion objective is nearly 0.25 μm. Refractive indices of some mounting media

No.	Medium	Refractive index (n)
1.	Distilled water	1.33
2.	Eucalyptus oil	1.46
3.	Carbon tetrachloride	1.46
4.	Olive oil	1.47
5.	Glycerol	1.47
6.	Euparal*	1.48
7.	Sandalwood oil	1.51
8.	Cedarwood (immersion) oil	1.51
9.	Canada balsam*	1.54
10.	Polystyrene*	1.59

* Permanent Mounting Media

(2) Eyepieces: A variety of eyepieces are manufactured and the specific type to be used depends upon the objectives fitted in the microscope. The main pm pose of ocular is to magnify the intermediate image and to correct certain aberrations produced by the objective. Ocular is composed of two or more lenses; the upper component or eye lens is the magnifier, while the lower compo¬nent is called the field lens. The commonly used eye pieces are Huygens (negative), Ramsden (positive), compensating and high eye-point eyepieces. They a;e available with various magnifications like lx, x, 5x, l0x, 15x.

(3) Condensers: The quality and method of use of condenser is a decisive factor for a good microscopy. Of course, a condenser is not an essential requisite for compound microscopy when low power objective is used. The primary function of condenser is to supply sufficient cone of light to fill the objective aperture to gain the maximum resolving power.

Therefore, it must be properly positioned during the microscopy. The Abbe condenser, aplanatic condenser and achromatic condensers are available for general purposes. Generally, condensers also incorporate iris diaphragm and filter holder. Iris diaphragm is used to control the light intensity.

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