Microscope in microbiology, Slides of Microbiology

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Microscopic observation of

microorganisms

Definition of microscope: It is an optical instrument by which small objects can be magnified several times, so that objects which are not visible to the naked eye become visible. Principle: In the optical microscope the specimen (object) is placed on a glass slide, is scanned by focus beam of light. Parts of the object that has a high refractive index or colored by a stain cast a potential image like a shadow in the beam of light which is magnified in two stages as it passes up the microscope into the eye. Microorganisms are much too small to be seen with the unaided eye; they must be observed with a microscope. The word microscope is derived from the Latin word micro, which means small, and the Greek word skopos, to look at.

Properties of a compound Microscope:

1.Magnification: It is the degree of enlargement of the object, measured by multiplying the objective lens magnification (power) by the ocular lens magnification (power).

2. Resolution: It is the microscope ability to reveal closely adjacent structural details as separate distinct.

Compound Light Microscopy

• In a compound microscope, the image from the objective lens is magnified again by the ocular lens
• Total magnification = objective lens magnification × ocular lens magnification

Compound Light Microscopy

• The refractive index is a measure of the light-bending ability of a medium
• The light may bend in air so much that it misses the small high-magnification lens
• Immersion oil is used to keep light from bending

Normally, when light waves travel from one medium into another, they bend. Therefore, as the light travels from the glass slide to the air, the light waves bend and scattered. The microscope magnifies this distortion effect. Also, if high magnification is to be used, more light is needed. For this reason we use immersion oil Reason for using immersion oil Immersion oil has the same refractive index as glass, therefore, provides an optically homogeneous path between the slide and the lens of the objective. Light waves thus travel from the glass slide, into glass-like oil, into the glass lens without being scattered or distorting the image. In other words, the immersion oil “traps” the light and prevents the distortion effect that is seen as a result of the bending of the light waves. The refractive index is a measure of the light-bending ability of a medium

In bright field microscope, the microscopic field is brightly lighted and the organisms appear dark because they absorb light. It is a two lens system compound microscope with the objective lens nearer the object and the ocular lens nearer the eye. Bright Field Microscope Principle

Light passes through the objective lens, forming an inverted real image. This image serves as an object for the ocular lens which re-magnifies the image and forms the virtual image. The lens system of the eye perceives this image and captures it on the retina. Usually there are three objective lens in a light microscope, the low power ( X) the high power (40 X) and oil immersion lens. The ocular lens further magnifies the real image. Generally this type of microscope produces a useful magnification of about X 1,000 to X 2,000. Uses: This type of microscope is useful for observing stained microorganism.

• All stains kill the microbes, so living microbes must be observed unstained.
• Some microbes do not stain well.
• Staining procedures may distort the microbe or slightly alter the size. Limitation: Situations where staining is undesirable because Mechanism of working

Most of the light from the source of illumination is directed through a special kind of condenser. If the specimen is completely transparent & homogeneous, the light directed through the condenser does not enter the objective & the entire field of view is dark. However, if the transparent medium contains object that differ from it in refractive index, there will be reflection & refraction (scattering). The diffracted light will enter the objective and reach the eye; thus the object or microbial cell will appear bright in an otherwise dark microscopic field. Uses: This microscope is particularly valuable for the examination of unstained microorganisms suspended in fluid– wet-mount & hanging-drop preparation. Mechanism of working Limitations: 1.Darkfield microscopes may require serious fine tuning. 2.There are colors that are undistinguishable in the dark field mode. 3.Darkfield microscopes are limited to black and white.

Phase - Contrast Microscope This technique is based on the fact that light passing through one material to another material of a slightly different refractive index (the ratio of the velocity of light in a vacuum to its velocity in a specified medium) or thickness will undergo a change in phase. These differences in phase or wave front irregularities are translated into variations in brightness of the structures and hence are detectable by the eye. Principle

Fluorescence Microscope Many chemical substances absorb light. After absorbing light of a particular wavelength and energy, some substances will then emit light of a longer wavelength and lesser energy content. Such substances are called fluorescent (ZnS) and the phenomenon is termed fluorescence. Principle In fluorescence microscope, the microorganism are stained with fluorescent dye and illuminated with blue light; the blue light is absorbed & green light emitted by the dye. Uses:

• The principal use of fluorescence microscopy is a diagnostic technique called the fluorescent-antibody (FA) technique, or immunofluorescence.
• This technique can detect bacteria or other pathogenic microorganisms, even within cells, tissues, or other clinical specimens.

A high – intensity mercury lamp is used as the light source and emits white light. The exciter filter transmits only blue light to the specimen and blocks out all other colors. The blue light is reflected downward to the specimen by a dichroic mirror (which reflects light of certain colors but transmits light of other colors). Certain portions of the specimen which is stained with a fluorescent dye absorb blue light and emit green light. The green light passes upward, penetrates the dichroic mirror and reaches the barrier filter (which blocks blue light or other light emitted by the fluorescing specimen). Thus the eye perceives the stained portions of the specimen as glowing green against a jet black background. Mechanism of working