Bright field compound microscope
A microscope that forms dark image against bright field ( background ) is called as bright field microscope. Microorganisms absorb some light and appear faint dark. Staining increases the light absorption ability of microorganisms. The staining increases the contrast. The compound microscope consists of two sets of lenses, one is the most commonly used microscope in microbiology laboratory. Microscopes designed by different manufactures differ greatly in the details of their construction and mode of operation
Principle
The glass lenses focus light rays at a focal point and produce enlarged image of minute object. The object is placed outside the focal point of objective lens. The objective lens produces magnified real ( true ) and inverted image. The real and inverted image is placed inside the focal point of eyepiece lens and a magnified Virtual and erect image is obtained.
Components of microscope
The bright field compound microscope consists of three parts.
1. Mechanical parts, 2. Magnifying parts and 3. Illuminating parts
1. Mechanical parts : The mechanical parts support and adjust magnifying and illuminating parts. The mechanical parts include.
a. Base : The microscope consists of a broad base or foot, often horseshoe shaped. It gives stability to the microscope.
b. Pillar : A short stout pillar arises from the base. It supports an upright curved arm.
c. Arm : The arm is curved. At the lower end, it holds of illuminating parts and stage and at the upper end it holds of focus adjustment knobs, body tube and microscope.
d. Stage : The stage is a horizontal working platform. It has an opening of about 3 to 4 inches at center. The opening permits light to reach to object. Stage provides space for glass slide on which object to be examined is mounted.
e. Mechanical stage : Above the stage, mechanical stage is fixed. It allows forward, backward or rither side ways smooth movement of slide.
f. Focus adjustment knobs : Two focus adjustment knobs, the fine and coarse adjustment knobs are also located on arm. They are used to move the body tube to focus the image.
g. Body tube : The body tube at its upper end holds ocular or eyepiece. At the lower end, the body tube consists of nosepiece. The body tube provides sufficient space for image formation. The body tube is mounted on a rack and pinion mechanism for vertical coarse and fine adjustment.
h. Nosepiece : Three to four objectives ( usually 10X, 45X and 100X ) are fixed on the revolving nosepiece. It is possible to bring a desired objective into position under the body tube by resolving the nosepiece.
2. Magnifying parts : Magnifying parts enlarge minute objects. They include:
a. Objectives : The lens system in a compound microscope near the object is called objective. A typical microscope used in microbiology laboratory equipped with three objectives with different degree of magnifications such as 10X , 40X (45X) and 100X. These objectives are referred as low power (10X), high power (45X) and oil immersion (100X). The magnification provided by an objective is engraved on it.
Functions :
i. It forms real and inverted image of the object.
ii. It magnifies the object.
b. Eyepiece ( Ocular ): The lens system in a compound microscope nearest to the eye is called ocular . The microscope, which contains one ocular, is called monocular microscope. The microscope that contains two oculars is called binocular. The magnifying power of ocular is engraved on it. High power eyepiece gives higher magnification, but a brighter and sharper image is obtained with low power eyepiece. Therefore, most common ocular is 10X. However, oculars of 5X, 15X and 20X magnification are available. There types of ocluars are available.
Huygenian eyepiece : It is most common, plano-convex and used with low power objective.
Hyperplane eyepiece : It gives flat fields and used with high power objectives.
Compensating eyepiece : In contains three lenses, superior and used with higher power objectives.
Functions :
i. It produces virtual and erect image of the object.
ii. It further magnifies the object.
iii. It corrects some of the defects of the objective.
iv. It carries any scale or market to be used with microscope.
3. Illuminating parts : The illuminating parts include:
a. Mirror : It is plano - concave and reflects light towards the object. The flat surface of mirror is used with oil immersion objective whereas the concave side is used with low and high power objectives.
b. Iris diaphragm : It is mounted with condenser. A small lever opens it. It controls amount of light reaching the object. Opening or closing the iris diaphragm controls the mount of light entering the condenser. Observation in oil immersion objective needs fully opened iris diaphragm whereas partially closed with other objectives.
c. Condenser : It consists of a system of convex lenses. It is the third lens system of compound microscope. It is also referred as substage condenser because it it mounted below the stage. The extensively used condenser is Abbe's condenser. It is simple with two lenses. It has 1.5 numerical aperture and good light gathering ability. The condenser is mounted on a rack and pinion mechanism for vertical adjustment. Lowering the condenser reduces light whereas raising increases the light intensity. Observation in oil immersion objective needs raised condenser whereas suitably lowered with other objectives. Condenser concentrates light on the object.
Working :
The mirror reflects light towards the condenser. The condenser passes a cone of light towards the specimen or object. Microorganisms absorb some light, refract most light, and appear faint dark. Staining increases the light absorption ability of microorganisms. The staining increases the contrast. The objective collects refracted light and forms a magnified, real and invented image (primary image). Upper part of the body tube locates the primary image and forms a virtual and erect image. Virtual and erect image is a final image and is seen by retina. A ray diagram of path of light in bright field compound microscope.
Application :
Compound microscope is used :
1. For observation of microorganisms
2. To observe cell structure by special staining.
3. To observe motility of bacteria.
4. To study bacterial spore and it's position using special staining technique.
5. To study the structure of other microorganisms like algae, fungi and protozoa.
6. To measure the size of microorganisms.
7. For study of blood cell.