Cellular BiochemistryUnit 4

Basic techniques in Microbiology

B.Sc Biochemistry II

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

Why Microscope,,,

1

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

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.2

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.

Light microscope

• Light microscope uses diffused light from

the sun or artificial light to illuminate the

object to be observed.

Types of Microscope

• Types of microscope.

Light microscopeBright field microscope

The Dark-Field Microscope

The Phase-Contrast Microscope

The Fluorescence Microscope

Electron microscopeScanning Electron microscope

Transmission Electron Microscope

Light Microscope

3

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

1.Arm

• Supports the body

tube.

4

2. Base

• Supports and

stabilizes the

microscope.

5

3. Eyepiece / ocular

lens

• Magnifies image

produced

by objective lens.

6

4. Body Tube

• Maintains the

proper distance

between the

objective and

ocular lenses.

7

• Moves stage up

and down

a large amount for

FOCUSING.

5. Coarse

adjustment knob

8

• small, round knob on

the side of the

microscope used to

fine-tune the focus of

your specimen

• after using the coarse

adjustment knob

6. Fine adjustment

knob

8

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.

10

• Stage clips

- hold the slide in

place.

• Stage

- Supports the slide

being viewed.

9. Stage and stage

clips

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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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• 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.

10. Objective lenses

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

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Focus only with fine

focus. Hopefully, the

specimen will come

into focus easily. Do

not change focus

dramatically.

High power objective lenses

• Rotates to allow

use of

different power

objectives.

11. Revolving

nosepiece

• Supports the arm

and

controls the body

of the

microscope.

12. Inclination

joint

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.

Microscope Vocabulary

• Magnification: increase of an object’s apparent size

• Resolution: The limit up to which two small objects are

still seen as separate entities is used as a measure of

the resolving power of a microscope. The distance

where this limit is reached is known as the effective

resolution of the microscope

• power to show details clearly

Both are needed to see a clear image

. 30

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

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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

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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

Working Distance

•working distance— Distance between the front surface of

lens and surface of cover glass or

specimen

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.

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.15

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 and microbial 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 distinguishlive bacteria from dead bacteria by the color they fluoresce aftertreatment with a special mixture of stains

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

Basic Microscope Technique

Rules to Follow1. 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• Size of object: about 0.2 micro meter

• Object examine: viruses or the internal structures of cells

• Sources : Electrons is used instead of light.

• Resolving power: Greater than that of the other microscopes

• Images: Black and white, but they may be colored artificially to accentuate

certain details.

• Glasses: Electromagnetic lenses to focu s a beam of electrons onto a

specimen.

• Types of electron microscopes: Transmission electron microscope and

the scanning electron microscope.

Transmission Electron Microscope

• Limitation of light microscope: resolution limit of about 0.2

micrometer. The 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 tetroxidefixative 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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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.

What is SEM?

It is a microscope that produces an image by using

an electron beam that scans the surface of a

specimen inside a vacuum chamber.

The SEM is designed for direct studying of the surfaces

of solid objects.

Scanning electron microscope (SEM) is a microscope

that uses electrons rather than light to form an image.

There are many advantages to using the SEM instead of a

OM.

Scanning Electron Microscope– a Totally Different Imaging Concept

Instead of using the full-field image, a point-to-

point measurement strategy is used.

High energy electron beam is used to excite the

specimen and the signals are collected and

analyzed so that an image can be constructed.

The signals carry topological, chemical and

crystallographic information, respectively, of the

samples surface.

HOW THE SEM WORKS?

The SEM uses electrons instead of light to form an

image.

A beam of electrons is produced at the top of the

microscope by heating of a metallic filament.

The electron beam follows a vertical path through

the column of the microscope. It makes its way through

electromagnetic lenses which focus and direct the

beam down towards the sample.

Once it hits the sample, other electrons

( backscattered or secondary ) are ejected from the

sample. Detectors collect the secondary or

backscattered electrons, and convert them to a signal

that is sent to a viewing screen similar to the one in an

ordinary television or computer.

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Secondary electrons (SE)

Generated from the collision between the incoming electrons and the loosely bonded outer electrons

Low energy electrons (~10-50 eV)

Only SE generated close to surface escape (topographic information is obtained)

Number of SE is greater than the number of incoming electrons

We differentiate between SE1 and SE2

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SE1 The secondary electrons that are generated by the

incoming electron beam as they enter the surface

High resolution signal with a resolution which is only

limited by the electron beam diameter

SE2

The secondary electrons that are

generated by the backscattered

electrons that have returned to the

surface after several inelastic

scattering events

SE2 come from a surface area that

is bigger than the spot from the

incoming electrons resolution is

poorer than for SE1 exclusively

Sample

surface

Incoming electronsSE2

Backscattered electrons (BSE)

A fraction of the incident electrons is

retarded by the electro-magnetic field of

the nucleus and if the scattering angle

is greater than 180° the electron can

escape from the surface

High energy electrons (elastic

scattering)

Fewer BSE than SE

We differentiate between BSE1 and

BSE2

How do we get an image?

Image

Detector

Electron gun

Signals from the sample

Incoming electrons

Secondary electrons

Backscattered

electrons

Auger electrons

X-rays

Cathod-

luminescence (light)

Sample

How an Electron Beam is Produced?

Electron guns are used to produce a fine, controlled beam of electrons which are then focused at the specimen surface.

The electron guns may either be

thermionic gun or field-emission gun

Some comments on resolution

Best resolution that can be obtained: size of the electron spot on the sample surface The introduction of FEG has dramatically improved the

resolution of SEM’s

The volume from which the signal electrons are formed defines the resolution SE image has higher resolution than a BSE image

Scanning speed: a weak signal requires slow speed to improve signal-to-

noise ratio

when doing a slow scan drift in the electron beam can affect the accuracy of the analysis

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.

Flowcytometry

Sorting the Cells

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Intro

• Flow cytometry is a laser-based, biophysical technology employed majorly in cell counting and cell sorting.

• By suspending cells in a stream of fluid and passing them by an electronic detection apparatus;

• It allows simultaneous multiparametricanalysis of the physical and chemical characteristics of up to thousands of particles per second.

61

• A flow cytometer is similar to

a microscope, except that, instead of

producing an image of the cell, flow

cytometry offers "high-throughput" (for a

large number of cells)

automated quantification of set

parameters.

• For analysis, a single-cell suspension

must first be prepared.62

History• The first impedance-based flow cytometry device,

using the Coulter principle, US patent 1953, by Wallace H. Coulter.

• The Coulter Counter is a vital constituent of today's hospital laboratory.

• Its primary function being the quick and accurate analysis of complete blood counts (often referred to as CBC). The CBC is used to determine the number or proportion of white and red blood cells in the body.

• Previously, this procedure involved preparing a blood cell stain and manually counting each type of cell under a microscope.

63

Five Components• A flow cytometer has five main components:

1. a flow cell - liquid stream (sheath fluid), which carries and aligns the cells so that they pass single file through the light beam for sensing

2. a measuring system - commonly used are measurement of impedance (or conductivity) and optical systems - lamps (mercury, xenon); high-power lasers (argon, krypton, dye laser); or diode lasers (blue, green, red, violet) resulting in light signals

3. a detector and Analogue-to-Digital Conversion (ADC) system - which generates FSC and SSC as well as fluorescence signals from light into electrical signals that can be processed by a computer

4. an amplification system

5. a computer for analysis of the signals

64

Fluorescence-activated cell sorting

(FACS)

• FACS is a specialized type of flow cytometry

• It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell.

• It is a useful scientific instrument as it provides fast, objective and quantitative recording of fluorescent signals from individual cells.

• Physical separation of cells of particular interest.

65

66

19

• The cell suspension is entrained in the center

of a narrow, rapidly flowing stream of liquid.

• A vibrating mechanism causes the stream of

cells to break into individual droplets.

• The system is adjusted so that there is a low

probability of more than one cell per droplet.

• The flow passes through a fluorescence

measuring station where the fluorescent

character of interest of each cell is measured.

67

• The charge is then provided by an

electrical charging ring based on the

immediately prior fluorescence intensity

measurement as it breaks from the

stream.

• The charged droplets then fall through an

electrostatic deflection system that diverts

droplets into containers based upon their

charge.68

69

20

Applications

• The technology has applications in a number of fields, including medicine, molecular biology, pathology, immunology, plant biology and marine biology.

• Flow cytometry is routinely used in the diagnosis of health disorders, especially blood cancers, but has many other applications in basic research, clinical practice and clinical trials.

• A common variation is to physically sort particles based on their properties, so as to purify populations of interest.

70

Measurable parametersThis list is very long and constantly expanding,

• used for confirming diagnosis of chronic lymphocytic leukemia

• volume and morphological complexity of cells

• cell pigments such as chlorophyll or phycoerythrin

• total DNA content (cell cycle analysis, cell kinetics, proliferation, ploidy, aneuploidy, endoreduplication, etc.)

• total RNA content

• DNA copy number variation (by Flow-FISH or BACs-on-Beads technology)

• chromosome analysis and sorting (library construction, chromosome paint)

• protein expression and localization

71

• Protein modifications, phospho-proteins

• transgenic products in vivo, particularly the Green fluorescent protein or

related Fluorescent Proteins

• cell surface antigens (Cluster of differentiation (CD) markers)

• intracellular antigens (various cytokines, secondary mediators, etc.)

• nuclear antigens

• enzymatic activity

• pH, intracellular ionized calcium, magnesium, membrane potential

• membrane fluidity

• apoptosis (quantification, measurement of DNA degradation, mitochondrial

membrane potential, permeability changes, caspase activity)

• cell viability

• monitoring electropermeabilization of cells

• characterising multidrug resistance (MDR) in cancer cells

• cell adherence (for instance pathogen-host cell adherence)

References • Images references:

1. https://www.gotoknow.org/posts/105470

2. http://polaros.com/fulminant-community-acquired-acinetobacter-baumannii-pneumonia-as-a-

distinct-clinical-syndrome-definition-of-terms.html

3. http://classroom.sdmesa.edu/eschmid/Lecture2-Microbio.htm

4-12. http://www.microscope-microscope.org/basic/microscope-parts.htm

13. https://lh5.ggpht.com/cpmDJKCYbRS3m-

1aZ4sebX0N8CVLomv91vEWuz3o1fgDbta2PRzWdypNFK_PepEUDh2d=s94

14.https://lh3.ggpht.com/_o1NDOt21DJ6iRN_HXPvlD33VgW9TgI4R8S1h8ZM5SJV6zTEymBszraZ8

Uw9WaJc_uZx_g=s85

15.https://lh3.ggpht.com/_o1NDOt21DJ6iRN_HXPvlD33VgW9TgI4R8S1h8ZM5SJV6zTEymBszraZ8

Uw9WaJc_uZx_g=s85

16.https://lh4.ggpht.com/rkbrNOAx19H0eYFDzacAQDb4uJJ3Iir6EdnOPFJaV_tt0XwCM6F658klSf9C

G8VOwZFd=s85

17.https://lh4.ggpht.com/H139WLhETH5qmKCJN4klUnXKqK8JRO1mmxSquqPXiXYIK1OVlucstd_

NjC7hsJ7zPbaoQw=s85

18.https://lh3.ggpht.com/x9ZN7zdwK4DtGWVV1_3d2Whjp2BQG8iaHAsMWOZ9mePvXsj3zUhS5k6

0LblAlbJ4Nz4qBA=s85

19. https://lh5.ggpht.com/2QC5nq87J8qpEdrdn6XJa-

H5jpDMSY7Z2JxU6JruwVIeAbP64DvFYGiiauwYnlkXd4HgxS8=s108

20. http://en.wikipedia.org/wiki/Flow_cytometry

• Reading references:

• Cell and Molecular Biology, 6th Ed By Karp

• Molecular Cell Biology by Lodish 5th Edition