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Chapter 2Observing the microbial cell

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Chapter Overview

● How microorganisms are observed

● The bright-field microscope

● Staining bacterial cells

● The dark-field and phase-contrast

● The fluorescence and electron microscopes

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IntroductionSince 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

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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 m (1/7 mm).

Figure 1.1

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• Resolution is the ability of the lenses to distinguish two points.

• A microscope with a resolving power of 0.4 nm can distinguish between two points ≥ 0.4 nm.

• Shorter wavelengths of light provide greater resolution.

Microscopy: The Instruments

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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

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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 m

- Prokaryotes

- Bacteria, Archaea

- 0.4–10 m

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Figure 2.4

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Microbial Shapes

Certain shapes of bacteria are common to many taxonomic groups.

- Bacilli = Rods

- Cocci = Spheres

- Spiral forms

- Spirochetes

- Spirilla

Figure 2.6

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Unusual Shapes of microorganisms

Stella Haloarcula

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Microscopy for Different Size Scales

Different microscopes are required to resolve various cells and subcellular structures.

Figure 2.7

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Optics and Properties of LightLight 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 medium2. Wavelength smaller than the object3. A detector with sufficient resolution for the given wavelength

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Figure 2.8

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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.

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Figure 2.9

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Wave fronts of light shift direction as they enter a substance of higher refractive index.

Figure 2.10

Parabolic lenses bring light rays to a focal point.

Figure 2.11

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Generating an Image with a Lens

Figure 2.12

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Microscopes

• Compound light microscope White light • Fluorescence microscope UV light• Confocal microscope Laser light• Electron microscopeBeam of electrons

• Magnification • Resolution

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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 objectiveEmpty magnification = Magnification without an

increase in resolution

The Compound Microscope

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• 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

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Figure 2.17

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Compound Light Microscope

Applications: Bright field microscopy

Dark field microscopy

Phase-contrast microscopy

Fluorescence microscopy

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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)

Bright-Field Microscopy

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Figure 2.15

Figure 2.16

NA = n sin

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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.

Fixation and Staining

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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

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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

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Figure 2.22

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Basic Dyes used in Bacterial Staining

Safranin Eosin YCrystal Violet

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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

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• Negative staining is useful for capsules.

• Heat is required to drive a stain into endospores.

• Flagella staining requires a mordant to make the flagella wide enough to see.

Special Stains

Figure 3.12a-c

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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

Dark-Field Microscopy

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Figure 2.27 Figure

2.26

Figure 2.25

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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

Phase-Contrast Microscopy

Figure 2.28

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

Fluorescence Microscopy

Figure 2.31

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• Uses UV light.

• Fluorescent substances absorb UV light and emit visible light.

• Cells may be stained with fluorescent dyes (fluorochromes).

Fluorescence Microscopy

Figure 3.6b

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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

Confocal Microscopy

Figure 2.35Figure

2.34

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• Uses fluorochromesand a laser light.

• The laser illuminates each plane in a specimen to produce a 3-D image.

Confocal Microscopy

Figure 3.7

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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.

Electron Microscopy

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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

Electron Microscopy

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

Figure 2.38

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The SEM is arranged somewhat differently from the TEM.

Figure 2.39

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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.

Sample Preparation

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Figure 2.40

Figure 2.41

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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

Cryo-Electron Microscopy

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Chapter Summary● 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

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Chapter Summary● Electron microscopes use beam of electrons

instead of light rays.

- TEM: Provides internal details in 2-D

- SEM: Provides external details in 3-D