Microbiology lab 2

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MICROSCOPY

A Brief Overview

Dr Saleh M.Y.

Practical Lab Experiment -1-

MBBS-Phase II

19/10/2010

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- Introduction

- Definition

- Historical Background

- Varibles Used In Microscopy

- Compound Microscope - Structure And Function

- Use Of Microscope

- Various Types Of Microscopes

- Care Of Microscope

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INTRODUCTION TO

MICROSCOPY

- Understanding the

optical principles and

construction of

microscopes

- Role of microscopy

- Microscopic

techniques and

application

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DEFINITION

- A microscope (Greek: micron = small and scopos =

to look)

- MICROSCOPE: Is an instrument for viewing

objects that are too small to be seen by the naked

or unaided eye

- MICROSCOPY: The science of investigating

small objects using such an instrument is called

microscopy

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HISTORICAL BACKGROUND

1590 - Hans Janssen and his son Zacharias Janssen,

developed first microscope.

1609 - Galileo Galilei - occhiolino or compound

microscope.

1620 - Christian Huygens, another Dutchman,

developed a simple 2-lens ocular system that was

chromatically corrected.

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Antony van Leeuwenhoek

(1632-1723)- Anton van Leeuwenhoek is

generally credited with bringing

the microscope to the attention of

biologists.

- A tradesman of Delft, Holland.

1661 - He discovered bacteria, free-living and

parasitic microscopic protists, sperm cells, blood

cells, microscopic nematodes etc.

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Microscope used by

Anton von Leeuwenhoek

An old pocket Microscope

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VARIABLES

USED IN

MICROSCOPY

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MAGNIFICATION

Degree of enlargement

No of times the length, breadth or diameter,

of an object is multiplied.

MAGNIFICATION VS SHARPNESS

USEFUL MAGNIFICATION AND EMPTY

MAGNIFICATION

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RESOLUTION: Ability to reveal closely

adjacent structural details as separate and

distinct

LIMIT OF RESOLUTION (LR): The min

distance between two visible bodies at

which they can be seen as separate and

not in contact with each other

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Types of microscope Resolving power

Compound Microscope 200 nanometers

Scanning Electron Microscope 10 nanometers

Transmission Electron Microscope 0.2 nanometers

LR = 0.61 x W W = Wavelength

NA = Num apertureNA

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NUMERICAL APERTURE(NA)

- Ratio of diameter of lens to its focal length

- NA = n Sin θ/2

n = refractive index,

θ = angle of aperture (CAD)

θ/2

A

B

DC

n of air = 1

n of oil = 1.5

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DEFINITION

- Capacity of an objective to render outline

of the image of an object clear and distinct

- Depends of elimination of Spherical and

Chromatic aberration

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ABERRATION

- Chromatic aberration

- Correction of aberration – Achromatic

objective and Apochromatic objectives.

Blue focusRed focus

Incident

light

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Spherical aberration

Focus of marginal rays

Focus of axial rays

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TYPES OF MICROSCOPE

- Simple microscope

- Compound microscope

- Phase Contrast

Microscope

- Dark Ground Microscope

- Fluorescent Microscope

- Electron Microscope

- Others

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COMPOUND

MICROSCOPE

Compound microscope made

by John Cuff 1750

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PARTS OF COMPOUND MICROSCOPE

- Ocular (Eye piece)

- Body or Tube

- Coarse focusing knob

- Fine focusing knob

- Objective Lens

- Movable stage

- Condenser Lenses

- Field (Iris) Diaphragm

- Mirror and light source

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OBJECTIVE LENS

It forms magnified real image.

• Mounted on Nose piece

• Magnification of objective

= Optical Tube length

Focal Length

• Scan - 4X

• Low Power - 10X

• High Power - 40X

• Oil immersion - 100X

TYPES

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OIL IMMERSION OBJECTIVE

- Highest magnification

- Oil prevents refraction of light outwards and

allows it to pass straight in to objective

GLASS

OIL

AB

C

D

E

G

F

FBEG - OILABCD - AIR

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EYE PIECE

- Forms magnified virtual & erect image

- TYPES

(a) Monocular

(b) Binocular

(c) Trinocular

or

(a) Huygenian

(b) Ramsden

(c) Compensating

What’s the power of this lens?

To calculate the power of magnification, multiply the power of the

ocular lens by the power of the objective, e..g.: 10x40=400 times

What are the powers of

magnification for each

of

the objectives we have

on our microscopes?

Fill in the table on

your worksheet.

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Comparing Powers of Magnification

Which of these images

would be viewed at a

higher power of

magnification?

We can see better details with higher the powers of

magnification, but we cannot see as much of the image.

10x 40x

Let’s give it a try ...1 – Turn on the microscope and then rotate the nosepiece to click the

red-banded objective into place.

2 – Place a slide on the stage and secure it using the stage clips. Use

the coarse adjustment knob (large knob) to get it the image into view

and then use the fine adjustment knob (small knob) to make it

clearer.

4 – When you are done, turn off the microscope and put up the

slides you used.

3 – Once you have the image in view, rotate the nosepiece to view it

under different powers. Draw what you see on your worksheet!

Be careful with the largest objective! Sometimes there is

not enough room and you will not be able to use it!

How to make a wet-mount slide …

1 – Get a clean slide and coverslip from your teacher.

2 – Place ONE drop of water in the middle of the slide. Don’t use

too much or the water will run off the edge and make a mess!

3 – Place the edge of the cover slip on one side of the water drop.

You do not need to use the stage clips

when viewing wet-mount slides!

5 – Place the slide on the stage and view it first with the red-banded

objective. Once you see the image, you can rotate the nosepiece to

view the slide with the different objectives.

4 - Slowly lower the cover slip on top of the drop.

Cover

SlipLower slowly

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ILLUMINATION - Lamp,

sunlight, battery operated

lamp, 60 W bulb, Quartz

halogen light.

FILTERS - Blue, Green,

Heat absorbing filters,

Barrier filters.

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Multiple step operation employed to attain

optimal illumination:

1. Remove any diffusing filter.

2. Put a slide on the stage and focus.

3. Completely close the field diaphragm.

4. Move the condenser until the border

or the iris hexagon is neat and clear.

5. Center if necessary.

6. Open the field diaphragm until the tip

of the hexagon touches the field limit

KOHLER’S ILLUMINATION

The Parts of a Microscope

Body Tube

Objective

Lenses

Stage

Clips

Diaphragm

Light Source

Ocular Lens

Arm

Stage

Coarse Adj

Fine Adjustment

Base

Nose Piece

The Parts of a Microscope

Body Tube

The body tube holds the objective lenses

and the ocular lens at the proper distance

Diagram

Nose Piece

The Nose Piece holds the objective lenses

and can be turned to increase the

magnification

Diagram

Objective Lenses

The Objective Lenses increase

magnification (usually from 10x to 40x)

Diagram

Stage Clips

These 2 clips hold the slide/specimen in

place on the stage.

Diagram

Diaphragm

The Diaphragm controls the amount of light

on the slide/specimen

Turn to let more light in or to

make dimmer.

Diagram

Light Source

Projects light upwards through the

diaphragm, the specimen and the lenses

Some have lights, others have mirrors

where you must move the mirror to reflect

light

Diagram

Ocular Lens/Eyepiece

Magnifies the specimen image

Diagram

Arm

Used to support the microscope when

carried. Holds the body tube, nose piece

and objective lenses

Diagram

Stage

Supports the slide/specimen

Diagram

Coarse Adjustment Knob

Moves the stage up and down (quickly) for

focusing your image

Diagram

Fine Adjustment Knob

This knob moves the stage SLIGHTLY to

sharpen the image

Diagram

Base

Supports the microscope

Diagram

Magnification

To determine your magnification…you

just multiply the ocular lens by the

objective lens

Ocular 10x Objective 40x:10 x 40 = 400

Objective Lens have

their magnification

written on them.

Ocular lenses usually magnifies by 10x

So the object is 400 times “larger”

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HOW A MICROSCOPE WORKS ?

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OPTICAL PATH IN COMPOUND

MICROSCOPE

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Method of using Compound

Microscope

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1. Grasp the microscopes arm with

one hand and place your other

hand under the base.

2. Place the microscope on a bench.

Adjust seat

3. Clean Lenses.

4. Turn the coarse adjustment knob

to raise the body tube

armarm

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5. Revolve the nose piece to set low-power objective lens.

6. Adjust the Condenser lenses and diaphragm .

7. Place a slide on the stage and secure with stage clips.

8. Switch on the light at low intensity and then increase intensity.

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9. Turn the coarse adjustment knob to lower the body tube until the low power objective reaches its lowest point.

10. Looking through the eyepiece, very slowly move

the coarse adjustment knob until the specimen

comes into focus.

11. Adjust distance between eye piece.

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12. Switch to the high power objective lens only

after adjusting condenser and iris diaphragm.

13. Place a drop of oil over specimen before using

oil immersion objective.

14. Lower the objective until oil makes contact with

objective.

15. Looking through the eyepiece, very slowly

focus the objective away from the slide i.e by

raising the objective lens.

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How to observe a slide ?

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Causes of Error in Focusing

- Revolving Nose Piece is off centre

- Preparation is upside down

- Thick cover slip

- Dirt or Dried oil over Lens

- Air bubble in immersion oil

- Poor illumination – Condenser not fully

racked up

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PHASE CONTRAST MICROSCOPE

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Phase Contrast Microscope

- First described in 1934 by Dutch physicist

Frits Zernike

- Produces high-contrast images of

transparent specimens

- Advantage - Living cells can be examined

in their natural state

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Principle Of Phase Contrast

Microscopy

- Unstained bacteria have constituents of

different refractive index .

- Diffraction of light

- Phase contrast microscope employs an

optical mechanism to translate minute

variations in phase into corresponding

changes in intensity of image.

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Requisite For Phase Contrast

Microscopy

- Annular Diaphragm

- Phase Plate

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Condenser Annulus

- The condenser annulus or annular

diaphragm is opaque flat-black (light

absorbing) plate with a transparent

annular ring.

- Produces hollow cone of light.

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Condenser

annulus

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Phase Plate

- Placed in back focal plane of objective.

- Function:

1. Enhances phase difference by retarding diffracted wave front by one quarter of wavelength .

2. Reduces intensity of direct rays and equalizes it with diffracted rays intensity.

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Phase plate

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Images of Phase Contrast

Microscpy

Clostridium botulinumSpirilium volutans

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Comparision of Images of Bright Field

and Phase Contrast Microscopy

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Uses of Phase Contrast Microscopy

- Phase contrast enables visualization of

internal cellular components.

- Diagnosis of tumor cells .

- Examination of growth, dynamics, and

behavior of a wide variety of living cells in

cell culture

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Dark Ground Microscope

- Optical system to enhance the contrast of

unstained bodies .

- Specimen appears gleaming bright against

dark background

PRINCIPLE OF DGI

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Optical path in Dark Ground

Microscopy

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Requisites for Dark Ground

Microscopy

- Dark ground

condenser

- High intensity

lamp

- Funnel stop

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Uses of Dark Ground Microscopy

Treponema pallidum

Useful in demonstrating

-Treponema pallidum

- Leptospira

- Campylobacter jejuni

- Endospore

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Fluorescence Microscopy

PRINCIPLE

UV light

Fluorochrome

Visible

radiation

FITC EX - 495 nm EM - 520nm

TRITC EX – 540 nm EM – 590 nm

Texas Red Ex – 600 nm EM – 615 nm

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- UV rays passes through exciter filter

- Dark ground condenser

- Micro organisms stained with fluorescent

dye, when examined under microscope

with ultraviolet light are seen as bright

object against dark background

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Use of Fluorescence Microscopy

- Auramine Rhodamine – Yellow

Fl - Tubercle bacilli

- Acridine Orange R - gives

orange red Fl with RNA and

yellow green Fl with DNA

- QBC

- IMMUNOFLUORESCENCE

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Electron

Microscope

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ELECTRON MICROSCOPE

-Electron Microscopes uses a beam of highly

energetic electrons to examine objects on a

very fine scale. This examination can yield the

info about

- Topography

- Morphology

- Composition

- Crystallographic structure

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TYPES OF EM

- Transmission Electron Microscope (TEM)

- Scanning Electron Microscope (SEM)

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Transmission Electron

Microscope (TEM)

- Stream of electrons is formed

- Accelerated using a positive electrical potential

- Focused by metallic aperture and Electro magnets

-Interactions occur inside the irradiated sample which are detected and transformed into an image .

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TEM (Cont)

-Projector Lens forms

image on Fluorescent

viewing screen

- 2D Image

- Magnification

10,000 X to 100,000 X

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- Scan a gold-plated specimen to give a 3-D

view of the surface of an object which is

black and white.

- Used to study surface features of cells and

viruses.

- Scanning Electron microscope has resolution

1000 times better than Light microscope .

Scanning Electron Microscope

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Working of SEM

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SEM IMAGES

Vibrio cholerae with

polar flagellaTreponema pallidum

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INVERTED MICROSCOPE

- Used in metallurgy

- Examination of cultures in flat bottom

dishes

- Micro dissection

- Examination of parasites

- Observation of agglutination in serology

OTHER MICROSCOPES

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STEREO MICROSCOPE- Double Microscope

- Produces 3D images

POLARIZING MICROSCOPE

- Uses two Polariser

- Gives info about Birefringence of a body

- Used in Crystallography, Urine

examination

- Apple Green Birefringerence in

AMYLODOSIS

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- Uses a laser beam to illuminate

a specimen whose image is

then digitally enhanced for

viewing on a computer monitor.

- Laser beam scans single plane

of 1µm thickness.

CONFOCAL SCANNING

LASER MICROSCOPE

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Comparison of Depth of Light

Collection and Image clarity

Light Microscope Confocal Scanning

Laser Microscope

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PRINCIPLE OF CONFOCAL MICROSCOPY

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USES OF CONFOCAL

MICROSCOPE

- Observing cellular morphology in

multilayered specimen

- Eg. used in diagnosing Ca cervix

- Evaluation and diagnosis of basal cell

carcinoma of skin

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What is the advantage of using a

confocal microscope?

- By having a confocal pinhole, the

microscope is really efficient at rejecting

out of focus fluorescent light so that very

thin section of a sample can be analyzed.

- By scanning many thin sections through a

sample, one can build up a very clean

three-dimensional image .

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NEWER MICROSCOPE

- SCANNING PROBE MICROSCOPE -

Class of Microscope that measures surface

features by moving a sharp probe over

object surface. Used to visualize atoms and

molecules

- Scanning Tunneling Microscope

(STM)

- Atomic Force Microscope (AFM)

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CARE OF THE MICROSCOPE

- Handling

- Proper storage

- Care of Lenses

- Care of oil emersion objective

- Care of lamp

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