chapter 4 microscopy

Immunofluorescence:

Fluorescent antibodies can be used to detect specific antigens. Very useful for the rapid diagnosis of specific diseases (e.g.: syphilis).

Fluorescence Microscopy

Fluorochrome: Fluorescent dye. •Image: Luminescent bright object against a dark background.

What accounts for the differential staining betweenGram-positive and Gram-negative cells?

Gram positive cells have very thick peptidoglycancell walls, whereas gram-negative cells have very thin cell walls. Crystal violet easily penetrates both cell types. •Because of its larger size, the crystal violet-iodine complex [CV-I] is not easily removed from gram-positive cells, due to their thick cell wall. The CV-I complex is readily washed out of gram-negative cells with alcohol. •Counterstainonly colors gram-negative cells.

History of CL Microscopes-

First developed by ZacchariasJanssen, Dutch spectacle maker in 1600. •Poor quality •Could not see bacteria

2.Fixing

Process that kills microorganisms and attaches them to a microscope slide. Fixing preserves and minimizes distortion of cells.

3. Decolorizing:

Slide is washed with alcohol, which will remove stain from Gram-negative cells but not from Gram-positive cells.

Natural Fluorescence:

Some microorganisms fluoresce naturally under UV light (Pseudomonas).

Smear

Spread a thin film of material containing microorganisms over slide surface. Allow to air dry.

B. Atomic Force Microscopy (AFM)-

Uses a diamond and metal probethat scans surface of specimen.

A. Scanning Tunneling Microscope (STM)

Uses a thin metal probethat scans the surfaceof a specimen.

Ultraviolet (UV) light

has a shorter wavelength (100 to 400 nm), and can resolve distances as small as 110 nm.

Immersion oil

has the same index of refraction as glass slide, preventing light loss from refraction.

Magnification can be very high, but

image quality can get very blurry

objective lens

magnify the image, light enters through this lens. Closest lenses to the specimen.

The image of the specimen is magnified once again by the-

ocular lensor eyepiece(10 X).

Stains are

salts composed of a positive ion (cation) and a negative ion (anion). The colored ion is called - chromophore.

Preparation of Specimens for Light Microscopy

smear, fix, stain

White light

thas a relatively long wavelength (550 nm), and cannot resolve structures less than 220 nm (0.2 um)apart.

The smaller the distance between objects at which they can be distinguished as separate

the greater the resolving power.

When two substances have a different index of refraction,

the light will bendas it passes from one material to another.

If wavelength is too long to pass between objects,

they will appear as one.

•Staining

will increase the contrast between a cell and its background

Visible light

400-700 nanometers

Electron beams

0.01-0.001 nm

Steps of Gram Stain

1. Primary stain - Cover a heat fixed smear with a basic dye (crystal violet). •All cells, gram-positive and gram-negative, are stained with crystal violet (appear purple). 2. Mordant:After smear is rinsed with water, an iodine mordantsolution is applied. •Crystal violet-iodine [CV-I] complex forms

Ultraviolet light

100-400 nm

Index of refraction

A measure of the speed at which light passes through a material. Refract can be changed by - staining, which increases contrast between specimen and surrounding medium.

•There are two important differential stains used in microbiology

A. Gram stain B. Acid-Fast stain

Two types of electron microscope:

A. Transmission Electron Microscope (TEM) B. Scanning Electron Microscope (SEM)

Resolution (Resolving power)

Ability of microscope to see two items as separate and discrete units.

Fluorescence:

Ability of substances to absorb short wavelengths of light (ultraviolet light) and emit them at a longer wavelength.

Resolution (Resolving Power)

Ability to distinguish between objects that are close together.

4. Counterstain

Alcohol is rinsed off. Safraninis applied, which will stain cells that were decolorized.

Magnification

Apparent increase in size of an object Occurs when radiation bends (refracts) as it passes through a lens

Refraction

Bending of light as it passes from one medium to another of different density.

Acridine orange:

Binds to nucleic acids, colors cells orange, green, or yellow depending on light source.

Staining

Coloring microorganisms with a dye that emphasizes certain structures. Before staining a sample, it must be fixed.

Image

Direct rays and reflected light rays come together, forming an image with many shades of gray to black.

condenser lenses-

Light originates from an illuminator passes through , which direct light onto the specimen.

Basic unit of length

Meter.

Simple Microscopes

Only have one lens, similar to a magnifying glass.

Units of Measurement

The metric systemis used to measure microorganisms.

Wavelength of radiation

The smaller the wavelength used, the higher the magnification and resolution of the microscope.

Microscopy

The technology of making very small things visible to the naked eye.

Leeuwenhoeck's simple microscopes

allowed him to magnify images from 100 to 300 X.

Use visible light

as a source of illumination.

Light must pass between two objects in order for them to

be seen as separate.

Applications and Limitations of the Gram stain

chemotherapy and limitations

Useful magnification

depends on resolution and contrast

Joseph Jackson Lister (Lister's father)-

developed improved compound light microscope in 1830s. •Basis for modern microscopes

1. Simple Stains

•Aqueous or alcohol solution of a single basic dye. •Primary purpose is to stain entire microorganism to view cell shape and basic structures. •Procedure: •Stain is applied for a certain time, and then washed off. •Slide is dried and examined. •Mordant - May be used to increase stain intensity. Increases affinity of stain for specimen. •Examples: Safranin, methyleneblue, crystal violet, and carbolfuchsin.

Basic dye

•Chromophoris in positive ions. •Most commonly used dyes. •Bacteria are slightly negatively charged at pH 7, therefore they stain with basic dyes. •Examples: •Crystal violet •Methyleneblue •Saffranin

Acidic dyes

•Color is in negativeions. •Stain the background: negative staining. •Bacteria do not stain with acidic dyes. •Used to observe cell shape, size, and capsules. •Minimal distortion because heat fixing is not necessary an dye is not taken up by cells. •Examples: •Eosin •Nigrosin •India ink.

Scanning Tunneling Microscopy and Atomic Force Microscopy (AFM )

•Developed in the 1980s.•Used to observe structure and surface of biological molecules and silicon computer chips.

Drawbacks of TEM:

•Due to limited penetrating power of electrons, can only view very thin slices (70-90 nm) of specimen. •Must slice, fix, dehydrate, and view specimen under a vacuum. Staining may be used to enhance image contrast. •Treatments kill specimen and may cause shrinkage and distortion of cells (artifacts).

Electron Microscopy

•Electron microscopes were first developed in 1932, and became widely available in 1940s. •Use a beam of electronsinstead of a beam of light. •Wavelength of electron beam is about 100,000 times smallerthan visible light. •Used to examine structures too small to be resolved with a light microscope.

Scanning Electron Microscope (SEM)

•Gives excellent view of external surface. •Magnification: 10,000 X or more. •Resolving power:20 nm or better. •Three dimensional image. •More recent invention than TEM. Used mainly to observe the surfaces of cells and viruses. •Specimens are covered with a layer of heavy metal (gold or palladium). •A narrow beam of electrons (primary electron beam) is swept across specimen surface. •Electrons on the specimen surface are knocked out, creating a secondary electron beam which is collected and amplified to produce an image.

Transmission Electron Microscope (TEM)

•Gives excellent view ofinternal structures. •Magnification: 100,000 X or more. •Resolving power:2.5 nm or better. •Two dimensional image.

What are the colors of gram negative and gram positive once they are counterstained?

•Gram-negativecells are stained pink.•Gram-positivecells remain purple.

What are the colors of gram positive and gram negative cells once they are decolorized?

•Gram-negativecells will be decolorized. •Gram-positivecells will remain purple.

Chemotherapy

•Gram-positive cells with their very thick peptidoglycancell walls, are susceptible to penicillinsand cephalosporins. •Gram-negative cells with their thin cell walls and lipopolysaccharidelayer are resistant to these antibiotics.

Two main methods of fixation

•Heat fixation: Pass over Bunsen burner flame several times. •Chemical fixation: Cover with methanol for 1 minute.

Advantages of both microscopes STM AND AFM

•Higher resolving power than electron microscopes •No special specimen preparation required

Limitations of light microscopy

•Magnification: Up to 2000 X. •Resolving Power: Up to 0.2 um. Because of the limits of magnification and resolving power,viruses and most internal structures of cells cannot be seen with a light microscope.

Acid-Fast Stain (Ziehl-Nielsen Stain)-

•Modification of a method developed in 1882 by Paul Ehrlich. •Used to detect tuberculosis and leprosy causing organisms of the genus Mycobacterium and pathogens of the genus Nocardia. •These bacteria have waxy cell walls

Limitations

•Not all bacterial cells stain well with the Gram-stain. •Gram-stain only works well on young bacterial cultures, that are actively growing. Therefore it is best to use cultures that are 18 to 24 hours old. •Older cultures (over 24-48 hours), are often gram-variable.

2. Differential Stains

•React differently to different types of bacteria. •Can be used to distinguish among different groups of bacteria.

sizes of objective lens

•Scanningobjective lens: 4 X •Low powerobjective lens: 10 X •High powerobjective lens: 40-45 X •Oil immersionlens: 95-100 X

Contrast differences in intensity between

•Two objects •An object and its background

Darkfield Microscope

•Useful to examine live or unstainedspecimens. •Light sensitive organisms •Specimens that lack contrast with their background. •condenser with opaque discblocks light that would enter objective lens directly •Light reflects off specimen at an angle. •Only light reflected by specimen enters objective lens. •No direct background light. •Image: Light specimen against dark background.

Phase Contrast Microscopy

•Useful to examine livespecimens: •Doesn't require fixing or staining, which usually kill and/or distort microorganisms. •Permits detailed examination of internalstructures. •Special objective lenses and condenser with ring shaped diaphragm accentuate small differences in refractive indexes of internal structures.

What two factors depend on Resolution?

•Wavelength of electromagnetic radiation •Aperture of lens: Ability to gather light Resolution Distance- .61 x wavelength/ numerical