The Microscope

THE MICROSCOPE

Antony van Leeuwenhoek (1632-1723)

• Inventor of the first microscope

• Leeuwenhoek earned his living as a draper, but spent much of his spare time constructing simple microscopes composed of double convex glass lenses held between two silver plates (figure below)• his microscope could magnify around 50 to 300 times,

• A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy

There are many types of microscopes, the most common two main type,1. light microscope 2. electron microscope .

MicroscopyLight Microscopy•Bright-field Microscopes•Dark-field Microscopes•Phase -contrast Microscopes•Fluorescence Microscopes

Electron Microscopy•SEM•TEM

Light Microscope

In the light microscopy there is a use of visible light and glass lenses for the magnification. Microbiologist currently employ a variedly light microscopes in their work. The most commonly used basic types of microscopes include;

a. Bright-field microscopes,b. Dark-field microscope,c. Phase-contrast microscopesd. Fluorescence microscope

• Modern microscopes are all compound microscopes, that is the magnified image formed by the objective lens is further enlarged by one or more additional lenses like eyepiece (ocular) . A compound microscope with a single eyepiece is said to be monocular; one with two eyepiece is said to be binocular

•The part of a modern compound microscope and the part light takes through it are shown in the above figure. light enters the microscope from a light source in the base and often passes through a blue filter, which filter out the base and often passes light, leaving the shorter wavelengths and improving resolution. It then goes through a condenser, which gathering the light beams so that they pass through a specimen. The iris diaphragm controls the amount of light that passes though the specimen and into the objective lens. The higher the magnification, the greater the amount of light needed to see the specimen clearly. The objective lens magnifies the image before it passes though the body tube to the ocular lens in the eyepiece. The ocular lens (eyepiece) further magnifies the image. A mechanical stage allows precise control of moving the slide , which is especially useful in the study of microbes. The focusing mechanism consist of a coarse adjustment knob, which changes the distance very slowly. The coarse adjustment knob is used to locate the specimen. The fine adjustment knob is used to bring it into sharp focus.

Resolution

The resolving power(RP) of a lens is a numerical measurement of the that can be obtained with that lens. We can calculate the RP of a lens if we know its numerical aperture(NA) and the wavelength(λ) of the electromagnetic radiation used. The formula for calculating resolving power is;

RP = λ/2NA

Numerical Apertures

The numerical aperture is more difficult to understand. That is defined as half aperture angle, the angle of the cone of light entering an objective,

θθ

Thus, NA = ½ n sinθ

But, the angle of the cone of light that can enter a lens depends on the refractive index(n) of the medium in which the lens work.The refractive index for air is 1.00 in case of dry objective.

a. Bright-field microscopes

Special features:-Use visible lightSimple to useLeast expensive

Appearance:-Coloured or clear specimen on light back ground

Uses:-Observation of dead stained organisms or live ones with sufficient natural colour contrast. 

Special features:-Uses visible light with special condenser that causes light rays to reflect off specimen at an angle

Appearance:-Bright specimen on dark background

Uses:-Observation of unstained living or difficult to stain organisms. Allows one to see motions

b. Dark-field microscope

Special features:-Use visible plus phase shifting plate in objective with a special condenser that causes some light rays to strike specimen out of phase with each other

Appearance:-specimen has different degrees of brightness and darkness

Uses:-Detail observation of internal structure of living unstained organisms

c. Phase-contrast microscopes

d. Fluorescence microscope

Special features:-Uses ultraviolet light to excite molecules to emit light of different wave lengths, often brilliant colour, because UV can burn eyes, special lens materials are used

Appearance:-Bright fluorescent colour specimen on dark back ground

Uses:-Diagnostic tool for detection of organism or antibiotic s in clinical specimens or for immunological studies

Electron Microscopy

Special features:- Uses electron beams and electromagnetic lenses, inexpensive, preparation requires considerable time and practice.

Appearance: -Three dimensional view of surfaces.

Uses:-Observation of exterior of cells or of internal surfaces

• To visualise the internal structure - TEM• Electrons instead of light• Greater resolving power• Disadvantages:

ExpensiveA high vacuum is requiredVery thin sections (<100 nm thick)

Figure:- Bacterial cells viewed with light microscope

Figure:- Bacterial cell viewed with electron microscope

STAINING OF BACTERIA

Why to stain bacteria?Because bacteria are semitransparent and difficult to see in the unstained state. It is necessary to make bacteria visible to find out their shape, arrangements and other morphological characters. Staining will make the cells more visible and help in revealing their internal structures.

Types of stains and dyesA large number of colored organic compounds are available for staining microorganisms. Based on the chemical behavior of the dye, these can be classified into three., acidic, basic or neutral. An acidic dye is one in which the change on the dye ion is negative. An basic dye is one in which the change on the dye ion is positive. A neutral dye is a complex salt of a dye acid with a dye base. Acid dyes generally stain basic cell components, and basic dyes generally stain acidic cell components.

Process of stainingthe process of staining may be involve ion-exchange reactions between the stain and active sites at the surface of or within the cell. The colored ions of dye may replace other ions on cellular components. For example the ionic exchange which takes place during staining can be represented by the following equation, in which the (MB ) action replaced the (NA ) action in the cell.

(Bacterial cell ˉ) (NA ) + (MB ) (Clˉ) ------- (Bacterial cell ˉ) (MB ) + (NA ) (Clˉ)

In this MB is methylene blue dye, which is actually methylene blue chloride.

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+ +++

Staining methods can be divided into two groups. 1. simple staining and 2. differential staining.

Under differential staining comes.• gram’s staining• acid-fast staining• endospore staining• capsule staining• flagella staining• cytoplasmic inclusion staining• giemsa staining