Chapter 2 bio 300 obe

Chapter 2 :Techniques in Histology1

Chapter 2 :Techniques in Histology

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BIO 300BIO 300BIOLOGICAL TECHNIQUES AND SKILLSBIOLOGICAL TECHNIQUES AND SKILLS

SARINI BINTI AHMAD WAKIDSARINI BINTI AHMAD WAKIDFACULTY OF APPLIED SCIENCEFACULTY OF APPLIED SCIENCE

BIO 300BIO 300BIOLOGICAL TECHNIQUES AND SKILLSBIOLOGICAL TECHNIQUES AND SKILLS

SARINI BINTI AHMAD WAKIDSARINI BINTI AHMAD WAKIDFACULTY OF APPLIED SCIENCEFACULTY OF APPLIED SCIENCE


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CHAPTER 2CHAPTER 2TECHNIQUES IN HISTOLOGYTECHNIQUES IN HISTOLOGY


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HISTOLOGICAL TECHNIQUEA. Histology involves the preparation of tissues for examination with a

microscope.

1. Basic methods of histological preparation of tissues.

a. Fix tissue (e.g. 4 % paraformaldehyde + buffer)

b. Dehydrate tissue (alcohol series followed by toluene)

c. Embed tissue in “hard” medium (e.g. wax)

d. Section embedded tissue on a microtome

e. Mount sections on a supportive structure (e.g. slide) that can be placed on a microscope stage

f. Usually remove the embedding medium

g. Stain tissue (e.g. hematoxylin-eosin)

h. Examine tissue with microscope.

Chapter 2 :Techniques in Histology 5

1. Preparative techniques are often “harsh” and can traumatize and change the natural structure of a tissue.

2. As a result, what you see is not always real !

3. Tissue/cellular structures that are “created” by preparative techniques for histological specimens are called “artifacts.”

Artifacts


Fixation Need to prevent autolysis of tissue samples by

enzymes/bacteria present Fixation may be chemical or less frequently,

physical methods chemical fixatives stabilize or cross-linking agents

need small to get complete penetration of tissue – intravascular perfusion

routine fixative is 4% formalin, gluteraldehyde – interact with amine groups of proteins

EM requires additional fixation – gluteraldehyde and OsO4 (stains lipids and proteins)

physical fixation – freezing sample


TISSUE FIXATION Fixation is a complex series of chemical events that differ for the different groups of

substance found in tissues. Main purpose: to preserve material in a life-like manner.

The aim of fixation:

1- To prevent autolysis and bacterial attack.

2- To fix the tissues so they will not change their volume and shape during processing.

3- To prepare tissue and leave it in a condition which allow clear staining of sections.

4- To leave tissue as close as their living state as possible, and no small molecules should be lost.

Fixation is coming by reaction between the fixative and protein which form a gel, so keeping every thing as their in vivo relation to each other.

The fixation solutions used for microscopy intended to: Penetrate rapidly to prevent post mortem

changes in the cells Coagulate the cell contents into insoluble

substances Protect tissues against shrinkage and

distortion during subsequent processing Allow cell parts to become selectively and

clearly visible when stained.



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Factors affect fixation:

- PH.- Temperature.- Penetration of fixative.- Volume of tissue.According to previous factors we can determine the

concentration of fixative and fixation time.

Types of fixative:Acetic acid, Formaldehyde, Ethanol, Glutaraldehyde, Methanol

and Picric acid.


Why fix tissue?

Preserve structure. Essentially to make the structural components of the tissue more durable so that the tissue can be manipulated in various ways.

• Fixed material is dead. You want to preserve the structure (chemical and morphological) of the living material so that it appears the same as it was in life.

• It will never be exactly the same. Important to choose fixative that does the best job. Fixative used will depend on type of tissue to be fixed.

Why dehydrate the fixed tissue

Most fixatives are water soluble, most embedding media are non-polar and are not miscible with water. So, you have to move the tissue from a polar (water-based) medium to a non-polar medium (e.g. toluene) that is miscible with the embedding medium.


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TISSUE PROCESSING

The aim of tissue processing is to embed the tissue in a solid medium firm enough to support the tissue and give it sufficient rigidity to enable thin sections to be cut , and yet soft enough not to damage the knife or tissue.

Stages of processing:1- Dehydration.2- Clearing.3- Embedding.


DehydrationTo remove fixative and water from the tissue and replace

them with dehydrating fluid.There are a variety of compounds many of which are

alcohols. Several are hydrophilic so attract water from tissue.

To minimize tissue distortion from diffusion currents, delicate specimens are dehydrated in a graded ethanol series from water through 10%-20%-50%-95%-100% ethanol.

In the paraffin wax method, following any necessary post fixation treatment, dehydration from aqueous fixatives is usually initiated in 60%-70% ethanol, progressing through 90%-95% ethanol, then two or three changes of absolute ethanol before proceeding to the clearing stage.


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Types of dehydrating agents:

Ethanol, Methanol, Acetone.

• Duration of dehydration should be kept to the minimum consistent with the tissues being processed. Tissue blocks 1 mm thick should receive up to 30 minutes in each alcohol, blocks 5 mm thick require up to 90 minutes or longer in each change. Tissues may be held and stored indefinitely in 70% ethanol without harm


Clearing Replacing the dehydrating fluid with a fluid that is totally miscible with

both the dehydrating fluid and the embedding medium.

Choice of a clearing agent depends upon the following:

- The type of tissues to be processed, and the type of processing to be undertaken.

- The processor system to be used. - Intended processing conditions such as temperature, vacuum and

pressure. - Safety factors. - Cost and convenience. - Speedy removal of dehydrating agent . - Ease of removal by molten paraffin wax . - Minimal tissue damage .


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• Some clearing agents:- Xylene.- Toluene.- Chloroform.- Benzene.- Petrol.


Embedding Is the process by which tissues are surrounded by a medium such as agar,

gelatin, or wax which when solidified will provide sufficient external support during sectioning.

Paraffin waxproperties :

Paraffin wax is a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils. It is about two thirds the density and slightly more elastic than dried protein. Paraffin wax is traditionally marketed by its melting points which range from 39°C to 68°C.

The properties of paraffin wax are improved for histological purposes by the inclusion of substances added alone or in combination to the wax:

- improve ribboning. - increase hardness. - decrease melting point - improve adhesion between specimen and wax


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Precaution while embedding in wax

• The wax is clear of clearing agent.• No dust particles must be present.• Immediately after tissue embedding, the wax must be

rapidly cooled to reduce the wax crystal size.


There are four main mould systems and associated embedding protocols presently in use :

1- Traditional methods using paper boats 2- Leuckart or Dimmock embedding irons or metal

containers 3- the Peel-a-way system using disposable plastic

moulds and 4- systems using embedding rings or cassette-bases

which become an integral part of the block and serve as the block holder in the microtome.


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Tissue processingEmbedding moulds:(A) paper boat; (B) metal bot mould; (C) Dimmock embedding mould; (D) Peel-a-way disposable mould; (E) base mould used with embedding ring ( F) or cassette bases (G)


General Embedding Procedure1- Open the tissue cassette, check against worksheet entry to ensure the correct number of tissue pieces are present.

2- Select the mould, there should be sufficient room for the tissue with allowance for at least a 2 mm surrounding margin of wax.

3- Fill the mould with paraffin wax.

4 Using warm forceps select the tissue, taking care that it does not cool in the air; at the same time.

5- Chill the mould on the cold plate, orienting the tissue and firming it into the wax with warmed forceps. This ensures that the correct orientation is maintained and the tissue surface to be sectioned is kept flat.

6- Insert the identifying label or place the labeled embedding ring or cassette base onto the mould.

7- Cool the block on the cold plate, or carefully submerge it under water when a thin skin has formed over the wax surface.

8- Remove the block from the mould.

9- Cross check block, label and worksheet.


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a. Tissue will be sectioned. Needs to be durable enough to withstand the sectioning process. Also, want components of tissue to remain in their natural positions. Don't want them to be moved to new positions.

b. Embedding in wax or plastic immobilizes structural components of tissue. Holds them in place as sectioning is done.

Why embed?


ORIENTATION OF TISSUE IN THE BLOCKCorrect orientation of tissue in a mould is the most important step in embedding. Incorrect placement of tissues may result in diagnostically important tissue elements being missed or damaged during microtomy.

elongate tissues are placed diagonally across the block

tubular and walled specimens such as vas deferens, cysts and gastrointestinal tissues are embedded so as to provide transverse sections showing all tissue layers

tissues with an epithelial surface such as skin, are embedded to provide sections in a plane at right angles to the surface (hairy or keratinised epithelia are oriented to face the knife diagonally)

multiple tissue pieces are aligned across the long axis of the mould, and not placed at random


Processing methods and routine schedules

Machine processing

manual processing


Sectioning

Use a microtome to section embedded samples 1-10 m thick

Float on surface of warm water to remove wrinkles and load on to slide

Frozen samples are cut with a cryostat use this method when wanting to study enzyme

activity which can be damaged by fixation


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CUTTING

• using the microtome


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• A microtome is a mechanical instrument used to cut biological specimens into very thin segments for microscopic examination. Most microtomes use a steel blade and are used to prepare sections of animal or plant tissues for histology. The most common applications of microtomes are :


1- Traditional histological technique:

Tissues are hardened by replacing water with paraffin. The tissue is then cut in the microtome at thicknesses varying from 2 to 25 micrometers thick. From there the tissue can be mounted on a microscope slide, stained and examined using a light microscope


2- Cryosection:Water-rich tissues are hardened by freezing and cut frozen; sections are stained and examined with a light microscope. This technique is much faster than traditional histology (5 minutes vs. 16 hours) and are used in operations to achieve a quick diagnosis. Cryosections can also be used in immunohistochemistry as freezing tissue does not alter or mask its chemical composition as much as preserving it with a fixative.


3- Electron microscopy: After embedding tissues in epoxy resin, a microtome equipped with a

glass or diamond knife is used to cut very thin sections (typically 60 to 100 nanometers). Sections are stained and examined with a

transmission electron microscope. This instrument is often called an ultramicrotome.

4- Botanical microtomy: hard materials like wood, bone and leather require a sledge microtome.

These microtomes have heavier blades and cannot cut as thin a regular microtomy.

Microtome blades are extremely sharp, and should be handled with great care. Safety precautions should be taken in order to avoid any contact with the cutting edge of the blade.


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Microtome knives

• STEEL KNIVES• NON-CORROSIVE KNIVES FOR

CRYOSTATS • DISPOSABLE BLADES• GLASS KNIVES • DIAMOND KNIVES


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Why section?

a. Allows you to see internal structure of tissue.b. Allows stains, or specific markers such as

antibodies to more easily infiltrate the tissues.c. Allows light to pass through tissue making

structure visible.d. While sectioning is useful in many instances, in

some cases tissues are stained and examined without sectioning.


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STAINING

Purpose1. Add contrast to the image –

separation of bacteria in terms of morphological characteristics and cellular structures.

2. Identify chemical components of interest (visualize)

3. Locate particular tissues, cells or organelles (differentiation)


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Staining

Need to stain samples to impart contrast to various structures, without hard to identify tissues

Dyes will stain more or less selectively – usually acids or bases and form electrostatic linkages with tissue components basic dyes – attach to acid components, basophilic

toluidine blue, methylene blue and hematoxylin acid dyes – attach to basic components, acidophilic

orange G, eosin, acid fuchsin


Stains Hemotoxylin and Eosin – (H&E) most common

stain for general morphology and structures nucleus, RNA-rich areas in cytoplasm and matrix of

hyaline cartilage is blue while cytoplasm and collagen are pink

Trichromes – additional dyes that will differentiate other structures

Counterstains – use to give general structure when doing immunohistochemistry – do not want to overpower the complex attached to what we are looking for


H & E is a charge-based, general purpose stain. Hematoxylin stains acidic molecules shades of blue. Eosin stains basic materials shades of red, pink and orange. H & E stains are universally used for routine histological examination of tissue sections.

Hematoxylin and Eosin (H & E)


Staining machine

Chapter 2 :Techniques in Histology 39http://www.ipass.net/grc/dimpg9.htm

Why stain the tissue?

a. Creates higher contrast that allows observation of structure that is not visible in unstained tissue.

b. May reveal differences in chemical nature of regions of the tissue.


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Staining

Dyes create contrast by imparting a color to cells or cell parts

Basic dyes – cationic, positively charged chromophore

Acidic dyes – anionic, negatively charged chromophore

Positive staining – surfaces of microbes are negatively charged and attract basic dyes

Negative staining – microbe repels dye, the dye stains the background


Staining reactions of dyes

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Staining Simple stains – one dye is used; reveals

shape, size, and arrangement Differential stains – use a primary stain

and a counterstain to distinguish cell types or parts (examples: Gram stain, acid-fast stain, and endospore stain)

Structural stains – reveal certain cell parts not revealed by conventional methods: capsule and flagellar stains


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Microbiological stains

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Crystal Violet Gram Stain Capsule Stain

Methylene Blue Acid Fast – Red Fast, blue non

Flagella Stain

Spore Stain - green


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Simple StainsBacteria have nearly the same refractive index as water, therefore, when Bacteria have nearly the same refractive index as water, therefore, when they are observed under a microscope they are opaque or nearly invisible they are observed under a microscope they are opaque or nearly invisible to the naked eye.to the naked eye.

Different types of staining methods are used to make the cells and their Different types of staining methods are used to make the cells and their internal structures more visible under the light microscope.internal structures more visible under the light microscope.

Simple stainsSimple stains use one dye that stains the cell wall. use one dye that stains the cell wall.

The cells are then visible against a light background.The cells are then visible against a light background.

Steps:Steps:1.1. Place the slide on the staining rack.Place the slide on the staining rack.

2.2. Flood the slide with a basic stain: either crystal violet (1 min.), Safranin Flood the slide with a basic stain: either crystal violet (1 min.), Safranin (2 min.), or Methylene blue (2 min.).(2 min.), or Methylene blue (2 min.).

3.3. Wash the stain off the slide with deionized water.Wash the stain off the slide with deionized water.

4.4. Blot the slide with bibulous paper.Blot the slide with bibulous paper.


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Differential Staining Differential StainsDifferential Stains use two or more stains and allow the cells to be use two or more stains and allow the cells to be

categorized into various groups or types.categorized into various groups or types. Both techniques allow the observation of cell morphology, or shape, Both techniques allow the observation of cell morphology, or shape,

but differential staining usually provides more information about the but differential staining usually provides more information about the characteristics of the cell wall (Thickness).characteristics of the cell wall (Thickness).

The most common differential stain used in microbiology is the Gram The most common differential stain used in microbiology is the Gram StainStain..

Basic stains, due to their positive (+) charge will bind electrostatically Basic stains, due to their positive (+) charge will bind electrostatically to negatively charged molecules such as many polysaccharides, to negatively charged molecules such as many polysaccharides, proteins and nucleic acids.proteins and nucleic acids.

Acid stains ( - ) bind to positively charged molecules which are much Acid stains ( - ) bind to positively charged molecules which are much less common, meaning acidic stains are used only for special less common, meaning acidic stains are used only for special purposes. purposes.

Some commonly encountered basic stains are crystal violet, safranin Some commonly encountered basic stains are crystal violet, safranin (a red dye) and methylene blue. (a red dye) and methylene blue.

Basic stains may be used alone (a simple stain) or in combination Basic stains may be used alone (a simple stain) or in combination (differential stain) depending on the experiment involved. (differential stain) depending on the experiment involved.

Mounting sections

• Wet mounts – to observe fresh specimens1. Isolate the specimen

2. Place the specimen in the small droplet of the relevant fluid (fresh water, seawater) on a microscope slide.

3. Gently lower a coverslip onto the droplet, using forceps of two needles with absorbent paper.

4. Remove any excess water on or around the coverslip with absorbent paper.


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Mounting sections

• Temporary mounts

-involve wet mounting in a mountant with a short useful life – for identification purposes.


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Mounting sections• Permanent mounts – protect sections during

examination and allow storage without deterioration.Procedure

1. Apply little mountant to a coverslip of appropriate size.

2. Turn the coverslip over and place on its edge to one side of the sections.

3. Lower the coverslip slowly down onto the sections so as to displace all the air and sandwich the sections between the slide and the coverslip.

4. Press firmly form the centre outwards to distribute the mounting medium evenly.

5. Allow the solvent to evaporate – best results come from slow drying when time allows, but many synthetic mountant will tolerate brief heating when speed is essential.


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The Gram Stain Developed by Christian Gram in 1884 A staining procedure that differentiates between

bacteria based on the structure of their cell walls. Thick Cell Wall

Penicillin and derivatives of penicillin are used on these bacteria because it attacks peptidoglycan (cell wall) synthesis.

Thin Cell Wall with Outer Membrane Penicillin is ineffective on these bacteria because

they have a lipid membrane outside of their cell wall


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Gram Staining The Gram Stain is a differential stain.The Gram Stain is a differential stain. Four different reagents are used and the results are based on differences in Four different reagents are used and the results are based on differences in

the bacterial cell wall.the bacterial cell wall. Gram PositiveGram Positive bacteria have a relatively thick cell wall composed of a bacteria have a relatively thick cell wall composed of a

special carbohydrate called special carbohydrate called PeptidoglycanPeptidoglycan.. Gram NegativeGram Negative bacteria have a much thinner cell wall composed of the same bacteria have a much thinner cell wall composed of the same

carbohydrate, carbohydrate, PeptidoglycanPeptidoglycan, but with certain chemical differences, such as , but with certain chemical differences, such as the presence of the presence of LipopolysaccharidesLipopolysaccharides (LPS). (LPS).

Notice that both Gram Positive and Gram Negative bacteria have a cell wall Notice that both Gram Positive and Gram Negative bacteria have a cell wall composed primarily of Peptidoglycan.composed primarily of Peptidoglycan.

Gram Staining StepsGram Staining Steps1.1. Crystal violetCrystal violet acts as the primary stain. Crystal violet may also be used acts as the primary stain. Crystal violet may also be used

as a simple stain because it dyes the cell wall of any bacteria.as a simple stain because it dyes the cell wall of any bacteria.2.2. Gram’s iodineGram’s iodine acts as a mordant (Helps to fix the primary dye to the cell acts as a mordant (Helps to fix the primary dye to the cell

wall).wall).3.3. DecolorizerDecolorizer is used next to remove the primary stain (crystal violet) from is used next to remove the primary stain (crystal violet) from

Gram Negative bacteria (those with LPS imbedded in their cell walls). Gram Negative bacteria (those with LPS imbedded in their cell walls). Decolorizer is composed of an organic solvent, such as, acetone or Decolorizer is composed of an organic solvent, such as, acetone or ethanol or a combination of both.)ethanol or a combination of both.)

4.4. Finally, a counter stain (Finally, a counter stain (SafraninSafranin), is applied to stain those cells (Gram ), is applied to stain those cells (Gram Negative) that have lost the primary stain as a result of decolorization.Negative) that have lost the primary stain as a result of decolorization.


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Gram Staining Procedure


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Gram Staining Results When reporting the results of the Gram stain you indicate the type of When reporting the results of the Gram stain you indicate the type of

stain used, the reaction, and the morphology of the cells observed.stain used, the reaction, and the morphology of the cells observed. Round (spherical), purple (or dark blue) cells are reported as Gram Round (spherical), purple (or dark blue) cells are reported as Gram

positive cocci (GPC).positive cocci (GPC). Rod-shaped, purple (or dark blue) cells are reported as Gram Rod-shaped, purple (or dark blue) cells are reported as Gram

positive bacilli (GPB).positive bacilli (GPB). The standard abbreviations for the four types of Gram stain and The standard abbreviations for the four types of Gram stain and

morphology are:morphology are:1.1. Gram Positive Cocci (GPC)Gram Positive Cocci (GPC)2.2. Gram Positive Bacilli (GPB)Gram Positive Bacilli (GPB)3.3. Gram Negative Cocci (GNC)Gram Negative Cocci (GNC)4.4. Gram Negative Bacilli (GNB)Gram Negative Bacilli (GNB)

Notice that both Gram Positive and Gram Negative bacteria have a Notice that both Gram Positive and Gram Negative bacteria have a cell wall composed primarily of Peptidoglycan.cell wall composed primarily of Peptidoglycan.

Spiral-shaped bacterial cells do not Gram stain well and are usually Spiral-shaped bacterial cells do not Gram stain well and are usually observed using dark-field microscopy. There are no standard observed using dark-field microscopy. There are no standard abbreviations for Gram stain reactions of the spirilla of medical abbreviations for Gram stain reactions of the spirilla of medical importance.importance.

Certain other types of bacteria may not Gram stain well, such as, Certain other types of bacteria may not Gram stain well, such as, Acid-fast Mycobacteria (Acid-fast Mycobacteria (Mycobacterium tuberculosisMycobacterium tuberculosis).).


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Bacterial Cell Shapes Bacteria can have several different shapes, but the primary shapes we will Bacteria can have several different shapes, but the primary shapes we will

be observing are:be observing are:1.1. Spherical or round cells – cocci (plural) or coccus (singular)Spherical or round cells – cocci (plural) or coccus (singular)

2.2. Rod shaped – bacilli (plural) or bacillus (singular)Rod shaped – bacilli (plural) or bacillus (singular)

3.3. Spiral shaped – spirillaSpiral shaped – spirilla

Some bacteria have characteristic clustering or arrangements, usually due Some bacteria have characteristic clustering or arrangements, usually due to how the cells divide and whether they remain attached together when to how the cells divide and whether they remain attached together when they divide.they divide.

Diplococci – divide in one plane and remain attached together after cell division.Diplococci – divide in one plane and remain attached together after cell division. Streptococci – divide in one plane and form long chains of attached cells.Streptococci – divide in one plane and form long chains of attached cells. Staphylococci – divide in many planes and remain attached together forming a Staphylococci – divide in many planes and remain attached together forming a

“grape-like cluster”“grape-like cluster”


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The Microscope

Key characteristics of a reliable microscope are:

Magnification – ability to enlarge objects Anybody have cheap telescope or binoculars?

Resolving power – ability to show detail


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Magnification in most microscopes results from interaction between visible light waves and curvature of the lens

Angle of light passing through convex surface of glass changes – refraction

Depending on the size and curvature of the lens, the image appears enlarged

Extent of enlargement – magnification


Light Microscopy

Mechanical and optical components make up microscope

3 systems of lenses condenser – collects and focuses light onto object objective – enlarge and project the illuminated

image in direction of eyepiece eyepiece – further magnification and project to the

retina or detector


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Principles of Light Microscopy

Magnification occurs in two phases – The objective lens forms the magnified real image The real image is projected to the ocular where it is magnified

again to form the virtual image Total magnification of the final image is a product of the separate

magnifying powers of the two lenses

power of objective x power of ocular = total magnification


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ResolutionResolution defines the capacity to distinguish or separate two adjacent

objects – resolving power Function of wavelength of light that forms the image along with

characteristics of objectives Visible light wavelength is 400 nm–750 nm Numerical aperture of lens ranges from 0.1 to 1.25 Oil immersion lens requires the use of oil to prevent

refractive loss of light Shorter wavelength and larger numerical aperture will

provide better resolution Oil immersion objectives resolution is 0.2 μm Magnification between 40X and 2000X


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Variations on the Optical Microscope Bright-field – most widely used; specimen is darker than

surrounding field; live and preserved stained specimens Dark-field – brightly illuminated specimens surrounded by dark

field; live and unstained specimens Phase-contrast – transforms subtle changes in light waves

passing through the specimen into differences in light intensity, best for observing intracellular structures


Three views of a basic cell

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Bright Field

Dark Field

Phase Contrast


Phase Contrast and DIC Microscope

The differential interference microscope is similar to the phase contrast but has more refinements

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Phase-Contrast and Differential Interference Microscopy Use phase-contrast for viewing images of

transparent samples light changes speed when passing thru cellular

and extracellular structures with different refractive indices

use to view living cells


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2 Types of Electron Microscopes

Transmission electron microscopes (TEM) – transmit electrons through the specimen. Darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts.

Scanning electron microscopes (SEM) – provide detailed three-dimensional view. SEM bombards surface of a whole, metal-coated specimen with electrons while scanning back and forth over it.


Transmission E.M.

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Electrons pass through the sample


Scanning EM

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Scanning looks at surface only


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TECHNIQUES OF MICROBIOLOGY

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Chapter 2 bio 300 obe

Technology