Chapter 2 Ppt

Chapter 2

The Study of Microbial Structure:

Microscopy and Specimen Preparation

Microscopy

• microorganisms range in size from the smallest, viruses which are measured in nanometers (nm), to the largest, which are about 200 micrometers (µm).

Table 2.1

Lenses and the Bending of Light

• light is refracted (bent) when passing from one medium to another

• refractive index– a measure of how greatly a substance slows

the velocity of light

• direction and magnitude of bending is determined by the refractive indices of the two media forming the interface

Figure 2.1

Lenses

• focus light rays at a specific place called the focal point

• distance between center of lens and focal point is the focal length

• strength of lens related to focal length– short focal length ⇒⇒⇒⇒more

magnification

Figure 2.2

The Light Microscope

• many varieties– bright-field microscope– dark-field microscope– phase-contrast microscope– fluorescence microscope– confocal microscope

• are compound microscopes– image formed by action of ≥≥≥≥2 lenses

The Bright-Field Microscope

• produces a dark image against a brighter background

• has several objective lenses– parfocal microscopes remain in focus

when objectives are changed

• total magnification– product of the magnifications of the

ocular lenses and the objective lenses

Figure 2.3

Microscope Resolution

• ability of a lens to separate or distinguish small objects that are close together

• wavelength of light used is major factor in resolutionshorter wavelength ⇒⇒⇒⇒ greater resolution

Figure 2.4

Figure 2.5

If air is replaced with immersion oil, many light rays that did not enter the objective due to reflection and refraction at the surfaces of the objective lens and slide will now do so. This results in an increase in resolution and numerical aperture.

•working distance— distance between the front surface of lens and surface of cover glass or specimen when it is in sharp focus

Table 2.2

The Dark-Field Microscope• image is formed by light reflected or

refracted by specimen• produces a bright image of the object

against a dark background• used to observe living, unstained

preparations– has been used to observe internal structures

in eukaryotic microorganisms– has been used to identify bacteria such as

Treponema pallidum, the causative agent of syphilis

Figure 2.6

Figure 2.7

The Phase-Contrast Microscope• converts slight differences in refractive index

and cell density into easily detected variations in light intensity.

• some light rays from hollow cone of light passing through an unstained cell are retarded and out of phase and dark compared to the bright background

• excellent way to observe living cells– studying microbial motility– detecting bacterial structures such as endospores

and inclusion bodies that have refractive indices different from that of water

Figure 2.9

Figure 2.10

Figure 2.8

The Differential Interference Contrast Microscope (DIC)

• creates image by detecting differences in refractive indices and thickness of different parts of specimen

• excellent way to observe living cells– live, unstained cells appear brightly colored

and three-dimensional

– cell walls, endospores, granules, vacuoles, and nuclei are clearly visible

Figure 2.11

The Fluorescence Microscope• developed by O. Shimomuram, M. Chalfie,

and R. Tsien• exposes specimen to ultraviolet, violet, or

blue light• specimens usually stained with

fluorochromes• shows a bright image of the object resulting

from the fluorescent light emitted by the specimen

• has applications in medical microbiology and microbial ecology studies

Figure 2.12

Table 2.3

Fluorescence Microscopy

• essential tool in microbiology– fluorochrome-labeled probes, such as

antibodies, or fluorochromes tag specific cell constituents for identification of unknown pathogens

– localization of specific proteins in cells

Figure 2.13

Figure 2.14

Preparation and Staining of Specimens

• increases visibility of specimen• accentuates specific morphological

features

• preserves specimens

Fixation• preserves internal and external structures

and fixes them in position• organisms usually killed and firmly

attached to microscope slide– heat fixation – routine use with bacteria and

archaea• preserves overall morphology but not internal

structures

– chemical fixation – used with larger, more delicate organisms

• protects fine cellular substructure and morphology

Dyes and Simple Staining• dyes

– make internal and external structures of cell more visible by increasing contrast with background

– have two common features• chromophore groups

– chemical groups with conjugated double bonds

– give dye its color

• ability to bind cells

Dyes and Simple Staining

• dyes• ionizable dyes have charged groups

– basic dyes have positive charges

– acid dyes have negative charges

• simple stains– a single stain is used

– use can determine size, shape, and arrangement of bacteria

Figure 2.17

Differential Staining

• divides microorganisms into groups based on their staining properties– e.g., Gram stain– e.g., acid-fast stain

• differential stain used to detect presence or absence of structures– endospores, flagella, capsules

Gram Staining

• most widely used differential staining procedure

• divides bacteria into two groups, Gram positive and Gram negative, based on differences in cell wall structure

Figure 2.18

Acid-Fast Staining

• particularly useful for staining members of the genus Mycobacterium

e.g., Mycobacterium tuberculosis – causes tuberculosis

e.g., Mycobacterium leprae – causes leprosy

– high lipid content in cell walls (mycolic acid) is responsible for their staining characteristics

Staining Specific Structures• endospore staining

– heated, double staining technique– bacterial endospore is one color and vegetative cell

is a different color

• capsule stain used to visualize capsules surrounding bacteria– negative stain - capsules may be colorless against a

stained background

• flagella staining– mordant applied to increase thickness of flagella

Figure 2.19

Electron Microscopy

• electrons replace light as the illuminating beam

• wavelength of electron beam is much shorter than light, resulting in much higher resolution

• allows for study of microbial morphology in great detail

Figure 2.20

A Comparison of Light and Electron Microscopy (Rhodospirillum rubrum)

The Transmission Electron Microscope (TEM)

• electrons scatter when they pass through thin sections of a specimen

• transmitted electrons are under vacuum which reduces scatter and are used to produce clear image

• denser regions in specimen, scatter more electrons and appear darker

Figure 2.22

Figure 2.23

Table 2.4

Specimen Preparation• analogous to procedures used for

light microscopy• for transmission electron

microscopy, specimens must be cut very thin

• specimens are chemically fixed and stained with electron dense materials, such as heavy metals, that differentially scatter electrons

Other Preparation Methods• negative stain

– heavy metals do not penetrate the specimen but render dark background

– used for study of viruses, bacterial gas vacuoles

• shadowing– coating specimen with a thin film of a heavy

metal only on one side

– useful for viral morphology, flagella, DNA

Figure 2.24

Other Preparation Methods

• freeze-etching– freeze specimen then fracture along

lines of greatest weakness (e.g., membranes)

– allows for 3-D observation of shapes of intracellular structures

– reduces artifacts

Freeze-Etching (Thiobacillus kabobis)

The Scanning Electron Microscope

• uses electrons reflected from the surface of a specimen to create detailed image

• produces a realistic 3-dimensional image of specimen’s surface features

• can determine actual in situ location of microorganisms in ecological niches

Figure 2.26

Scanning Electron Micrograph of Mycobacterium tuberculosis

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