Observing microorganism9 06 (1)


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  • Now, over the years the microscope has evolved and scientists have developed different kinds of microscopes to adequately examine different organisms/specimens.
  • Dark field microscopy is a very simple yet effective technique and well suited for uses involving live and unstained biological samples, such as a smear from a tissue culture or individual water-borne single-celled organisms. Considering the simplicity of the setup, the quality of images obtained from this technique is impressive.
  • Phase contrast microscopy is an optical microscopy illumination technique in which small phase shifts in the light passing through a transparent specimen are converted into amplitude or contrast changes in the image. A phase contrast microscope does not require staining to view the slide. This type of microscope made it possible to study the cell cycle . Phase contrast images have a characteristic grey background with light and dark features found across the sample. Light and dark fringes appear around regions with a change in optical density, for example the boundary between water and a cell. This normally manifests as a light halo around a dark object.
  • is an optical microscopy illumination technique used to enhance the contrast in unstained, transparent samples . DIC works on the principle of interferometry to gain information about the optical path length of the sample, to see otherwise invisible features. A relatively complex lighting scheme produces an image with the object appearing black to white on a grey background. This image is similar to that obtained by phase contrast microscopy but without the bright diffraction halo.
  • A fluorescence microscope is an optical microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption
  • Direct fluorescent antibody ( DFA or dFA ) (also known as "Direct immunofluorescence" [1] ) is a laboratory test that uses antibodies tagged with fluorescent dye that can be used to detect the presence of microorganisms . This method offers straight-forward detection of antigens using fluorescently labeled antigen-specific antibodies. Because detection of the antigen in a substrate of patient sample (cellular smear, fluid or patient- inoculated culture medium) is the goal, DFA is seldom quantitative.
  • An electron microscope is a type of microscope that uses a particle beam of electrons to illuminate the specimen and produce a magnified image. Electron microscopes (EM) have a greater resolving power than a light-powered optical microscope , because electrons have wavelengths about 100,000 times shorter than visible light ( photons ), and can achieve better than 50 pm resolution [1] and magnifications of up to about 10,000,000x, whereas ordinary, non-confocal light microscopes are limited by diffraction to about 200 nm resolution and useful magnifications below 2000x.
  • Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes
  • In staining for microscopic examination for diagnosis or research acid dyes are used to color basic tissue proteins in contrast to basic dyes, which are used to stain cell nuclei and some other acidic components of tissues
  • A simple stain consists of a solution of a single dye. Some of the most commonly used dyes are methylene blue, basic fuchsin, and crystal violet. Simple stains allow one to distinguish the shape (morphology) of the bacteria. For example, E. coli and Bacillus Subtillus are bacilli or rod-shaped bacteria. Many bacilli occur singularly, but chains may also be observed. Bacilli very greatly in length and diameter. Staphylococcus aureus and Streptococcus pneumoniae are cocci or spherical bacteria. Cocci may occur singularly, in pairs (as in Streptococcus pneumoniae) , or in clusters (as in Staphylococcus aureus) . R. rubrum is a spirillum or curved bacterium, Spirilla always occur singularly.
  • Differential stains are more complex than simple ones and use more than one stain to differentiate cellular components. They are used to examine structural differences between bacterial groups or to provide contrast to different structures within the same organism
  • As mentioned, Gram-negative bacteria generally possess a thin layer of peptidoglycan between two membranes (diderms). Most bacterial phyla are Gram-negative, namely the cyanobacteria , spirochaetes and green sulfur , most Proteobacteria (the exceptions in the Proteobateria are some members of the Rhickettsiales and the insect-endosymbionts of the Enterobacteriales ) and many other phyla. [5] [14] [ edit ] Gram-positive bacteria Main article: Gram-positive bacteria On the other hand, Gram-positive bacteria have generally a single membrane (monoderm) surrounded by a thick peptidoglycan The Gram stain procedure uses 3 different stains. These are crystal violet, Gram’s iodine, and safranin. The cells are first stained with crystal violet, then Gram’s iodine. Following a rinse in alcohol, to de-colorize the cells, the cells are then stained with safranin. The Gram stain procedure separates almost all bacteria into two large groups: the Gram-positive bacteria that stain blue and the Gram-negative bacteria that stain pink. Bacteria take up the Gram stain differently because they differ in cell wall composition. Gram-positive bacteria have a thick cell wall layer. Alcohol does not readily penetrate to decolorize the cell wall of the previously applied crystal violet stain. Gram-negative cells have a thinner cell wall through which the alcohol readily penetrates. The crystal violet is removed from these cell walls that are then stained with the safranin counterstain
  • It is helpful in diagnosing Mycobacterium tuberculosis since its lipid rich cell wall makes it resistant to Gram stain . It can also be used to stain few other bacteria like Nocardia . The reagents used are Ziehl–Neelsen carbolfuchsin , acid alcohol and methylene blue . Acid-fast bacilli will be bright red after staining
  • Negative staining is an established method, often used in diagnostic microscopy , for contrasting a thin specimen with an optically opaque fluid. For bright field microscopy , negative staining is typically performed using a black ink fluid such as nigrosin . The specimen, such as a wet bacterial culture spread on a glass slide, is mixed with the negative stain and allowed to dry. When viewed with the microscope the bacterial cells, and perhaps their spores , appear light against the dark surrounding background
  • Observing microorganism9 06 (1)

    1. 1. Yasmine Kanaan, Ph.D. Tel. 202 806 9540 Cancer Center, Rm # 410 Observing Microorganisms
    2. 2. Microorganisms? Much too small to be seen with unaided eyes
    3. 3. Units of Measurement <ul><li>The standard unit of length is the meter (m) </li></ul><ul><li>Microorganisms are measured in micrometers, µm (10 -6 m), </li></ul><ul><li>& in nanometers, nm (10 -9 m) </li></ul>
    4. 5. Microscope <ul><li>Is derived from the Latin word: </li></ul><ul><li>Micro: small </li></ul><ul><li>And the Greek word: </li></ul><ul><li>Skopos: to look at </li></ul>
    5. 6. Antoni van Leeuwenhoek is called &quot;the inventor of the microscope,&quot; <ul><li>1676 </li></ul><ul><li>The first clear mention </li></ul><ul><li>microorganisms and their discovery. </li></ul><ul><li>Leeuwenhoek uses of </li></ul><ul><li>a self-made, hand-held, single lens </li></ul><ul><li>microscope </li></ul><ul><li>magnifying to about 100X, </li></ul><ul><li>Many drawings are highly indicative of species still known to be present in the materials he examined. </li></ul>
    6. 7. Kinds of Microscopes <ul><li>Light Microscopy </li></ul><ul><li>Electron Microscopy </li></ul><ul><li>Scanned-Probe Microscopy </li></ul>
    7. 8. A. Light Microscopy <ul><li>Refers to use any kind of microscope that uses visible light to observe specimens. </li></ul><ul><li>Several types: </li></ul><ul><li>Compound light microscopy </li></ul><ul><li>Darkfield Microscopy </li></ul><ul><li>Phase-Contrast Microscopy </li></ul><ul><li>Differential Interference Contrast Microscopy </li></ul><ul><li>Flourescence Microscopy </li></ul><ul><li>Confocal Microscopy </li></ul>
    8. 9. 1. Compound Light Microscope <ul><li>The total magnification of an object is calculated by multiplying the magnification of the objective lens by the magnification of the ocular lens. </li></ul>40X,10X, 100x 10x
    9. 10. <ul><li>Resolution : resolving power: is the ability of the lenses to distinguish fine details and structure. </li></ul><ul><li>Refractive index : is a measure of the light-bending ability of a medium. </li></ul>
    10. 11. 2.Darkfield Microscopy <ul><li>Uses a special condenser w/ an opaque disc that block light from entering the objective lens directly . </li></ul><ul><li>Shows a light silhouette of an organism against a dark background. </li></ul><ul><li>It is most useful for detecting the presence of extremely small organisms. </li></ul><ul><li>Examine unstained microorganisms suspended in a media. </li></ul>
    11. 12. 3. Phase-Contrast Microscopy <ul><li>Brings direct & reflected of diffracted light rays together(in phase) to form an image of the specimen on the ocular lens. </li></ul><ul><li>It allows the detailed observation of living organisms ( internal structures ). </li></ul><ul><li>No staining required </li></ul>
    12. 13. 4. Differential interference contrast (DIC) Microscopy <ul><li>Uses 2 beams of light separated by a prisms. </li></ul><ul><li>Provides a colored, 3-dimensional image of the object being observed. </li></ul><ul><li>It allows detailed observations of living cells. </li></ul><ul><li>No staining is required </li></ul>
    13. 14. 5. Fluorescence Microscopy <ul><li>Specimens are stained w/fluorochromes and then viewed through a compound microscope by using ultraviolet light source. </li></ul><ul><li>The microorganism appear as bright objects against a dark background. </li></ul>
    14. 15. 5 cont. Fluorescence Microscopy <ul><li>FM is used primarily in a diagnostic procedure called fluorescent-antibody (FA) technique, </li></ul><ul><li>or immunofluorescence to detect and identify microbes in tissues or clinical specimens. </li></ul>
    15. 16. 6. Confocal Microscopy <ul><li>Uses laser light to illuminate one plane of a specimen at a time </li></ul><ul><li>To obtain 2 and 3-dimensional images of cells for biomedical applications. </li></ul>
    16. 17. B. Electron Microscopy <ul><li>Uses a beam of electrons, instead of light. </li></ul><ul><li>Electromagnets, instead of glass lenses, control focus, illumination, and magnification. </li></ul>
    17. 18. B cont .Transmission Electron Microscopy <ul><li>To examine viruses or the internal ultra structure in thin sections of cells. </li></ul><ul><li>Electrons pass through the specimen. </li></ul><ul><li>Magnification: 10,000-10,000X </li></ul><ul><li>The image produced is 2-dimensional. </li></ul>
    18. 19. B cont .Scanning Electron Microscopy <ul><li>The e- beam is directed at the intact specimen from the top, rather than passing through a section, </li></ul><ul><li>e- leaving the surface of the specimen (2ry e-) are viewed on a television-like screen. </li></ul><ul><li>To study the surface features of cells and viruses. </li></ul><ul><li>Magnification 1000-10,000X </li></ul>
    19. 20. C. Scanned-Probe Microscopy/ Scanning tunneling <ul><li>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 </li></ul><ul><li>Detailes views of molecules such as DNA </li></ul>
    20. 21. C. Scanned-Probe Microscopy/ Atomic force <ul><li>Uses a metal-and-diamond probe gently forced down along the surface of the specimen. </li></ul><ul><li>Produces a 3-dimensional image. </li></ul>
    21. 22. Staining Techniques <ul><li>Simple </li></ul><ul><li>Differential </li></ul><ul><li>Special </li></ul>
    22. 23. Staining Reaction <ul><li>Stains - salts composed of a positive and negative ion, one of which is colored (chromophore) </li></ul><ul><li>Basic Dyes - chromophore is the positive ion </li></ul><ul><ul><li>dye+ Cl- </li></ul></ul><ul><li>Acid Dyes - chromophore is the negative ion </li></ul><ul><ul><li>Na+ dye- </li></ul></ul>
    23. 24. Bacteria are slightly negative, so are attracted to the positive chromophore of the BASIC DYE <ul><li>Common Basic Dyes </li></ul><ul><ul><li>crystal violet </li></ul></ul><ul><ul><li>methylene blue </li></ul></ul><ul><ul><li>safranin </li></ul></ul><ul><ul><li>Malachite green </li></ul></ul>
    24. 25. <ul><li>Common acidic dyes </li></ul><ul><li>Eosin </li></ul><ul><li>Acid fuchsin </li></ul><ul><li>nigrosin </li></ul>
    25. 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.
    26. 27. Simple Stains <ul><li>A simple stain is an aqueous or alcohol solution of a single basic dye (methylene blue, carbolfuchsin, crystal violet, safranin). </li></ul><ul><li>To visualize shapes and arrangements of cells. </li></ul><ul><li>A mordant may be used to improve bonding between the stain and the specimen. </li></ul>
    27. 28. Differential Stains <ul><li>React differently with different types of bacteria </li></ul><ul><li>2 Most Common </li></ul><ul><ul><li>Gram Stain </li></ul></ul><ul><ul><li>Acid-Fast Stain </li></ul></ul>
    28. 29. Gram Stain <ul><li>1884 Hans Christian Gram </li></ul><ul><li>most important stain used in Bacteriology </li></ul><ul><li>Divides all Bacteria into 2 groups: </li></ul><ul><ul><li>Gram (+) </li></ul></ul><ul><ul><li>Gram (-) </li></ul></ul>
    29. 30. Differential Stains <ul><li>Such as the Gram stain and the acid-fast stain, differentiate bacteria according to their reactions to the stain. </li></ul><ul><li>Gram stain procedure uses a purple stain (crystal violet), iodine as a mordant, an alcohol decolorizer, and a red counterstain. </li></ul>
    30. 32. Results <ul><li>Gram (+) Purple </li></ul><ul><li>Gram (-) Red </li></ul><ul><li>Difference - due to structure of cell wall </li></ul><ul><ul><li>Gram (+) Thick cell wall </li></ul></ul><ul><ul><li>Gram (-) Thin cell wall </li></ul></ul>
    31. 33. Differential Stains/ Acid-Fast <ul><li>Acid-fast microbes, such as members of the genera Mycobacterium & Nocardia, retain carbolfuchsin after acid alcohol decolorization & appear red </li></ul><ul><li>non-acid-fast microbes take up the methylene blue counterstain and appear blue. </li></ul>
    32. 34. Special Stains <ul><li>Special stains are used to color and isolate specific parts of microorganisms, such as </li></ul><ul><li>Endospores </li></ul><ul><li>Flagella </li></ul><ul><li>To reveal the presence of capsules. </li></ul>
    33. 35. Negative staining for capsules <ul><li>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. </li></ul><ul><li>Capsules do not accept most stains, & appear as unstained halos around bacterial cells & stand out against a dark background. </li></ul>
    34. 36. Special Stains/Endospore staining <ul><li>Schaeffer-Fulton endospore stain. </li></ul><ul><li>Uses Malachite green, the 1ry stain, heat, wash, add safranin (counterstain) to stain portions of the cells other than endospores. </li></ul>
    35. 37. Special Stains/ Flagella staining <ul><li>Use a mordant & and the stain carbolfuchsin to build up the diameters of the flagella until they become visible under the light microscope. </li></ul>
    36. 38. Any Questions?? If you have any Qs, please don’t hesitate to stop by my office