Bio: Microscopy

Source:  Bio: Microscopy    Tag:  uses for visible light waves

Microscope

Microscope:- It is an instrument that is being used in lab to visualize the ultra fine structure and specific morphological features (size and shape) of various microorganisms.


Types of dyes used in Microscopy:-

·        Acidic dye
·        Basic dye
·        Neutral Dye



Basics of Microscope:

Limit of resolution : - It is the smallest distance by which two different points can be separately visualized and distinguished. The greater limit of resolution of light microscope is obtained with shorter wavelength (λ) of the visible light and objective with larger Numerical Aperture (NA).

Limit= λ/(2*NA)    NA=ŋ*Sinθ



Types of microscopes:-

·        Light based:-
·        Bright field,
·        Dark field,
·        Fluoresce &
·        Phase contrast microscope

·        Electron Based:-
·        Scanning tunneling microscope
·        Transmission electron microscope



Light Based Microscope


Light Based: - In it the image is obtained via optical lenses and light waves.

·   Bright Field Microscope: -

In it the microscopic field is brightly enlightened & microorganism appears because they absorb some of the light. Ordinarily microorganisms don’t absorb light but staining them with dye greatly increases the light absorbing ability resulting in greater contrast and color differentiation. Magnification range is from X1000 to X2000.

Limitations: - Lack of resolving power





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Dark field microscope:-

It has dark background in comparison to illuminated specimen. It is accompanied by a special type of condenser that transmits a hollow cone of light from the source of illumination. Mostly light directed through the condenser does not enter the objective. Field is dark essentially. Some of light ray are scattered, if the transparent media contains microbial cell. The diffracted light enters objective & reaches the eye. So the microbial cell appears bright in the dark microscopic field. It is valuable for unstained microorganisms suspended in a fluid.











Fluorescence Microscope: -

Most of the chemical substances absorb light. Some substance after absorbing the light of a particular energy emits light of lower energy. Such substances are called fluorescent and the phenomenon is called fluorescence. It is the basis of fluorescence microscopy. When specimen is stained in fluorescence dye and is illuminated with blue light, blue light is absorbed and green light is emitted. Two light filters used are Exciter filter & Barrier filter.



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Phase Contrast Microscope: -

It is valuable for unstained cells. It uses a conventional light microscope fitted with phase contrast objective and phase contrast condenser. The special optic system makes it possible to distinguish unstained structure with in the cells that differ only slightly in refractive index (µ) & thickness. It is based on the fact that light passing through one medium to another undergoes a phase change. The differences in phase irregularities are translated into variations in the brightness of the structure and are detected by eye. In phase contrast microscope it is possible to differentiate in cell and its structure which is not possible with other light based microscopes.



Electron Microscope




Electron Microscope: - It uses a beam of electron in place of light waves to produce us an ultra fine image of specimen. It can be done either by transmission of the electron along the specimen or by scanning the specimen


·        Transmission Electron Microscope: -


It differs from normal optical microscope. It obtains tremendous magnification and high resolution with extremely short wavelength of electron beam. It employs 60-80 KeV of energy and λ=0.5Ǻ. The resolving power is 100 times that of simple light microscope and produces a useful magnification of X400,000. In TEM the specimen is extremely thin dry film and is introduced into machine at a point between the magnetic condenser and magnetic objective. The magnified view can be observed on fluorescent tube kept in a closed window and is recorded on a photographic plate by an inbuilt camera.







Ways of visualizing Image by TEM



1.    Shadow Casting: - in it an extremely thin layer of platinum is used at oblique angle to specimen. So that the specimen cast its topographical shadow. Specimen is dried on special grid which is placed in vacuum tube. Heavy metal atoms are projected at an angle that produces the shadow behind the particle.




2.   Negative Staining:- in it Phosphotungstic acid (an electron dense substance) is used to outline the object. It forms a thick deposition on the cervices. Fine details of virus and bacteria can be viewed using this method.





3.   Ultra thin Sectioning:- It is used to observe intra cellular structures that are extremely thin in comparison to microbial cell. In it the specimen is sectioned into fine slices to be viewed.



4.   Location of enzyme:-  Light electron microscope is developed to locate the position of an enzyme within the specimen cell. Like Isocytrate Dihydrogenase of E-Coli. An enzyme is a bio-molecule that enhances the rate of reaction within the cell and remains unchanged. An enzyme has its own affinity and specificity for a particular substrate. An enzyme has the affinity throughout it which binds it to substrate and carries the required conversion.



5.   Autoradiography:- it is a cytochemical method in which location of a particular chemical constituent is determined by observing the site at which the radioactive material becomes positioned. Cells are exposed to radioactive rays and then it is covered with a layer of photographic emulsion and is stored in dark. The ionizing radiations emitted during decay are imaged.



·        Scanning electron microscope:-


Specimen is subjected to narrow electron beam of light which rapidly moves over the surface of specimen. This causes a release of shower of electron and other types of radiation from the specimen surface. The intensity of secondary electron is collected by the detector and generates the electronic signal. The signals are scanned in a manner so as to produce an image on cathode ray tube. It lacks resolving power in comparison to the TEM but have an advantage of 3D picture.

   

Cell Count


Quantitative measurement of bacterial growth:-



·        Cell Count:- it is done directly by microscope or by using electron particle counter or indirectly by colony count.

1.    Plate count method:- it determines the number of cells that will multiply under the certain defined conditions. A measured amount of bacterial suspensions is introduced into the petridish after which agar medium (45°C) is added and is thoroughly mixed by rotating the plate. When medium solidifies, the microbial cells get trapped into the gel. Each viable cell reproduces itself to give a visible mass of colony. In it the sample is to be diluted to get cell count in range of 30-300 to make count easier and accurate, and also to reduce interference. Colonies are observed using dark field illumination under large magnified lenses.



2.   Membrane filter count method:- a useful modified method based on the use of molecular membrane filter. This filter has the known uniform porosity of predetermined size. That is sufficient to filter off the present microbial cells. This technique is valuable for determining number of bacteria. The membrane got after filtering the sample is then placed in a special plate containing an appropriate medium. Special medium and dye is used to make it easier to detect microorganism in the conventional plate count method. During incubation each viable cell grows into colony that appears on membrane surface.






How to go serial dilution?

Here we take 1 ml of sample and transfer it to 99 ml of blank medium to form 1d. Again add 1 ml of 1d to 99 ml to form 2d and so on.

After adding the 1 ml of sample to the plate, 15-20ml of agar medium is poured in each of this plate and the plate is gently rotated for a thorough out distribution of specimen. And at last is incubated for 24hrs.



No. of bacteria/ml= (no. of colonies)*(dilution of sample)



Limitation:- only viable bacteria that grows in the medium and incubator are countable.


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