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3: Observing Microorganisms Through a Microscope

LEARNING OBJECTIVES	CHECK YOUR UNDERSTANDING
3-1   List the metric units of measurement that are used for microorganisms.	If a microbe measures 10 µm in length, how long is it in nanometers?
3-2   Diagram the path of light through a compound microscope.	Through what lenses does light pass in a compound microscope?
3-3   Define total magnification and resolution.	What does it mean when a microscope has a resolution of 0.2 nm?
3-4   Identify a use for darkfield, phase-contrast, differential interference contrast, fluorescence, confocal, two-photon, and scanning acoustic microscopy, and compare each with brightfield illumination.	How are brightfield, darkfield, phase-contrast, and fluorescence microscopy similar?
3-5   Explain how electron microscopy differs from light microscopy.	Why do electron microscopes have greater resolution than light microscopes?
3-6   Identify one use for the TEM, SEM, and scanned-probe microscopes.	For what is TEM used? SEM? Scanned-probe microscopy?
3-7   Differentiate an acidic dye from a basic dye.	Why doesn’t a negative stain color a cell?
3-8   Explain the purpose of simple staining.	Why is fixing necessary for most staining procedures?
3-9   List the steps in preparing a Gram stain, and describe the appearance of gram-positive and gram-negative cells after each step.	Why is the Gram stain so useful?
3-10  Compare and contrast the Gram stain and the acid-fast stain.	Which stain would be used to identify microbes in the genera Mycobacterium and Nocardia?
3-11  Explain why each of the following is used: capsule stain, endospore stain, flagella stain.	How do unstained endospores appear? Stained endospores?

 

CHAPTER SUMMARY

Units of Measurement (p. 55)

1. The standard unit of length is the meter (m).
2. Microorganisms are measured in micrometers, µm (10^–6 m), and in nanometers, nm (10^–9 m).

Microscopy: The Instruments (p. 55)

 

  

1. A simple microscope consists of one lens; a compound microscope has multiple lenses.

Light Microscopy (pp. 56, 58–62)

    

Compound Light Microscopy (pp. 56, 58–59)

2. The most common microscope used in microbiology is the compound light microscope (LM).
3. The total magnification of an object is calculated by multiplying the magnification of the objective lens by the magnification of the ocular lens.
4. The compound light microscope uses visible light.
5. The maximum resolution, or resolving power (the ability to distinguish two points) of a compound light microscope is 0.2 µm; maximum magnification is 2000x.
6. Specimens are stained to increase the difference between the refractive indexes of the specimen and the medium.
7. Immersion oil is used with the oil immersion lens to reduce light loss between the slide and the lens.
8. Brightfield illumination is used for stained smears.
9. Unstained cells are more productively observed using darkfield, phase-contrast, or DIC microscopy.

Darkfield Microscopy (p. 59)

10. The darkfield microscope shows a light silhouette of an organism against a dark background.
11. It is most useful for detecting the presence of extremely small organisms.

Phase-Contrast Microscopy (pp. 59–60)

12. A phase-contrast microscope brings direct and reflected or diffracted light rays together (in phase) to form an image of the specimen on the ocular lens.
13. It allows the detailed observation of living organisms.

Differential Interference Contrast (DIC) Microscopy (p. 60)

14. The DIC microscope provides a colored, three-dimensional image of the object being observed.
15. It allows detailed observations of living cells.

Fluorescence Microscopy (pp. 61–62)

16. In fluorescence microscopy, specimens are first stained with fluorochromes and then viewed through a compound microscope by using an ultraviolet light source.
17. The microorganisms appear as bright objects against a dark background.
18. Fluorescence microscopy is used primarily in a diagnostic procedure called fluorescent-antibody (FA) technique, or immunofluorescence.

Confocal Microscopy (p. 62)

19. In confocal microscopy, a specimen is stained with a fluorescent dye and illuminated with short-wavelength light.
20. Using a computer to process the images, two-dimensional and three-dimensional images of cells can be produced.

Two-Photon Microscopy (p. 62)

21. In TPM, a live specimen is stained with a fluorescent dye and illuminated with long-wavelength light.

Scanning Acoustic Microscopy (p. 63)

22. Scanning acoustic microscopy (SAM) is based on the interpretation of sound waves through a specimen.
23. It is used to study living cells attached to surfaces such as cancer cells, artery plaque, and biofilms.

Electron Microscopy (pp. 63–65)

   

24. Instead of light, a beam of electrons is used with an electron microscope.
25. Instead of glass lenses, electromagnets control focus, illumination, and magnification.
26. Thin sections of organisms can be seen in an electron micrograph produced using a transmission electron microscope (TEM). Magnification: 10,000–100,000x. Resolving power: 2.5 nm.
27. Three-dimensional views of the surfaces of whole microorganisms can be obtained with a scanning electron microscope (SEM). Magnification: 1000–10,000x. Resolving power: 20 nm.

Scanned-Probe Microscopy (p. 65)

28. Scanning tunneling microscopy (STM) and atomic force microscopy (AFM) produce three-dimensional images of the surface of a molecule.

Preparation of Specimens for Light Microscopy (pp. 68–72)

  

  

   

  Preparing Smears for Staining (pp. 68–69)

        

  

1. Staining means coloring a microorganism with a dye to make some structures more visible.
2. Fixing uses heat or alcohol to kill and attach microorganisms to a slide.
3. A smear is a thin film of material used for microscopic examination.
4. Bacteria are negatively charged, and the colored positive ion of a basic dye will stain bacterial cells.
5. The colored negative ion of an acidic dye will stain the background of a bacterial smear; a negative stain is produced.

Simple Stains (p. 69)

    

6. A simple stain is an aqueous or alcohol solution of a single basic dye.
7. It is used to make cellular shapes and arrangements visible.
8. A mordant may be used to improve bonding between the stain and the specimen.

Differential Stains (pp. 69–71)

  

  

  

9. Differential stains, such as the Gram stain and acid-fast stain, differentiate bacteria according to their reactions to the stains.
10. The Gram stain procedure uses a purple stain (crystal violet), iodine as a mordant, an alcohol decolorizer, and a red counterstain.
11. Gram-positive bacteria retain the purple stain after the decolorization step; gram-negative bacteria do not and thus appear pink from the counterstain.
12. Acid-fast microbes, such as members of the genera Mycobacterium and Nocardia, retain carbolfuchsin after acid-alcohol decolorization and appear red; non–acid-fast microbes take up the methylene blue counterstain and appear blue.

Special Stains (pp. 71–72)

   

  

13. Negative staining is used to make microbial capsules visible.
14. The endospore stain and flagella stain are special stains that color only certain parts of bacteria.