Lab assignment #1

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Microscopy, Staining, and Classification

General principles of microscopy

Wavelength of radiation

Resolution

Contrast

Magnification

Wavelength of radiation

Distance between two corresponding parts of a wave of radiation (from crest to crest or trough to trough)

visible light or electromagnetic, including X-rays, microwaves and radio waves)

The shorter the wave length of radiation, the stronger the resolving power

Microscopy, Staining, and Classification
The electromagnetic spectrum

Microscopy, Staining, and Classification

Resolution (resolving power)

Ability to distinguish between objects that are close together

Resolution is determined by the wavelength of light used and numerical aperture of lens. Resolution distance is dependent on wave length of light, electron beam and/or numerical aperture of the lens

Modern microscopes use shorter wave length radiation and have lenses with larger numerical apertures

Limit of resolution for light microscope is about 0.2 µm.

Contrast

Difference in intensity between two objects or between an object and its background

Important in determining resolution (clarity of an image)

Staining increases contrast

Resolution and contrast determine the magnification of a microscope

Use of light that is in phase increases contrast

Microscopy, Staining, and Classification

Magnification

An increase in size of an object.

Results when a beam of radiation bends as it passes through a lens

Curved lenses refract light and magnetic fields (magnetic lenses) refract electron beams

Lenses refract (bend) radiation because they are optically dense compared to other media (air or water)

Magnification depends on the thickness of the lens, its curvature and the speed of light through its medium (substance such as glass, lens, air or water)

Lenses and the bending of light

When a ray of light passes from one medium to another, refraction occurs (the light is bent at the interface).

The refractive index (n) is a measure of how greatly a substance slows the velocity of light. The direction and magnitude of bending are determined by the refractive indices of the two media forming the interface.

Refraction

Light beam enters head on

Light beam enters glass at angle to normal

Air

n = 1

Air

n = 1

Air

n = 1

Air

n = 1

Glass

n = ~1.5

Glass

n = ~1.5

Dashed line depicts the normal

Light

Bending of light through a rism

Prism

Air

Glass

Normal

Light

n = 1

n = ~1.5

Slowed down

Sped up

Can also say the air is less optically dense than glass.

F = focal point

The Convex Lens

f = focal length

Lens

Air

Glass

strength of lens related to focal length short focal length more magnification

Refractive Properties of Lenses

Flat glass

Convex lens (less round)

Convex lens (more round)

Concave lens

short focal length more magnification

Microscopy, Staining, and Classification
Light refraction and image magnification

Units of Measurement

Range of Light and Electron Microscopes

Light

Electron

Rhodospirillum rubrum

Photoionization microscopy

These are all things you absolutely can not see without microscopes. Know that viruses are smaller than a um and can not be seen with light microscope. Know that bacteria are in the um in sizes and can typically be seen with light microscope. Thin section TEM at bottom right.

Types of microscopes

Light microscopes include:

Bright-field
Dark-field
Phase-contrast
Fluorescence
Confocal

Modern microscopes use visible light to illuminate cells and are compound, meaning that they have two sets of lenses.

Bright-field microscopes aren’t ideal for viewing unpigmented and unstained cells due to lack of contrast. What if you need to see living cells?

These light microscopes are more useful:

Dark-field microscope
Phase-contrast microscope
Differential interference (DIC) microscope.

Types of microscopy

Light Microscopy

Bright-field microscopes
Simple
Contain a single magnifying lens
Similar to magnifying glass
Leeuwenhoek used simple microscope to observe microorganisms

Types of microscopy

Light Microscopy
Bright-field microscopes
Compound
Series of lenses for magnification
Light passes through specimen into objective lens
Oil immersion lens increases resolution
Have one or two ocular lenses
Total magnification = magnification of objective lens X magnification of ocular lens
Most have condenser lens (direct light through specimen)

A bright-field, compound light microscope.

Coarse focusing knob

Moves the stage up and

down to focus the image

Illuminator

Light source

Diaphragm

Controls the amount of

light entering the condenser

Condenser

Focuses light

through specimen

Stage

Holds the microscope

slide in position

Objective lenses

Primary lenses that

magnify the specimen

Body

Transmits the image from the

objective lens to the ocular lens

using prisms

Ocular lens

Remagnifies the image formed by

the objective lens

Line of vision

Ocular lens

Path of light

Prism

Body

Objective

lenses

Specimen

Condenser

lenses

Illuminator

Fine focusing knob

Base

Arm

Routinely used in microbiology to examine both stained and unstained specimens. Specimens are visualized because of differences in contrast (density) between specimen and surroundings.

Named for its ability to form a dark image against a brighter background.

Parfocal – specimen remains in focus as you change objectives.

Multiply objective and ocular magnification to obtain total magnification.

The effect of immersion oil on resolution

Glass cover slip

Slide

Specimen

Light source

Without immersion oil

Lenses

Immersion oil

Glass cover slip

Slide

Light source

With immersion oil

Microscope

objective

Refracted light

rays lost to lens

Microscope

objective

More light

enters lens

Immersion oil redirects light rays by minimizing refraction and prevents reflection, resulting in increased numerical aperture and resolution.

Dark-Field Microscope

Produces detailed images of living, unstained specimens by changing the way in which they are illuminated.

Unreflected and unrefracted rays do not enter the objective.

Object appears bright on black background.

Dark-Field Microscopy

Treponema pallidum (syphilis)

Useful for study of internal structure of eukaryotic microorganisms and for observing motility.

S. Cerevisiae

Microscopy

Light microscopy

Phase microscopes

Used to examine living organisms or specimens that would be damaged or altered by attaching them to slides or staining them

These microscopes treat one set of light rays differently from another set

Light rays in phase produce brighter image, while light rays out of phase produce darker image

Contrast is created because light waves are ½ wavelength out of phase

Two types

Phase Contrast Microscope: produce shapely defined images in which fine structures can be seen in living cells; useful for observing cilia and flagella

Differential Interference Contrast Microscope(Nomarski microscopes): Create phase interference patterns; gives the image a three-dimensional or shadowed appearance

Phase-Contrast Microscope

Four kinds of light microscopy

Fluorescent microscopy

Fluorophores are molecules that absorb energy and emit light, this is the basis of fluorescence microscopy. When some molecules absorb radiant energy, they become excited and release much of their trapped energy as light (emission). Fluorescence light is emitted very quickly by the excited molecule as it gives up its trapped energy and returns to a more stable state.

Explain how antibodies are used in fluorescence microscopy. Mbl is a cytoskeletal protein of Bacillis subtilis.

Immunofluorescence

Staining

Most microorganisms are difficult to view by bright-field microscopy
Coloring specimen with stain increases contrast and resolution
Specimens must be prepared for staining
Thin smear (film) of microorganisms on glass slides is made prior to staining
Smear is allowed to air-dry and then heat-fixed to glass surface

Staining

Principles of Staining
Microbiological stains/dyes used as stains are usually salts composed of cation and anion and contain one colored substance (chromophore)

Acidic dyes (anionic chromophores) stain alkaline structures (positively charged molecules). Acidic dyes are also used in negative staining

Basic dyes (cationic chromophores) stain acidic structures (negatively charged molecules). They are used more commonly in microbiology because most microbial cells are negatively charged.

Types of staining

Simple stains

Differential stains
Gram stain
Acid-fast stain
Endospore stain

Special stains
Negative (capsule) stain
Flagellar stain

Simple Staining

Commonly used and easy.

Fixed smear is covered with a single basic dye such as crystal violet, excess stain is washed off with water, and blotted dry.

Used to determine the size, shape, and arrangement of bacterial and archaeal cells.

Differential Stains

Distinguish organisms based on their staining properties.

For example, the Gram stain, developed in 1884 by the Danish physician Christian Gram, is the most widely employed staining method in bacteriology.

Gram stain divides most bacteria (but not archaea) into two groups – those that stain gram negative and those that stain gram positive.

Acid-fast stain

Mixed stain: Gram positive (purple) Acid-fast stain

and Gram negative stain (pink)

The Gram Staining Procedure

Special stains: Preparation and staining of specimens

Most dyes are used to directly stain the cell or object of interest to make internal and external structures of the cell more visible.

Some dyes (special stains, e.g., India ink) are used in negative staining, where the background but not the cell is stained. The unstained cells appear as bright objects against a dark background.

Negative stain (Capsule stain)

Flagella

Flagellar stain of Proteus vulgaris

Staining and microscopy

Staining

Staining for Electron Microscopy

Chemicals containing heavy metals used for transmission electron microscopy
Stains may bind molecules in specimens or the background
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

Electron Microscopy

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

Wavelength of an electron in a TEM can be as short as 2.5 pm as in picometers as in 2.5 x 10-12 m

That’s ~100,000 times shorter wavelength than a light microscope uses.

Transmission electron microscope (TEM)

Specimen is coated with plastic and cut really thin 20-100 nm thick slices.

The Scanning Electron Microscope

Uses electrons reflected from the surface of a specimen that is coated in metal to create detailed image

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

Resolution of 7 nm.

Can determine actual in situ location of microorganisms in ecological niches

Scanning Electron Microscope (SEM)

Mycobacterium tuberculosis

Classification and Identification of Microorganisms

Classification and identification of microorganisms

Taxonomy is the science of classifying and naming organisms

Taxonomy consists of:

classification (assigning organisms to taxa based upon similarities)

Nomenclature (rules of naming organisms) and

Identification (determining which individual organism or population belongs to a particular taxa)

Enables scientists to organize large amounts of information about organisms

Make predictions based on knowledge of similar organisms

Classification and Identification of Microorganisms

Linnaeus, Whittaker, and taxonomic categories

Linnaeus

Linnaeus provided system that standardized the naming and classification of organisms based on characteristics they have in common

Grouped similar organisms that can successfully interbreed into categories called species

Used binomial nomenclature in his system

Binomial Nomenclature (assigning two names to every organism)

Linnaeus proposed only two kingdoms: animalia and plantae

Whitaker proposed taxonomic approach based on five kingdoms: Animalia, Plantae, Fungi, Protista, and Prokaryotae (widely accepted)

Classification and Identification of Microorganisms

Taxonomic categories

Linnaeus’s goal was classifying and naming organisms as a means of cataloging them

Today, more modern goal of understanding relationships among groups of organisms

Major goal of modern taxonomy is to reflect phylogenetic hierarchy (derivation from common ancestors)

Greater emphasis on comparisons of organisms’ genetic material led to proposal to add a new, most inclusive taxon, the domain

Classification and Identification of Microorganisms

Domains

Taxonomists compare nucleotide sequences of the smaller rRNA subunits of both prokaryotes and eukaryotes

Carl Woese compared nucleotide sequences of rRNA subunits. rRNA molecules are present in all cells and changes in their nucleotide sequence presumably occur rarely

Proposal of three domains as determined by ribosomal nucleotide sequences: Bacteria, Archaea and Eukarya

Cells in the three domains also differ with respect to many other characteristics

Levels in Linnaean taxonomic scheme

Whittaker’s five-kingdom taxonomic scheme

Classification and Identification of Microorganisms

Taxonomic and identifying characteristics

Main criteria and laboratory techniques used for classifying and identifying microorganisms are:

Macroscopic and microscopic examination

Differential staining

Growth (cultural ) characteristics

Serological tests - microbial interaction with antibodies

Phage typing - microbial susceptibility to viruses

Nucleic acid analysis

Biochemical tests and microbial environmental requirements (temperature and pH).

Two biochemical tests for identifying bacteria

An agglutination test, one type of serological test

Phage typing

Classification and Identification of Microorganisms

Taxonomic Keys

Dichotomous keys

Series of paired statements where only one of two “either/or” choices applies to any particular organism

Key directs user to another pair of statements, or provides name of organism

Use of dichotomous taxonomic key