1. Chapter 2 Observing the microbial cell

2. Chapter Overview ● How microorganisms are observed
● The bright-field microscope
● Staining bacterial cells
● The dark-field and phase-contrast
● The fluorescence and electron microscopes

3. Introduction
Since Leeuwenhoek’s time, powerful microscopes have been devised to search for microbes in unexpected habitats.
- Example = The human stomach
- Microscopy revealed the presence of Helicobacter pylori, the cause of stomach ulcers.
Figure 2.1

4. Observing Microbes
The size at which objects become visible depends on the resolution of the observer’s eye.
Resolution is the smallest distance between two closely placed objects that can still be distinguished.
The resolution of the human retina is about 150 mm (1/7 mm).
Figure 1.1

6. Detection is the ability to determine the presence of an object (can not resolve individual objects)
Magnification means an increase in the apparent size of an image to resolve smaller separations between objects.
Figure 2.3

7. Microbial Size
Microbes differ in size, over a range of a few orders of magnitude, or powers of ten.
- Eukaryotic microbes
- Protozoa, algae, fungi
- 10–100 mm
- Prokaryotes
- Bacteria, Archaea
- 0.4–10 mm

8. Figure 2.4 8

9. Microbial Shapes
Certain shapes of bacteria are common to many taxonomic groups.
- Bacilli = Rods
- Cocci = Spheres
- Spiral forms
- Spirochetes
- Spirilla
Figure 2.6

10. Unusual Shapes of microorganisms
Stella
Haloarcula

11. Microscopy for Different Size Scales
Different microscopes are required to resolve various cells and subcellular structures.
Figure 2.7 11

12. Optics and Properties of Light
Light is part of the spectrum of electromagnetic radiation.
- Wavelength of visible light = 400–750 nm
For electromagnetic radiation to resolve an object, certain conditions must exist:
1. Contrast between object and its medium
2. Wavelength smaller than the object
3. A detector with sufficient resolution for the given wavelength 12

13. Figure 2.8 13

14. Light Interacts with an Object
Absorption means that the photon’s energy is acquired by the absorbing object.
Reflection means that the wave front bounces off the surface of an object.
Refraction is the bending of light as it enters a substance that slows its speed.
Scattering occurs when the wave front interacts with an object smaller than the wavelength of light. 14

15. Figure 2.9 15

16. Parabolic lenses bring light rays to a focal point.
Wave fronts of light shift direction as they enter a substance of higher refractive index.
Parabolic lenses bring light rays to a focal point.
Figure 2.10
Figure 2.11 16

17. Generating an Image with a Lens
Figure 2.12

18. Microscopes Compound light microscope  White light
Fluorescence microscope  UV light
Confocal microscope  Laser light
Electron microscopeBeam of electrons
Magnification
Resolution

19. The Compound Microscope
A system of multiple lenses designed to correct or compensate for aberration
- Ocular lens
- Objective lens
- Needs to be parfocal
Total magnification = Magnification of ocular multiplied by that of the objective
Empty magnification = Magnification without an increase in resolution

20. Compound Microscope • In a compound microscope the image from the
objective lens is
magnified again by
the ocular lens.
Total magnification =
objective lens
ocular lens
Compound Microscope
Figure 3.1b

21. Figure 2.17

23. Bright-Field Microscopy
Generates a dark image of an object over a light background
To increase resolution:
- Use shorter wavelength light
- Improve contrast
- Use immersion oil
- Use wider lens closer to specimen
- Higher numerical aperture (NA) 23

24. Figure 2.15
Figure 2.16
NA = n sin 24

25. Fixation and Staining
The detection and resolution of cells under a microscope are enhanced by:
- Fixation = Cells are made to adhere to a slide in a fixed position
- Staining = Cells are given a distinct color
- Most stains have conjugated double bonds or aromatic rings, and one or more positive charges. 25

26. - Most commonly used stain is methylene blue.
A simple stain adds dark color specifically to cells, but not to the external medium or surrounding tissue.
- Most commonly used stain is methylene blue.
Figure 2.20 26

27. A differential stain stains one kind of cell but not the other.
- Gram stain differentiates between two types of bacteria.
- Gram-positive retain the crystal violet stain because of their thicker cell wall.
- Gram-negative bacteria do not.
Figure 2.21 27

28. Figure 2.22 28

29. Basic Dyes used in Bacterial Staining
Safranin
Eosin Y
Crystal Violet

30. Other differential stains
- Acid-fast stain = Carbolfuchsin used to stain Mycobacterium species (M. tuberculosis and M. leprae
- Spore stain = Malachite green used to detect spores of Bacillus and Clostridium
- Negative stain = Colors the background, which makes capsules more visible
Figure 2.24 30

32. Dark-Field Microscopy
Dark-field optics enables microbes to be visualized as halos of bright light against darkness.
Light shines at oblique angle.
- Only light scattered by sample reaches objective.
- Makes visible objects below resolution limit
- Flagella, very thin bacteria 32

33. Figure 2.25
Figure 2.27
Figure 2.26 33

34. Phase-Contrast Microscopy
Superimposes refracted light and transmitted light shifted out of phase
- Reveals differences in refractive index as patterns of light and dark
- Can be used to view live cells and cellular organelles
Figure 2.28 34

35. Fluorescence Microscopy
In fluorescence microscopy, incident light is absorbed by the specimen and reemitted at a lower energy, thus longer wavelength.
Figure 2.31 35

38. Confocal Microscopy
In confocal laser scanning microscopy, both excitation light and emitted light are focused together.
-Can visualize cells in three dimensions
-Allows observation of live microbes in real time
Figure 2.35
Figure 2.34 38

40. Electron Microscopy Electrons behave like light waves.
- Very high frequency
- Allows very great resolution
- A few nanometers
Sample must absorb electrons.
- Coated with heavy metal
Electron beam and sample are in a vacuum.
- Lenses are magnetic fields. 40

41. Electron Microscopy Two major types
- Transmission electron microscopy (TEM)
- Electrons pass through the specimen.
- Reveals internal structures
- Scanning electron microscopy (SEM)
- Electrons scan the specimen surface.
- Reveals external features in 3-D 41

43. The TEM closely parallels the design of the bright-field microscope.
Figure 2.38
The TEM closely parallels the design of the bright-field microscope. 43

44. The SEM is arranged somewhat differently from the TEM.
Figure 2.39
The SEM is arranged somewhat differently from the TEM. 44

45. Sample Preparation
The specimens for electron microscopy can be prepared in several ways.
- Embedded in a polymer for thin sections
- Microtome is used to cut slices.
- Sprayed onto a copper grid
The specimen is then treated with a heavy-metal salt such as uranyl acetate.
Note: For SEM, specimen is coated with heavy metal and it is not sliced. 45

46. Figure 2.40
Figure 2.41 46

47. Cryo-Electron Microscopy
In cryo-EM, or electron cryo-microscopy, the specimen is flash-frozen.
- Suspended in water and frozen rapidly in a refrigerant
Cryo-electron tomography, or electron cryotomography, avoids the need to physically slice the sample for thin-section TEM.
- Generates high-resolution models of virus particles 47

48. Chapter Summary - Bright-field: Employs various stains
● When observing microbes, resolution and magnification are paramount.
● Different kinds of microscopes are required to resolve cells and subcellular structures:
- Bright-field: Employs various stains
- Dark-field: Detects unresolved objects
- Phase-contrast: Exploits differences in refractive indices
- Fluorescence: Employs fluorophores for labeling
- Confocal: Visualizes cells in 3-D 48

49. Chapter Summary - TEM: Provides internal details in 2-D
● Electron microscopes use beam of electrons instead of light rays.
- TEM: Provides internal details in 2-D
- SEM: Provides external details in 3-D 49