Microscopy and Staining

Course : B.Sc. Microbiology

Sem I

Sub: Basic Techniques in Microbiology

Unit 1

Microscopy• What is microscopy?

• Why Microscope required?

• How to use microscope?

• Principle of microscope.

• Parts of Microscope.

• Types of microscope.

– Light microscope• Bright field microscope

• The Dark-Field Microscope

• The Phase-Contrast Microscope

• The Fluorescence Microscope

– Scanning and electron microscope

Replica of Marshall

Microscope, c. 1700, by John

Marshall (1663-1725).

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Why Microscope,,,

History of the Micro(organism)scope

• 1590 –first compound

microscope

Discovery of Microorganisms.

Anton van

Leeuwenhoek (1632-

1723)

– first person to

observe and describe

micro-organisms

accurately

The way how he found micro-organism’s….

2

Microscope• Microscope is a tool

which can help you see tiny objects and living organisms. It makes them look bigger.

• This ability of the microscope is called its magnifying power or magnification.

3

Microscope• The microscope also has the capacity to

distinguish small gaps between two separate points which humans cannot distinguish. It is called its resolving power or resolution.

4

Types of Microscope

• Types of microscope.

Light microscopeBright field microscope

The Dark-Field Microscope

The Phase-Contrast Microscope

The Fluorescence Microscope

Scanning and electron microscope

Light microscope

• Light microscope uses diffused light from the sun or artificial light to illuminate the object to be observed.

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Parts of Microscope

• Ocular (eyepiece)

• Body

• Arm

• Coarse focus

• adjustment knob

• Fine focus

• adjustment knob

• Stage adjustment knobs

• Interpupillary adjustment

• Nosepiece

• Objective lens (4)

• Mechanical stage

• Substage condenser

• Aperture diaphragm control

• Base with light source

• Field diaphragm lever

• Light intensity control

Microscopy

What are the parts of the microscope?

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1. Arm

• Supports the body tube.

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2. Base

• Supports and stabilizes the

microscope.

7

3. Eyepiece / ocular lens

• Magnifies image produced

by objective lens.

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4. Body Tube

• Maintains the proper distance between the objective and ocular lenses.

7

5. Coarse adjustment knob

• Moves stage up and down

a large amount for

FOCUSING.

7

6. Fine adjustment knob

• small, round knob on the side of the microscope used to fine-tune the focus of your specimen

• after using the coarse adjustment knob

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7. Light source

• (lamp or mirror) Provides

light for viewing the slide.

• Projects light UPWARDS

through the diaphragm,

the SPECIMEN, and

the LENSES.

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8. Diaphragm

• Controls the amount of

light passing through

the slide.

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9. Stage and stage clips

• Stage clips

- hold the slide in place.

• Stage

- Supports the slide

being viewed.

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Lens

Objective lens Condenser Lens

Usually you will find 3 or 4 objective lenses on

a microscope

It consist of 4X, 10X, 40X and 100X powers.

When coupled with a 10X (most common)

eyepiece lens, we get total magnifications of

40X (4X times 10X), 100X , 400X and 1000X

The purpose of the condenser lens is to focus

the light onto the specimen

Condenser lenses are most useful at the highest

powers (400X and above).

Microscopes with in stage condenser lenses

render a sharper image than those with no lens

(at 400X)

If the microscope has a maximum power of

400X, you will get the maximum benefit by

using a condenser lenses rated at 0.65 NA or

greater

10. Objective lenses

• Focus and magnify light coming

through the slide.• Usually you will find 3 or 4

objective lenses on a microscope. They almost

• always consist of 4X, 10X, 40X and 100X powers. When coupled with a 10X (most common)

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10. Objective lenses

• eyepiece lens, we get total magnifications of 40X (4X times 10X), 100X , 400X and 1000X. The shortest

• lens is the lowest power, the longest one is the lens with the greatest power. Lenses are color coded.

• The high power objective lenses are retractable (i.e. 40XR). This means that if they hit a slide, the end of the lens will push in (spring loaded) thereby protecting the lens and the slide.

High power objective lenses

Rotate so that the 100x oil immersion objective touches the oil and clicks into place.

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Place a small drop of oil on the slide in the center of the lighted area. (Take care not to dribble on the stage.)Put the small drop of oil directly over the area of the specimen to be Examined.

High power objective lenses

Focus only with fine focus. Hopefully, the specimen will come into focus easily. Do not change focus dramatically.

11. Revolving nosepiece

• Rotates to allow use of different power objectives.

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12. Inclination joint

• Supports the arm and

controls the body of the

microscope.

7

Microscope Vocabulary

• Magnification: increase of an object’s

apparent size

• Resolution: power to show details clearly

Both are needed to see a clear image

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 ,

where c is the speed of light in vacuum and v is the speed of light in the substance

• Direction and magnitude of bending is Determined by the refractive indexes of the two media forming the interface

. 29

Focal point and Focal length

• 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

30

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Microscope Resolution

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

together

• Wavelength of light used is major factor in resolution

shorter wavelength greater resolution

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Working Distance

•working distance— Distance between the front surface of

lens and surface of cover glass or

specimen

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Bright Field microscope

• The ordinary microscope is called a bright-field microscope because it forms a dark image against a brighter background. The microscope consists of a sturdy metal body or stand composed of a base and an arm to which the remaining parts are attached

• A light source, either a mirror or an electric illuminator, is located in the base. Two focusing knobs, the fine and coarse adjustment knobs, are located on the arm and can move either the stage or the nosepiece to focus the image.

Dark Field Microscope• Unstained cells and organisms can be observed by

simply changing the way in which they are illuminated.

• A hollow cone of light is focused on the specimen in such a way that unreflected and unrefracted rays do not enter the objective.

• Only light that has been reflected or refracted by the specimen forms an image.

• The field surrounding a specimen appears black, while the object itself is brightly illuminated.

• Because the background is dark, this type of microscopy is called dark-field microscopy. Considerable internal structure is often visible in larger eukaryotic microorganisms The dark-field microscope is used to identify bacteria like the thin and distinctively shaped Treponema pallidum (figure 2.8a), the causative agent of syphilis.

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Phase-Contrast Microscope

• Unpigmented living cells are not clearly visible in the brightfield microscope because there is little difference in contrast between the cells and water. Thus microorganisms often must be fixed and stained before observation to increase contrast and create variations in color between cell structures.

• A phase-contrast microscope converts slight differences in refractive index and cell density into easily detected variations in light intensity and is an excellent way to observe living cells.

• Phase-contrast microscopy is especially useful for studying microbial motility, determining the shape of living cells, and detecting bacterial components such as endospores and inclusion bodies that contain poly--hydroxybutyrate, olymetaphosphate, sulfur, or other substances.

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Fluorescence Microscope• The microscopes thus far considered produce

an image from light that passes through a specimen.

• An object also can be seen because it actually emits light, and this is the basis of fluorescence microscopy.

• When some molecules absorb radiant energy, they become excited and later release much of their trapped energy as light.

• Any light emitted by an excited molecule will have a longer wavelength (or be of lower energy) than the radiation originally absorbed.

• Fluorescent light is emitted very quickly by the excited molecule as it gives up its trapped energy and returns to a more stable state.

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Working of FM

• Fluorescence microscope exposes a specimen : ultraviolet, violet, or blue light and forms an image of the object with the resulting fluorescent light.

• Mercury vapor arc lamp or other source produces an intense beam

• Heat transfer is limited by a special infrared filter.

• The light passes through an exciter filter that transmits only the desired wavelength. A darkfield condenser provides a black background against which the fluorescent objects glow.

• Usually the specimens have been stained with dye molecules, called fluorochromes, that fluoresce brightly upon exposure to light of a specific wavelength, but some microorganisms are auto fluorescing. The microscope forms an image of the fluorochrome-labeled microorganisms

• A barrier filter positioned after the objective lenses removes any remaining

• ultraviolet light, which could damage the viewer’s eyes, or blue and violet light, which would reduce the image’s contrast.

Applications of FM• The fluorescence microscope has become an essential tool is as follow:

• Medical microbiology andmicrobial ecology.

• Bacterial pathogens (e.g., Mycobacterium tuberculosis, the cause oftuberculosis) can be identified after staining them with fluorochromes orspecifically labeling them with fluorescent antibodies usingimmunofluorescence procedures.

• The stained organisms will fluoresce orange or green and can be detected evenin the midst of other particulate material. It is even possible to distinguish livebacteria from dead bacteria by the color they fluoresce after treatmentwith a special mixture of stains

• Thus the microorganisms can be viewed and directly counted in arelatively undisturbed ecological niche.

Example of FM

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Basic Microscope TechniqueRules to Follow

1. If you must carry a microscope, always hold it with one hand on the arm and the other under the base.

2. Always lower the stage or raise the objectives all the way before placing a slide under the objectives.

3. Always begin working with the LOW POWER (shortest) objective first.

4. Observe the slide from the side, not looking through the eye piece, when using the coarse focus to avoid running the objective lens into the slide.

5. Never use the coarse focus adjustment when on the medium or high power objectives. Focus on low power first and then rotate the higher power objective into place. Make final focus adjustments with the fine focus adjustment.

Electron Microscope

• Transmission Electron Microscope

• Scanning microscope

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Transmission Electron Microscope• Limitation of light microscope: resolution limit of about 0.2 micrometer.

bacteria usually are around 1 micrometer in diameter,

• General shape and major morphological features are visible in the light microscope.

• The detailed internal structure of larger microorganisms also cannot be effectively studied by light microscopy.

• These limitations arise from the nature of visible light waves, not from any inadequacy of the light microscope

• itself.

Sample preparation on TEM

1 step

• Thickness: 20 to 100 nm

• 1⁄50 to 1⁄10 the diameter of a typical bacterium

2 step• Thin section required some support like plastic

3 step

• Fixation with chemical like glutaraldehyde or osmium tetroxide to stabilize cell struvtutr

4 step• Dehydrated with organic solvent( aceton or ethanol)

5 step

• specimen is soaked in unpolymerized, liquid epoxy plastic until it is completely permeated, and then the plastic is hardened to form a solid block.

6 step

• Thin sections are cut from this block with a glass or diamond knife using a special instrument called an ultramicrotome.

7 step• Cells usually must be stained before they can be seen clearly in the bright-field microscope;

8 step

• prepared for observation by soaking thin sections with solutions of heavy metal salts like lead citrate and uranyl acetate.

9 step

• The lead and uranium ions bind to cell structures and make them more electron opaque, thus increasing contrast in the material. Heavy osmium atoms from the osmium tetroxide fixative also “stain” cells and increase theircontrast.

9 step

• The lead and uranium ions bind to cell structures and make them more electron opaque, thus increasing contrast in the material. Heavy osmium atoms from the osmium tetroxide fixative also “stain” cells and increase their contrast.

10 step• The stained thin sections are then mounted on tiny copper grids and viewed

Working of TEM• Tungsten filament: generates a beam of

electrons that is then focused on the specimen by the condenser

• Magnetic lenses: are used to focus the beam

• The column containing the lenses and specimen must be under high vacuum to obtain a clear image because electrons are deflected by collisions with air molecules.

• Magnetic lenses : Form the Enlarged, visible image of the specimen on a fluorescent screen.

• Photographic film: The screen can also be moved aside and the image captured on photographic film as a permanent record.

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Images of TEM

Specimen Shadowing for the TEM. Examples ofspecimens viewed in the TEM after shadowing with uranium metal. (a) Proteus mirabilis (42,750); note flagella and fimbriae. (b) T4 coliphage (72,000). 9

Characteristics of TEM and LM

Scanning microscope

• Specification of SM:

• Examine the surfaces of microorganisms

• Principle: “The SEM differs from other electron microscopes in producing an image from electrons emitted by an object’s surface rather than from transmitted electrons”.

• Specimen preparation is easy,

• Air-dried material can be examined directly.

Method and sample preparation

1 step•Fixation

2 step•Dehydrated the cell

3 step•Dried to preserve surface structure and prevent collapse of the cells when they are exposed to the SEM’s high vacuum.

4 step

•Before viewing, dried samples are mounted and coated with a thin layer of metal to prevent the buildup of an electrical charge on the surface and to give a better image.

5 step

•specimen is soaked in unpolymerized, liquid epoxy plastic until it is completely permeated, and then the plastic is hardened to form a solid block.

6 step•The SEM scans a narrow, tapered electron beam back and forth over the specimen

7 step•When the beam strikes a particular area, surface atoms discharge a tiny shower of electrons called secondary electrons.

8 step•trapped by a special detector.

9 step

•Secondary electrons entering the detector strike a scintillator causing it to emit light flashes that a photomultiplier converts to an electrical current and amplifies.

Working of SEM

Scanning Electron Micrographs of Bacteria. (a) Staphylococcus aureus(32,000). (b) Cristispira, a spirochete from the crystalline

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Why Black and white image?

• The number of secondary electrons reaching the detector depends on the nature of the specimen’s surface. When the electron beam strikes a raised area, a large number of secondary electrons enter the detector.

• In contrast, fewer electrons escape a depression in the surface and reach the detector.

• Thus raised areas appear lighter on the screen and depressions are darker.

• A realistic three-dimensional image of the microorganism’s surface with great depth of focus results.

• The actual in situ location of microorganisms in ecological niches such as the human skin and the lining of the gut also can be examined

Application of SEM

• Human skin and the lining of the gut also can be examined.

• Fungal hyphal structures can be studied.

Why Stain Cells?

• The most basic reason that:

– Enhance visualization of the cell or certain cellular components under a microscope.

– Cells may also be stained to highlight metabolic processes or to differentiate between live and dead cells in a sample.

– Cells may also be enumerated by staining cells to determine biomass in an environment of interest.

How Are Cells Stained and Slides Prepared?

• Cell staining techniques and preparation depend on the type of stain and analysis used. One or more of the following procedures may be required to prepare a sample:

– Smear preparation

– Permeabilization

– Fixation

– Mounting

– Staining

Smears and Staining

• Bacteria must be stained (dyed) so they can be seen with the microscope

• Before staining a smear must be made

• A smear is just a film of bacteria on a glass slide

• After the smear dries it is heat fixed, this– Kills the bacteria

– Helps adhere the cells to the slide

– Makes the cells more receptive to the dye

Stains

• Stains are dyes

• Stains carry either a positive charge (basic dyes) or a negative charge (acidic dyes)

• Bacteria typically carry a slight negative charge on the cell surface so they attract a basic dye

• Most of the stains used in the lab are basic dyes

• A negative stain uses acidic dyes that do not stain the cell but rather the background

1. Basic dyes—methylene blue, basic fuchsin, crystal violet, safranin, malachite

green—have positively charged groups (usually some form of pentavalent nitrogen) and are generally sold as chloride salts. Basic dyes bind to negatively charged molecules like nucleic acids and many proteins. Because the surfaces of bacterial cells also are negatively charged, basic dyes are most often used in bacteriology.

2. Acid dyes—eosin, rose bengal, and acid fuchsin—possess negatively

charged groups such as carboxyls (— COOH) and phenolic hydroxyls (—OH). Acid dyes, because of their negative charge, bind to positively charged cell structures.

Staining Techniques

• Simple Stain– Uses only one basic dye

– Provides basic information about cell shape and arrangement

• Differential Stain– Uses more than one dye

– These procedures react differently with different kinds of bacteria

– Helps distinguish between different kinds of bacteria

– Most common and important differential stain is the GRAM STAIN

Gram Stain

• Most important differential staining technique

• Differentiates all bacteria based on cell wall composition

• Bacteria are either Gram + and stain blue or Gram- and stain red

• Gram stain is usually the first step in identifying an unknown bacteria

Gram Stain

Gram stain

Acid-fast Stain

• Differential stain

• Identifies bacteria with MYCOLIC ACID in their cell walls

• Very important human pathogens that can be identified with this stain is Mycobacterium tuberculosis

• All members of genus Mycobacterium are acid-fast

Acid-fast Stain

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Special stains

• Negative stain

• Acidic dye stains background, not cell

• Used to determine cell shape and size

• Spore stain

• Used to identify bacteria that can form spores

Special Stains

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References

Images:1. http://www.scienceheritage.com/wp-

content/uploads/wpsc/productimages/grandm.jpg2. http://micro.magnet.fsu.edu/primer/anatomy/introduction.html3. http://www.recyclind.it/dati/pagine/immagine_201210192337.jpg4. https://s10.lite.msu.edu/res/msu/botonl/b_online/library/biology107/terry/images/

micro/resolvpower.gif5. http://www.olympusmicro.com/primer/techniques/polarized/images/polmicroalign

mentfigure1.jpg6. http://www.microscopemaster.com/images/cm501microscopediagram.jpg7. http://www.microscope-microscope.org/images/BWScope.jpg8. http://www.scienceprofonline.com/images/science-image-

library/microbiology/equipment/microscope-objective-lenses-SIL.jpg9. http://classroom.sdmesa.edu/eschmid/Lecture2-Microbio.htm10. https://classconnection.s3.amazonaws.com/553/flashcards/661553/jpg/picture1132

8380497708.jpg11. http://microbialworld.blog.com/files/2011/09/P.png12. http://php.med.unsw.edu.au/cellbiology/images/0/0d/Phase_contrast_microscopy.j

pg

13.http://www.dermpedia.org/files/images/histioid_leprosy_Fite_oil.jpg

14.https://lh6.ggpht.com/PZTVqr-v7Set_4U8p5il7UU-yrRPMENYClH9-jc1MZYbw0miu6xFdoVo6RvfE-hrdjsEhA=s117

Books:

1. Microbiology by pelczar

References

Thank You