Microscopy

1

Course: B.Sc. Biochemistry

Sem II

Sub: Biophysics and Instrumentation

Unit 4

Replica of Marshall

Microscope, c. 1700, by John

Marshall (1663-1725).

1.

Why Microscope

2.

History of the Microscope

• 1590 –first compound

microscope

Discovery of

Microorganisms.

Anton van

Leeuwenhoek (1632-

1723)

– first person to

observe and describe

micro-organisms

accurately

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3.

Discovered by Robert Hooke,

1665 and Observed cells of

corkCurrent Tools

• Light Microscope

• Electron Microscope

• Atomic Force

Microscope (AFM)

• Scanning Tunnelling

Microscope (STM)

IMAGING

4.

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.

5.

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.

6.

Light microscope

• Light microscope uses diffused light from

the sun or artificial light to illuminate the

object to be observed.

7.

Types of Microscope

• Types of microscope.

Light microscopeBright field microscope

The Dark-Field Microscope

The Phase-Contrast Microscope

The Fluorescence Microscope

Scanning and tunneling electron microscope

Light Microscope

8.

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?

9

1.Arm

• Supports the body

tube.

10.

2. Base

• Supports and

stabilizes the

microscope.

11.

3. Eyepiece / ocular

lens

• Magnifies image

produced

by objective lens.

12.

4. Body Tube

• Maintains the

proper distance

between the

objective and

ocular lenses.

13.

• Moves stage up

and down

a large amount for

FOCUSING.

5. Coarse

adjustment knob

14.

• 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

15.

7. Light source

• (lamp or mirror) Provides

light for viewing the slide.

• Projects light UPWARDS

through the diaphragm,

the SPECIMEN, and

the LENSES.

16.

8. Diaphragm

• Controls the

amount of

light passing

through

the slide.

• Stage clips

- hold the slide in

place.

• Stage

- Supports the slide

being viewed.

9. Stage and stage

clips

Lens

Objective lens Condenser Lens

Usually you will find 3 or 4 objective lenses on

a microscope

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

When coupled with a 10X (most common)

eyepiece lens, we get total magnifications of

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).

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 10X, 40X

and 100X powers. When

coupled with a 10X (most

common)

17.

• 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. 18.

19.

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

20.

Focus only with fine

focus. Hopefully, the

specimen will come

into focus easily. Do

not change focus

dramatically.

High power objective lenses

21.

• Rotates to allow

use of

different power

objectives.

11. Revolving

nosepiece

22.

• Supports the arm

and

controls the body

of the

microscope.

12. Inclination

joint

23.

Microscope Vocabulary

• Magnification: increase of an object’s

apparent size

• Resolution: power to show details clearly

Both are needed to see a clear image

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.

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

24.

Gram stain

25.

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

26.

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

27.

28.

Image References

1.http://saxonianfolkways.wordpress.com/page/16/

2. quizlet.com/20103976/test-2-flash-cards/

3. www.visioneng.com/resources/history-of-the-microscope

4.commons.wikimedia.org/wiki/File:Hooke_Microscope.jpg

5. www.acinetobacterbaumannii.org/

6. en.wikipedia.org/wiki/Resolving_power

7.http://www.trekearth.com/gallery/Europe/Portugal/South/Lisboa/Mass

ama/photo133016.htm

8.http://learning.hccs.edu/faculty/joy.marshall/microbiology-power-

points/chapter-3-lecture-power-point

Image References

9. http://www.microscopemaster.com/parts-of-a-compound-

microscope.html

10-17,22,23. https://www.microscopeworld.com/t-parts.aspx

18.http://biology.clc.uc.edu/fankhauser/Labs/Microscope/Oil_Immersion

.htm

19. wswx.taru.edu.cn/source/whole/html/chapter2.htm

20.http://biology.clc.uc.edu/fankhauser/labs/microscope/Oil_Immersion/

21.http://biology.clc.uc.edu/fankhauser/Labs/Microscope/Oil_Immersion

.htm

24. http://faculty.mc3.edu/jearl/ML/ml-5.htm

Image References

25. http://astro.temple.edu/~jasoncg/ID/microreporting.htm

26. http://classroom.sdmesa.edu/eschmid/lecture17-microbio.htm

27. http://en.wikipedia.org/wiki/Negative_stain

28. http://www.cram.com/flashcards/exam-1-lab-4704350

References

• The Microscope and How to Use It by Dr. Georg

Stehli

• Practical Microscopy by J E Marson

• The World of the Microscope by Chris Oxlade

• The Microscope Book by Shar Levine

• http://www.microrao.com/simple_staining.htm

• http://www.cliffsnotes.com/sciences/biology/micr

obiology/microscopy/staining-techniques