1. Honors Microbiology: Chapter 3 Microscopy and Staining

2. I. Principles of Microscopy
A. Properties of light: wavelength and resolution
Wavelength – length of a light ray
Resolution – ability to see two items as separate and discrete
Resolving power (RP) numerical measure of the resolution of the lens
Numerical aperture (NA) – widest cone of light that can enter a lens

3. B. Properties of light: light and objects
Reflection: when light strikes an object and bounces back, giving the object color
Transmission: passage of light through an object
Absorption: when light rays are taken up by an object

4. Refraction: bending of light as it passes from one medium to another of different density
Diffraction: when light waves are broken up into bands of different wavelengths as they pass through a small opening

5. Light Travel
Reflection
Transmission
Absorption

6. II. Light Microscopy
A. Basic feature: uses visible light to make specimens observable
B. Compound light microscope: has more than one lens

7. Compound Light Microscopy
Condenser
Iris diaphragm
Objective lenses
Ocular lens(es)
Stage
Focusing knobs
Total Magnification

8. Brightfield Microscopy

9. C. Darkfield microscopy – has a condenser that prevents light from being transmitted through the specimen. It causes light to reflect off the specimen at an angle, showing a light object against a dark background.

10. Darkfield Microscopy

11. D. Phase- contrast microscopy: has a condenser that accentuates small differences in the refractive index of various structures within the cell, causing parts of the cell to display different degrees of brightness.

12. Phase-contrast Microscopy

13. E. Differential interference contrast microscopy (Nomarski) – uses differences in refractive index to visualize structures, producing a nearly 3-D image

14. Nomarski microscopy

15. F. Fluorescence microscopy: ultraviolet light is used to excite molecules so they release light of different colors.

16. Fluorescence microscopy

17. III. Electron Microscopy
A. Basic features: uses a beam of electrons rather than light, and electromagnets, rather than glass lenses, to produce an image

18. Electron Microscopy (EM)
Transmission (TEM)
Scanning (SEM)
Scanning Tunneling

19. B. Transmission electron microscope (TEM):
Gives an excellent view of the internal structure of microbes, magnifying objects up to 500,000x. Very thin slices of specimens are used.

20. Transmission Electron Microscopy

21. C. Scanning electron microscope (SEM): used to create images of the surfaces of specimens, magnifying objects up to 50,000x
D. Scanning tunneling microscopy (STM): used to create 3-D images and movies of individual molecules and atoms.

22. Scanning Electron Microscopy

23. IV. Techniques of light microscopy
A. Preparation of specimens for the light microscope:
1. Wet mounts: a drop of medium containing the organisms is placed on a slide, and living microbes can be observed.
2. Smears – microbes from a loopful of medium are spread onto the surface of a glass slide and heat-killed, so that killed microbes can be observed.

24. B. Principles of staining:
1. Simple stains – make use of a single dye and reveals basic cell shapes and arrangements
2. Differential stains – uses two or more dyes and distinguishes between two kinds of organisms or two different parts of an organism

25. C. Gram Stain – a differential stain
C. Gram Stain – a differential stain. Groups that can be distinguished with the Gram stain:
1. gram-positive, which stain violet
2. gram-negative, which stain pink
3. gram-nonreactive, which do not stain, or stain poorly
4. gram-variable, which stain unevenly

26. Gram Stain
Technique
Significance
Cell wall anatomy
Diagnosis

27. D. Ziehl-Neelsen acid-fast stain: used to detect tuberculosis, and leprosy-causing organisms

28. Acid fast Staining

29. E. Special Staining Procedures:
1. Negative staining
2. Flagellar staining
3. Endospore staining
4. Capsular staining

30. Negative Staining

31. Flagellar Staining

32. Endospore Staining

33. Capsular Staining