demonstration of microorganism

Microscopy

The use of microscope to magnify or visually enlarge objects that are too small to be visualized with the naked eye so that their characteristics are readily observable.

Resolution (resolving power)

Ability of a lens to separate or distinguish fine detail or structure between small objects that are close together.

Maximum resolution of LM

0.2 µm.

Contrast

Ability to make objects stand out from the background, usually achieved by staining techniques that highlight the organisms and allow them to be differentiated from one another and from background material and debris.

Refractive index

A measure of the light-bending ability of a medium.

Microscopical techniques

1. Bright field microscopy; 2. Dark-field microscopy; 3. Phase contrast microscopy; 4. Differential Interference Contrast Microscope; 5. Electron microscopy; 6. Fluorescence Microscopy; 7. Confocal Microscopy (Confocal Scanning Laser Microscopy); 8. Scanning probe microscopy.

Bright-field microscopy

The ordinary microscope is called the bright-field microscope because it forms a dark image against a brighter background.

Maximum magnification using oil-immersion objectives

Approximately 1000×.

Bacteria visualized using bright-field microscopy

Suitably stained bacteria as small as 0.2 µm in size can be visualized.

Dark-field microscopy

Allows a viewer to observe living, unstained cells and organisms by simply changing the way in which they are illuminated.

Dark-field microscopy principle

The scattering of light by fine microorganisms such as spirochetes suspended in liquid allows them to be observed against a dark background.

Dark-field microscope and eukaryotic microorganisms

Can reveal considerable internal structure in larger eukaryotic microorganisms.

Phase contrast microscopy

Employs a special condenser containing an annular (ring-shaped) diaphragm which allows direct light to pass through the condenser, focusing light on the specimen, and a diffraction plate in the objective lens.

Phase contrast microscopy principle

Converts slight differences in refractive index and cell density into easily detected variations in light intensity.

Major advantage of phase contrast microscopy

It is an excellent way to observe the details of living cells.

Differential Interference Contrast Microscope

Similar to PCM in that it creates an image by detecting differences in refractive indices and thickness.

DIC microscopy light

Two beams of plane-polarized light at right angles to each other are generated by prisms.

Appearance of live, unstained specimen in DIC microscopy

Brightly colored and 3-dimensional.

Electron microscopy

Uses beams of electrons in place of visible light to visualize small structures such as viruses.

Transmission Electron Microscopy (TEM)

Uses beams of electrons in place of visible light to visualize small structures such as viruses.

TEM specimen preparation

Specimens are placed on grids, negatively stained with electron-dense compounds such as potassium phosphotungstate and viewed as magnified images on a fluorescent screen.

Electron-dense compound used in TEM

Potassium phosphotungstate.

Magnification possible with modern TEM instruments

Greater than 100,000×.

TEM methods of viewing a specimen

1. Shadow casting; 2. Negative staining; 3. Ultra-thin sectioning; 4. Freeze etching; 5. Autoradiography.

Shadow casting

One method used in viewing a specimen with TEM.

Negative staining

One method used in viewing a specimen with TEM.

Ultra-thin sectioning

One method used in viewing a specimen with TEM.

Freeze etching

One method used in viewing a specimen with TEM.

Autoradiography

One method used in viewing a specimen with TEM.

Scanning Electron Microscopy (SEM)

Used to obtain three-dimensional views of microorganisms when coated with a thin film of heavy metal.

SEM magnification

Up to 100,000× is feasible.

Fluorescence Microscopy

Used for identifying microorganisms with specific antibodies conjugated to fluorochromes.

Fluorescent light

Emitted very quickly by the excited molecule as it gives up its trapped energy and returns to a more stable state.

Light used in fluorescence microscopy

Ultraviolet, violet, or blue light.

Fluorescence microscopy image

Forms an image of the object with the resulting fluorescent light.

Confocal Microscopy (Confocal Scanning Laser Microscopy)

Uses a LASER beam to illuminate a specimen, usually one that has been fluorescently stained.

Major component of confocal microscope

An aperture placed above the objective lens.

Function of the confocal microscope aperture

Eliminates stray light from parts of the specimen that lie above and below the plane of focus.

Scanning probe microscopy

Measures surface features by moving a sharp probe over the object's surface.

Scanning tunneling microscope

An excellent example of scanning probe microscopy; invented in 1980.

Magnification of scanning tunneling microscope

100 million.

What can the scanning tunneling microscope allow scientists to view?

Atoms on the surface of a solid.

Atomic Force Microscopy (AFM)

Moves a sharp probe over the specimen surface while keeping the distance between the probe tip and the surface constant.

AFM vs STM

Unlike STM, AFM can be used to study surfaces that do not conduct electricity well.

Demonstration of microorganisms

Living microorganisms can be directly examined with the light microscope, but they often must be fixed and stained to increase visibility, accentuate specific morphological features, and preserve them for future study.

Smear

A thin film of material containing the microorganisms spread over the surface of the slide; the film is allowed to air dry prior to staining.

Basic steps in preparing bacterial specimen for bright-field microscopy

1. Preparation of the bacterial smear; 2. Fixation of the smear on the glass slide; 3. Staining.

Preparation of the bacterial smear

First basic step in preparing bacterial specimen for bright-field microscopy.

Fixation of the smear on the glass slide

Second basic step in preparing bacterial specimen for bright-field microscopy.

Staining

Third basic step in preparing bacterial specimen for bright-field microscopy.

Sample from tissue lesions

The specimen is held firmly with sterile forceps and the scalpel is used to scrape deep into the material.

Tissue lesion sample on microscope slide

A small amount of the scrapings is placed on the cleaned microscope slide.

Liquid or semi-liquid specimens

A little of the sample is placed on the slide with a sterile swab or sterile inoculating loop.

Liquid/semi-liquid specimen smear

The contents of the swab are smeared over the surface of the slide, with the aim of having thick and thin areas of specimen present.

Fixation

The process by which the internal and external structures of cells and microorganisms are preserved and fixed in position.

Functions of fixation

1. Preserves and fixes internal and external structures of cells and microorganisms in position; 2. Inactivates enzymes that might disrupt cell morphology; 3. Toughens cell structures so they do not change during staining and observation.

Effect of fixation on microorganism

A microorganism usually is killed and attached firmly to the microscope slide during fixation.

Types of fixation

1. Heat fixation; 2. Chemical fixation.

Heat fixation

Routinely used to observe prokaryotes.

Heat fixation procedure

A film of cells (a smear) is gently heated as a slide is passed through a flame.

What does heat fixation preserve?

Overall morphology but not structures within cells.

Chemical fixation

Used to protect fine cellular substructure and the morphology of larger, more delicate microorganisms.

Chemical fixatives

Penetrate cells and react with cellular components, usually proteins and lipids, to render them inactive, insoluble, and immobile.

Common chemical fixative components

Ethanol, acetic acid, mercuric chloride, formaldehyde, and glutaraldehyde.

Staining

Simply means coloring the microorganisms with a dye that emphasizes certain structures.

Stains

Salts composed of a positive and a negative ion, one of which is colored and is called the chromatophore.

Chromatophore

The colored ion of a stain.

Purpose of stains

Used for differential coloring microorganisms to observe their structure more clearly under the microscope.

Purposes of staining

1. To see organisms better; 2. To differentiate one organism from another; 3. To determine particular structures.

Purpose of staining: to see organisms better

Enables the organism to be seen better in contrast with the background.

Purpose of staining: to differentiate one organism from another

Particularly evident in staining procedures which are called differential stains.

Purpose of staining: to determine particular structures

Special stains react only with certain structures such as spores, capsules, cell wall, nuclei, etc.

Types of stains

1. Direct or General stains; 2. Indirect stains; 3. Differential stains; 4. Selective/Special stains.

Direct or General stains

Use of simple stain, an aqueous or alcohol solution of a single basic dye.

Simple stain

An aqueous or alcohol solution of a single basic dye.

Aniline dyes

Are able to stain bacteria directly.

Examples of direct/general stains

Crystal violet, Congo red, Methylene blue, Carbolfuchsin, Crystal violet, and Safranin.

Mordant

A chemical added to the solution to intensify the stain.

Example of mordant

Iodine.

Functions of mordant

1. Increase the affinity of a stain for a biological specimen; 2. Coat a structure such as a flagellum to make it thicker and easier to see after it is stained with a dye.

Indirect stains

Acidic dyes that are repelled by the bacterial surface, so the stain colors the background instead.

Examples of indirect stains

Nigrosin or India ink.

Negative staining

Preparing colorless bacteria against a colored background.

Uses of negative staining

Valuable in observing cell shape, size, and capsules.

Differential stains

Stains that enable one to differentiate two different groups of bacteria in a mixture.

Principle of differential stains

React differently with various kinds of bacteria and thus can be used to distinguish among them.

Most frequently used differential stains for bacteria

Gram stain and acid-fast stain.

Gram stain

Developed in 1884 by Hans Christian Gram, a Danish bacteriologist; it classifies bacteria into two large groups: Gram positive (G+) and Gram negative (G−).

Hans Christian Gram

Danish bacteriologist who developed the Gram stain in 1884.

Gram-positive (G+)

One of the two large groups of bacteria classified by Gram staining.

Gram-negative (G−)

One of the two large groups of bacteria classified by Gram staining.

Importance of Gram staining

One of the most important staining techniques in medical microbiology.

Uses of Gram staining

1. To classify microorganisms on the basis of their Gram staining characteristic, size, shape and arrangement of cells; 2. To assist in the rapid presumptive diagnosis of an infectious disease; 3. To assess the quality of the clinical specimen based on the somatic cellular content.

Gram staining procedure

1. Primary stain; 2. Mordant; 3. Decolorizer; 4. Counterstain; 5. Dry and examine under microscope.

Gram stain primary stain

Gentian or crystal violet.

Gram stain primary stain duration

60 seconds.

What is done after crystal violet?

Wash the slide with tap water to remove excess crystal violet.

Gram stain mordant

Gram's iodine solution.

Gram's iodine duration

60 seconds.

What is done after Gram's iodine?

Wash the slide with tap water to remove excess iodine.