This document provides an overview of different types of microscopes used to observe microorganisms and describes various staining techniques. It discusses light microscopes like compound, darkfield, phase contrast, and fluorescence microscopes. Electron microscopes like transmission and scanning electron microscopes are also outlined. Different staining methods such as simple, differential, and special stains are summarized, with examples like Gram staining and acid-fast staining.

1. Yasmine Kanaan, Ph.D. Tel. 202 806 9540 Cancer Center, Rm # 410 Observing Microorganisms

2. Microorganisms? Much too small to be seen with unaided eyes

3. Units of Measurement The standard unit of length is the meter (m) Microorganisms are measured in micrometers, µm (10 -6 m), & in nanometers, nm (10 -9 m)

5. Microscope Is derived from the Latin word: Micro: small And the Greek word: Skopos: to look at

6. Antoni van Leeuwenhoek is called "the inventor of the microscope," 1676 The first clear mention microorganisms and their discovery. Leeuwenhoek uses of a self-made, hand-held, single lens microscope magnifying to about 100X, Many drawings are highly indicative of species still known to be present in the materials he examined.

7. Kinds of Microscopes Light Microscopy Electron Microscopy Scanned-Probe Microscopy

8. A. Light Microscopy Refers to use any kind of microscope that uses visible light to observe specimens. Several types: Compound light microscopy Darkfield Microscopy Phase-Contrast Microscopy Differential Interference Contrast Microscopy Flourescence Microscopy Confocal Microscopy

9. 1. Compound Light Microscope The total magnification of an object is calculated by multiplying the magnification of the objective lens by the magnification of the ocular lens. 40X,10X, 100x 10x

10. Resolution : resolving power: is the ability of the lenses to distinguish fine details and structure. Refractive index : is a measure of the light-bending ability of a medium.

11. 2.Darkfield Microscopy Uses a special condenser w/ an opaque disc that block light from entering the objective lens directly . Shows a light silhouette of an organism against a dark background. It is most useful for detecting the presence of extremely small organisms. Examine unstained microorganisms suspended in a media.

12. 3. Phase-Contrast Microscopy Brings direct & reflected of diffracted light rays together(in phase) to form an image of the specimen on the ocular lens. It allows the detailed observation of living organisms ( internal structures ). No staining required

13. 4. Differential interference contrast (DIC) Microscopy Uses 2 beams of light separated by a prisms. Provides a colored, 3-dimensional image of the object being observed. It allows detailed observations of living cells. No staining is required

14. 5. Fluorescence Microscopy Specimens are stained w/fluorochromes and then viewed through a compound microscope by using ultraviolet light source. The microorganism appear as bright objects against a dark background.

15. 5 cont. Fluorescence Microscopy FM is used primarily in a diagnostic procedure called fluorescent-antibody (FA) technique, or immunofluorescence to detect and identify microbes in tissues or clinical specimens.

16. 6. Confocal Microscopy Uses laser light to illuminate one plane of a specimen at a time To obtain 2 and 3-dimensional images of cells for biomedical applications.

17. B. Electron Microscopy Uses a beam of electrons, instead of light. Electromagnets, instead of glass lenses, control focus, illumination, and magnification.

18. B cont .Transmission Electron Microscopy To examine viruses or the internal ultra structure in thin sections of cells. Electrons pass through the specimen. Magnification: 10,000-10,000X The image produced is 2-dimensional.

19. B cont .Scanning Electron Microscopy The e- beam is directed at the intact specimen from the top, rather than passing through a section, e- leaving the surface of the specimen (2ry e-) are viewed on a television-like screen. To study the surface features of cells and viruses. Magnification 1000-10,000X

20. C. Scanned-Probe Microscopy/ Scanning tunneling Uses a thin metal probe that scans a specimen and produces an image revealing the bumps and depressions of the atoms on the surface of the specimen Detailes views of molecules such as DNA

21. C. Scanned-Probe Microscopy/ Atomic force Uses a metal-and-diamond probe gently forced down along the surface of the specimen. Produces a 3-dimensional image.

22. Staining Techniques Simple Differential Special

23. Staining Reaction Stains - salts composed of a positive and negative ion, one of which is colored (chromophore) Basic Dyes - chromophore is the positive ion dye+ Cl- Acid Dyes - chromophore is the negative ion Na+ dye-

24. Bacteria are slightly negative, so are attracted to the positive chromophore of the BASIC DYE Common Basic Dyes crystal violet methylene blue safranin Malachite green

25. Common acidic dyes Eosin Acid fuchsin nigrosin

26. Mordant - intensifies the stain or coats a structure to make it thicker and easier to see after it is stained Example: Flagella - can not normally be seen, but a mordant can be used to increase the diameter of the flagella before it is stained Salmonella typhosa Counterstain: a second stain applied to a smear, provides contrast to the primary stain.

27. Simple Stains A simple stain is an aqueous or alcohol solution of a single basic dye (methylene blue, carbolfuchsin, crystal violet, safranin). To visualize shapes and arrangements of cells. A mordant may be used to improve bonding between the stain and the specimen.

28. Differential Stains React differently with different types of bacteria 2 Most Common Gram Stain Acid-Fast Stain

29. Gram Stain 1884 Hans Christian Gram most important stain used in Bacteriology Divides all Bacteria into 2 groups: Gram (+) Gram (-)

30. Differential Stains Such as the Gram stain and the acid-fast stain, differentiate bacteria according to their reactions to the stain. Gram stain procedure uses a purple stain (crystal violet), iodine as a mordant, an alcohol decolorizer, and a red counterstain.

32. Results Gram (+) Purple Gram (-) Red Difference - due to structure of cell wall Gram (+) Thick cell wall Gram (-) Thin cell wall

33. Differential Stains/ Acid-Fast Acid-fast microbes, such as members of the genera Mycobacterium & Nocardia, retain carbolfuchsin after acid alcohol decolorization & appear red non-acid-fast microbes take up the methylene blue counterstain and appear blue.

34. Special Stains Special stains are used to color and isolate specific parts of microorganisms, such as Endospores Flagella To reveal the presence of capsules.

35. Negative staining for capsules Microbiologist mix the bacteria in a solution containing a fine colloidal suspension of colored particles (India ink or nigrosin) and then stain, w/ simple stain as safranin. Capsules do not accept most stains, & appear as unstained halos around bacterial cells & stand out against a dark background.

36. Special Stains/Endospore staining Schaeffer-Fulton endospore stain. Uses Malachite green, the 1ry stain, heat, wash, add safranin (counterstain) to stain portions of the cells other than endospores.

37. Special Stains/ Flagella staining Use a mordant & and the stain carbolfuchsin to build up the diameters of the flagella until they become visible under the light microscope.

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