Microscopy

Microscopy and use of different Microscopes in Microbiology

1. Dr. M ahendra Swamy MBBS, MD(Microbiology) Microscopy

2. Introduction Definition: Use of Microscope to magnify the objects that are too small to be visible to naked eye. Used to detect for the presence of Microorganisms ( Bacteria, Fungus, Parasites, Viruses) in the specimen. Direct smears: Prepared from the specimen. Indirect smears: Prepared from the growth of Microorganisms on culture plate.

3. Applications of Microscopy in Microbiology Rapid Inexpensive Preliminary identification of organisms. Final identification of organisms in some cases . (Ex: Identification of M tb , M leprae by ZN staining, Identification of C diphtheria by Albert staining) Detection of different organisms present in the specimen. Detection of organisms that are difficult to grow. Detection of late growing organisms. Detection of anaerobic bacteria where facilities for anaerobic culture is not available. Providing information on what organisms to be expected in culture.

4. Applications of Microscopy in Microbiology Helps in selection of Culture media for growing the organisms. Helps in initiation of empirical treatment with antibiotics. Helps in assessment of quality of specimen. Helps in deciding clinical significance of organisms. Helps in detailed study of organisms (Electron Microscope).

5. Types of Microscopes Bright field Microscope (Compound/Light Microscope) : Field of view is brightly illuminated. Dark field Microscope : Filed of view is dark. But organisms are illuminated using a special condenser. Phase contrast Microscope : Here the organisms are observed by enhancing their contrast. Mainly used for examination of live unstained organisms. Fluorescence Microscope : Fluorochromes are used here for staining the organisms. Electron Microscope : Beam of electrons are used for illuminating the organisms.

6. Bright field Microscopy Fluorescence Microscopy Dark field Microscopy Electron Microscopy Bacteria + + + _ Fungi + + _ _ Parasites + + _ _ Viruses _ + _ +

7. Phase contrast Microscope Principle: Instead of using stains to enhance the contrast, here the differences in refractive index (density) of different cell components are used to create contrast. When beam of light pass through cells, the differences between refractive index of different components of cell result in difference in light intensity thus creating contrast. Applications: Used for examination of live unstained organisms, for detecting microbial motility and for detection of Intracellular Inclusion bodies.

8. Dark field Microscope Filed of view is dark. But organisms are illuminated using a special condenser. Principle: Special condenser with a central opaque disc is used. This condenser doesn’t allow light to directly pass through the specimen, but directs the light to hit the specimen at an oblique angle. Only the light rays that are deflected by the specimen enter the objective lens. As a result specimen is illuminated and the background appears dark. Applications: Used for detecting thin bacteria like Spirochetes: Treponema pallidum

9. Spirochetes : Treponema pallidum in Dark field Microscope

10. Electron Microscope Principle: Here electrons are used instead of light and electromagnetic fields are used to focus the electrons. Because of increased resolution, the final magnification of the image will be >100000 times. Scanning electron microscope (SEM): It uses electron beam to scan the surface of an object and provide 3D view of surface structures. Transmission electron microscope (TEM): Here electron beam passes through the object and hence internal structures of an object can be studied. Applications: Used for detection of viruses, studying intracellular components of cell, studying thin structures like Fimbria & Flagella.

11. Fluorescence Microscope Certain dyes ( Fluorochromes ) can be raised to high energy level after absorbing UV rays. When they return to their original, low energy level, they release excess energy in the form of visible light. This process is called as Fluorescence . 2 types: Fluorochroming : Here F luorochromes are used directly to stain the smear. Immunofluorescence : Here F luorochromes are tagged with specific antibodies.

12. Principle:

13. Applications: Acridine orange: Used for detection of Malarial parasites and detection of cell wall deficient bacteria (Ex: Mycoplasma) Auramine-Rhodamine : Used for detection of Mycobacterium tuberculosis. Calcofluor white: Used for detection of Fungi . Immunofluorescence: Here dyes are tagged with antibodies that will bind with antigens present on the surface of cells. Mainly used for detection of bacteria that are difficult to grow or slow growing (Legionella, Bordatella , Chlamydia). Ex: Fluorescein isothiocyanate (FITC) is the dye commonly used for conjugation.

14. Bright field Microscope Compound/ light Microscope Here visible light is used Total magnification is product of magnification of O cular and Objective lenses . Magnification: Ocular lens: 10x Total magnification is product of Ocular and Objective lenses Objective lenses: Scanning lens: 4x Low power lens: 10x High power lens: 40x Oil immersion lens: 100x

15. Resolution: It is ability of Microscope to produce separate images of closely spaced objects. Resolving power of human eye: 0.2 mm Resolving power of light Microscope is 0.2 Oil used in Oil immersion lens gives very good resolution as its refractive index is almost same as glass slide Contrast: Because Microorganisms are almost transparent because of high water content, they can not be easily visible among the specimen. Hence to enhance their contrast, Staining techniques are used.  

16. Numerical aperture (NA): It is a measure of light gathering power of a lens. High NA of a lens indicates high resolving power. Objective lenses Numerical aperture (NA) 10x 0.25 40x 0.65 100x 1.25

17. Parts of Compound Microscope Base : It supports the Microscope and holds various parts like source of light, brightness adjustment knob, light switch. Arm : Backbone of the Microscope. Bears Adjusting knobs and Mechanical stage. Source of light : Halogen lamps are preferred over Tungsten lamps as the former emits white light, have higher luminosity and have long life. Coarse adjusting knob : To raise the stage and bring the slide near the objective lens. Fine adjusting knob : To focus the smear present on the slide.

18. Mechanical stage : Stage with stage clips hold the slide in place. Stage control knobs can be moved to move the slide during observation. Condenser : It focuses light on the slide. Filter : Filters may be used below the condenser. Ex: Blue/Green filters. Blue filter may be used to change the light from ordinary electric bulb into a natural white light. Iris diaphragm : Used to control the amount of light that enters condenser. Nose piece with Objective lenses: Scanning lens: 4x Low power lens: 10x High power lens: 40x Oil immersion lens: 100x

19. Microscope tube : It contains Prisms that bend light thus providing comfortable viewing angle. Eye piece with Ocular lens : Magnification of Ocular lens is 10x. Eye piece can be Mono ocular or Binocular. It can have a movable pointer inside to focus an area on the smear.

20. Care of the Microscope Keep the Microscope on a table away from chemicals/water source to avoid any kind of splashes. The table should not be shared by vibrating devices like centrifuge / refrigerator. Turn off the light of the Microscope after use. Dust should be cleaned with air brush/artist’s brush. Clean/ wipe the lens after use. Cover the Microscope with p olythene/plastic bag when not in use.

21. Cleaning of the lens: Clean the lens with lens cleaning fluid recommended by the manufacturer. Usually Ethyl ether/Xylene are used. Alcohol/Acetone are not used unless specified by the manufacturer. Use lens paper for cleaning. If it is not available, use Muslin cloth/thin silk cloth.

22. Storing of the Microscope: Microscope should generally be closed with cover after use and stored in well lit and ventilated area. If it is stored in a closed space, keep drying agents like Silica gel, rice grains, salt along with it which will absorb moisture. In humid areas store the Microscope every night in a cabinet fitted with an electric bulb (5W to 40W). Light is switched on at night to reduce the humidity.

23. Preventing fungal growth on the Microscope: Fungal growth on the Microscope is common in hot & humid areas. They generally grow on objective lenses, eye piece & prisms. To prevent fungal growth, store the Microscope in a cabinet fitted with light. Use hygroscopic materials like silica gel, regularly clean the Microscope and store in a well lit and ventilated areas. Film of fungus can be removed from the Microscope by using fungus cleaner/lens cleaner fluid.

24. Micrometry It is a process where the size of the Microorganisms are measured on the Microscope. Micrometer is used for this purpose. Ocular Micrometer is a circular disc with graduations that fits in the eye piece. Stage micrometer is clipped on the stage of the Microscope.

25. Thank you