Microbiology

Light Microscopy: Compound Microscope

Microscope is the main and important instrument of microbiology. One can say actual microbiology starts after the discovery of microscope. 

Initially scientists make the arrangement of lenses to enlarge image of any microorganisms. to study its feature, shape and effect towards its surroundings.

Moderne microscope has the capacity to enlarge the image up to 1000 to 2000 times, this can be achieved by the simplest microscope of today i.e. a light microscope or compound microscope.

First successful microscope was build by Antonie van Leeuwenhoek. For his this discovery he is called as father of microbiology, one of the first microbiologist, and microscopists. Microscopes developed by him has the power of enlargement up to 200 to 300 times. It was good enough to see some microorganisms under the microscope. The main organisms seen by this microscope include protista.

Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eyes (objects those are not in the resolution field of normal eyes).

Depending up on the principle on which magnification is based, microscopes are of two type. Optical (light) microscopes and electron microscopes.

Here we would discuss light microscope. Light microscope provide magnification depending upon the system of lenses and a light beam. Light microscope or optical microscope can be further divided in the following types: Bright field microscope, dark field microscope, fluorescence microscope, and phase-contrast microscope.

Optical or light microscopes involves visible light transmission through or reflected from the sample through a single lenses or multiple lenses and provide a magnified view of sample. The resultant image can be seen by eyes directly or can be taken on screen, or because of advancement in technology this image can be captured digitally.

Limitation Of Light Or Optical Microscopes

Limitations of light or optical microscopes can be discussed as (1) the technique can only magnify object in dark or strongly reflecting objects effectively. (2) there is limited resolution depending upon incident wavelength; in visible range, the resolution of optical microscopy is limited to approximately 0.2 micrometers and the practical magnification limit to ~1500x.

Bright Field Microscope

This is the most common type of microscope used in microbiology labs. It is the simplest of all the light microscopy techniques. A beam of white light is used to visualize the sample. In this microscope, sample is illuminated from below and seen from upside.

The simplicity of the technique and the minimal sample preparation makes these type of microscope a first choice of microbiologist. However, low contrast of most biological samples and low apparent resolution are some limitations of bright field microscope.

Dark Field Microscopy 

This type of microscope work on the technique to improve the illumination of unstained, transparent specimens. 

As the name indicate the microscope show the object (specimen) in dark background. This is achieved by equipping the light microscope with a special kind of condenser that transmit a hollow cone of light from the source of illumination. Most of the light directed through the condenser do not enter to the objective, this causes a dark field. However some of the light scattered (diffracted) if the sample contain some object like microbial cells. This diffracted light enter the objective and reach the eye. Thus the object present in sample will appear bright against a dark background.

Phase-Contrast Microscopy

Phase contrast microscopy is one of the most common and important techniques of microbiology as because of phase contrast microscopy we can visualize living unstained objects.

Phase contrast microscopy uses a conventional light microscope fitted with a phase-contrast objective, and a phase contrast condenser. This special optical system makes it possible to distinguish unstained structures within a cell which differ only slightly in there reflective indices or thickness.

If we talk about the principle of phase contrast microscopy, this technique is based on the fact that light passing through one material and into another material of a slightly different refractive index and / or thickness will undergo a change in phase. These change in phase , or wavefront irregularities, are translated into variation in brightness of the structure and hence are detectable by the eyes.With phase contrast microscopy it is possible to reveal differences in cells and their structure, this cannot be done by other microscopic methods.

Reference: wiki, Microbiology by pelczar

Gaurav Singh

Editor in Chief Medical Microbiology & Recombinant DNA Technology (RDT) Labs - RDT Labs Magazine

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