Observing Microorganisms Through a Microscope

1. Observing Microorganisms Through a Microscope Chapter 3

2. Units of Measurement

3. Sizes and Magnification

4. Check Your Understanding • Differentiate between the different metric units used to measure the sizes of microbes.

5. Microscopy: The Instruments

6. Light Microscopy • Uses light to illuminate a specimen to be viewed • Useful for objects larger than 0.2μm in size • Bacteria, eukaryotic cells • Allows scientists to view living specimens

7. Compound Light Microscopy • Visible light provides the source of illumination • Image passes from Objective to Ocular Lens • TotalMagnification • Resolution (Resolving Power) is ability of a lens to distinguish fine detail • 0.2 μm

8. Immersion Oil • Only used with 100X objective lens • Reduces refraction of light rays passing from one medium to another • Has same refractive index as a glass slide or lens

9. Brightfield Microscopy • Background is brightly illuminated • Condenser focuses light directly on specimen • Typical compound light microscope

10. Darkfield Microscopy • Useful for living specimens or that cannot be stained while maintaining integrity • Treponemapallidum • Condenser is opaque • Light is reflected off the speciment • Background is darkened

11. Phase-Contrast Microscopy • Allows visualization of large internal structures in living specimens • Structures are well-defined • Uses “in-phase” and “out-of-phase” light rays to produce areas of contrast

12. Differential Interference Contrast (DIC) Microscopy • Uses two light sources to produce differences in phase contrast • High resolution is possible • Image is bright and has a three-dimensional quality

13. Fluorescence Microscopy • Fluorescence is ability to absorb one wavelength of light and give off at another • Antibodies are immune particles produced against a specific molecule(Antigen) • Allows scientists to “paint” specific molecules to make them highly visible

14. Confocal Microscopy • Uses fluorescent antibodies in conjunction with light microscopy • Specimen is scanned in series of focal planes and then reassembled via computer • Produces a detailed, 3-dimensional image

15. Electron Microscopy • Uses a beam of electrons to resolve the object being “viewed” • Provides a highly detailed image for objects smaller than 0.2μm • Organelles and viruses

16. Transmission Electron Microscopy • Uses a focused beam of electrons that penetrate the specimen • Provides detailed images of internal structures • “Stains” produced by various metals can enhance the contrast of the image

17. Scanning Electron Microscopy • Electron beam is focused across the surface of the specimen • Gives a three-dimensional view of the specimen • Provides detailed images of external structures

18. Check Your Understanding • Describe the path of light through a compound microscope. • Explain the role of immersion oil in light microscopy. When is it used? • Define total magnification and resolution. What is the significance of a 0.2μm resolution for light microscopes. • Differentiate between electron and light microscopy. Why do electron microscopes have a greater resolution than light microscopes? • Differentiate between the different types of light and electron microscopy techniques available. When would each be used?

19. Preparation of specimens for light microscopy

20. Preparing Smears for Staining • Specimen are added to the slide using water and then spread to a thin film called a smear • Heat-fixing kills the microbes and adheres them to the slide BEFORE stains are applied • Performed only after the smear has completely air-dried • Staining requires adding dyes to a specimen to add contrast so it is more visible under the microscope • Stains (dyes) are liquids that can remove specimens

21. Types of Dyes Basic Dyes Acidic Dyes Chromophores are negatively charged Repelled by the the negatively charged cell walls of bacteria Cells appear colorless against a dark background Negative Staining Nigrosin • Chromophoresare positively charged • Attracted to the negatively charged cell walls of bacteria • Cells appear colored against a clear background • Crystal Violoet, Methylene Blue, Malachite Green, Saffranin

22. Simple Stain • A single dye is added to a specimen providing contrast • A mordant may be added to help the specimen retain the dye Bacillus subtilis

23. Gram Stain • Differential stain • Developed by Han Christian Gram (1884) • Helps to begin classifying bacteria • Differences are based on differences in the cell wall of bacteria • Gram (+) bacteria have a thick peptidoglycan (PG) cell wall • Gram (-) bacteria have a thin PG cell wall and outer layer of lipopolysaccharide (LPS)

24. Acid-Fast Stain • Differential stain • Dyes adhere strongly to the thick layer of mycolic wax found in some bacteria • Wax resists decolorization even with acid-alcohol • Useful in identifying bacteria in the genus Mycobacterium

25. Capsule Stain • Capsules enhance the virulence of an organism • Prevent its phagocytosis by leukocytes • A type of negative stain preventing removal of the capsule by excessive washes Klebsiellapneumoniae

26. Endospore Stain • Endospores are resistant structures containing the DNA of a bacteria • Formed under adverse environmental conditions • A type of differential stain • Uses heat to allow primary dye to penetrate the spore coat Bacillus megaterium

27. Flagella Stain • Flagella are normally too small to be seen with the light microscope • A type of simple stain • Uses a mordant and dye to build up the layers of dye on the flagella Proteus vulgaris

28. Check Your Understanding • What steps are needed to make a smear prep? Why is it necessary to heat-fix a specimen prior to staining? • Differentiate between an acidic and a basic dye. Why does a negative stain color the background and not the specimen? • What is a simple stain and what is its role in the microbiology lab? Why does a simple stain color the specimen and not the background? • Describe the steps in a Gram stain. Differentiate between a Gram-positive and a Gram-negative organism based on color. • Compare and contrast the Gram stain and the acid-fast stain. Provide two examples of organisms that can be identified with an acid-fast stain. • What is the purpose of a capsular stain, endospore stain, and flagellar stain? Give examples of organisms that could be identified with each type of staining technique.