Microscope and Specimen Preparation, Biology tutorial


Microbiology is basically the study of organisms too small to be seen clearly with the without the help eyes. The nature of this discipline makes the microscope of vital significance because the study of microorganisms is not possible devoid of the microscope. Microscopes give magnification that allows us to view microorganisms and study their structures. The magnification achieved by microscopes range from x100 to x400,000; moreover there are various kinds of microscopes and many methods have been developed through which specimens of microorganisms can be made for examination.

The Microscope

A microscope is a device or instrument for producing enlarged images of objects which are too small to be seen without help.

Types of Microscopes:

Microscopes are generally of two types: The Light Microscope and Electron Microscope.

The Light Microscope:

This is a kind of microscope in which magnification is achieved through a system of optical lenses by using light waves. It comprises:

1) Bright Field Microscope

2) Dark Field Microscope

3) Fluorescence Microscope

4) Phase Contract Microscope

Modern microscopes are the compound microscopes. That is, the magnified image prepared by the objective lens is further enlarged by one or more extra lenses.

The Bright Field Microscope:

a) The ordinary microscope is termed as a bright field microscope as it makes a dark image against a brighter background.

b) The microscope comprises of a sturdy metal body or stands made up of a base and an arm to which the remaining portions are attached.

c) A light source, either a mirror or the electric illuminator, is positioned at the base.

d) Two focusing knobs, that is, the fine and coarse adjustment knobs are positioned on the arm and can move either the phase or the nose piece to focus the image.

e) The phase is positioned around halfway up the arm and hold microscope slides through slide clips or a mechanical stage clip.

f) There is a sub-stage condenser mounted in or below the stage which mainly focuses a core of light on the slide.

g) The upper portion of arm of the microscope holds the body assembly to which a nose piece and one or more eyepieces and ocular lenses are joined.

h) Most advanced microscopes encompass eyepieces for both eyes and are termed as binocular microscopes.

i) The nose piece holds 3-5 objective lenses of dissimilar magnifying power and is easily rotated to place any objective.

j) The image you see if viewing a specimen is mainly focused by the objective and ocular lenses working altogether.

k) Light from the specimen that has been enlightened is focused by the objective lens making an enlarged image in the microscope. The ocular lens further magnifies the primary image.

l) The total magnification is computed by multiplying the objective and eye piece magnification altogether; example: if a 45x objective is employed by a 10x eyepiece, the total magnification of the specimen will be 450x.

The Dark-Field Microscope:

The dark field microscope is employed to examine living unstained cells and organisms as a result of change in the manner they are illuminated.

A hollow core of light is focused on the specimen in such a manner that un-reflected and un-refracted rays don't enter the objective only light which has been reflected or refracted through the image makes an image. 

The fields surrounding the specimen looks like black whereas the object itself is brightly illuminated.

The dark field microscope is helpful in revealing lots of internal structures in bigger eukaryotic microorganisms.

It is as well employment in the examination of unstained microorganisms suspended in fluids, example: wet mount and hanging drop preparation.

The Phase-Contrast Microscope:

This kind of microscope transforms slight differences in the refractive index and cell density into easily detected variations in light intensity and is employed to view the living cells.  The background made by the undeviated light is bright whereas the unstained objects come out dark and well defined. 

This microscope is much helpful for studying microbial motility, finding out the shape of living cells and detecting several bacterial components like endospores and inclusion bodies. It is as well employed in studying the eukaryotes.

The Fluorescent Microscope:

This kind of microscope depicts a specimen to ultraviolet, violet or blue light and makes an image of the object with resultant fluorescent light. The most generally employed fluorescence microscope light is epifluorescence microscope which is as well termed as incident light or reflected light microscope. Epifluoresence microscope uses an objective lens which as well acts as a condenser. A mercury vapor arc lamp or other source generates an intense beam of light which passes via an exciter filler. The exciter filler transmits on the desired wavelength of the excitation light. 

The excitation light is directed down the microscope through a speed minor termed as the dichromatic minor. This minor reflects light of shorter wavelength however lets light of longer wavelength to pass through. The excitation light carries on through the objective lens to specimen stained having spaced dye molecules termed as fluorochromes.

Microscope Resolution:

Resolution is the capability of a lens to separate or differentiate between small objects which are close altogether that is, the microscope should produce a clear image and not just a magnified one. It is as well termed as the resolving power.  

Resolution is explained mathematically through an equation in the year 1870 by Ernest Abbe, a German physicist. The Abbe equation defines that the minimal distance (d) among two objects which reveal them as separate entities based on the wavelength of light (λ) used to illuminate the specimen and on the numerical aperture of the lens (n sin θ) that is the capability of the lens to collect light.

d = (0.5 λ)/(n sin θ)

Since, d becomes smaller, the resolution rises and finer details can be distinguished in a specimen; d becomes smaller as the wavelength of light used reduces and as the numerical aperture (NA) rises. Therefore, the greatest resolution is achieved by using a lens having the largest NA and light by the shortest wavelength.

The relationship among NA and resolution can be stated as follows:

d = λ/2NA

Here, d = resolution and λ = wavelength of light. By employing the values 1.3 for NA and 0.5 μm, the wavelength of green light, for > λ, resolution can be computed as: 

d = (0.55)/(2 x 1.30) = 0.21μm.

From these computations, we might conclude that the smallest details that can be seen by the light microscope are those having dimensions of around 0.2μm.

Preparation for Light-Microscope examination:

There are two general techniques employed for preparing specimens for light-microscope examination.

1) The organisms are hanged or suspended in a liquid (that is, the wet-mount or the hanging drop method).

2) The organism is dried fixed and stained prior to observing beneath the microscope.

Electron Microscope:

This kind of microscope employs a beam of electron in place of light waves to generate the image. There are two kinds:

  • Scanning electron microscope
  • Transmission electron microscope

Transmission Electron Microscope:

Electron microscopes make use of a beam of electrons to illuminate and make magnified images of specimens. Electrons substitute light as the illuminating beam. They can be focused, much as light is in a light microscope; however their wavelength is around 0.005mm 1000,000 times shorter than that of visible light. Thus, electron microscopes encompass a practical resolution around 1,000 times better than the light microscope, with lots of electron microscopes point closer than 0.5 nm can be distinguished and the helpful magnification is well over 100,000x. In transmission electron microscope, the electron beam is transmitted via the specimen.

Scanning Electron Microscope:

The scanning electron microscope generates an image from electron discharged from atoms on an object's surface. It has been employed to observe the surfaces of microorganisms in great detail. Most of the SEM consists of a resolution of around 7nm or less.

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