Microscopy and staining techniques are important tools in microbiology. There are several types of microscopes that are used to view microorganisms, including light microscopes, electron microscopes, and fluorescent microscopes. Light microscopes such as brightfield, darkfield, and phase contrast microscopes use lenses and light to view stained specimens. Electron microscopes like the transmission electron microscope and scanning electron microscope use electron beams instead of light and allow viewing of unstained specimens at much higher magnifications. Different microscopy techniques provide complementary information about microbial structure and function.

1. Microscopy and Staining
Course : B.Sc. Microbiology
Sem I
Sub: Basic Techniques in Microbiology
Unit 1

2. 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

3. Replica of Marshall
Microscope, c. 1700, by John
Marshall (1663-1725).
1

4. Why Microscope,,,

5. 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

6. 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

7. 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

8. Types 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

9. Light microscope
• Light microscope uses diffused light from the
sun or artificial light to illuminate the object to
be observed.
5

10. 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

11. Microscopy
What are the parts of the
microscope?
6

12. 1. Arm
• Supports the body
tube.
7

13. 2. Base
• Supports and
stabilizes the
microscope.
7

14. 3. Eyepiece / ocular
lens
• Magnifies image
produced
by objective lens.
7

15. 4. Body Tube
• Maintains the
proper distance
between the
objective and
ocular lenses.
7

16. 5. Coarse adjustment
knob
• Moves stage up and
down
a large amount for
FOCUSING.
7

17. 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
7

18. 7. Light source
• (lamp or mirror) Provides
light for viewing the slide.
• Projects light UPWARDS
through the diaphragm,
the SPECIMEN, and
the LENSES.
7

19. 8. Diaphragm
• Controls the amount
of
light passing through
the slide.
7

20. 9. Stage and stage clips
• Stage clips
- hold the slide in
place.
• Stage
- Supports the slide
being viewed.
7

21. 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

22. 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)
7

23. 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.

24. High power objective lenses
Rotate so that the 100x oil immersion
objective touches the oil and clicks into
place.
8

25. 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.

26. 11. Revolving
nosepiece
• Rotates to allow use
of different power
objectives.
7

27. 12. Inclination joint
• Supports the arm
and
controls the body of
the
microscope.
7

28. Microscope Vocabulary
• Magnification: increase of an object’s
apparent size
• Resolution: power to show details clearly
Both are needed to see a clear image

29. 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

30. 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
9

31. 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
31

32. Working Distance
•working distance
— Distance between the front surface of
lens and surface of cover glass or
specimen
10

33. 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.

34. 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.
9

35. 11

36. 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.
12

37. 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.
9

38. 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.

39. 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 of
tuberculosis) can be identified after staining them with fluorochromes or
specifically labeling them with fluorescent antibodies using
immunofluorescence procedures.
• The stained organisms will fluoresce orange or green and can be detected even
in the midst of other particulate material. It is even possible to distinguish live
bacteria from dead bacteria by the color they fluoresce after treatment
with a special mixture of stains
• Thus the microorganisms can be viewed and directly counted in a
relatively undisturbed ecological niche.

40. Example of FM
9

41. Basic Microscope Technique
Rules 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.

42. Electron Microscope
• Transmission Electron Microscope
• Scanning microscope
9

43. 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.

44. 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.

45. 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

46. 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.
9

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

48. Characteristics of TEM and LM

49. 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.

50. 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.

51. Working of SEM
Scanning Electron Micrographs of Bacteria. (a) Staphylococcus aureus
(32,000). (b) Cristispira, a spirochete from the crystalline
9

52. 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

53. Application of SEM
• Human skin and the lining of the gut also can
be examined.
• Fungal hyphal structures can be studied.

54. 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.

55. 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

56. 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

57. 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

58. 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.

59. 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

60. 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

61. Gram Stain

62. Gram stain

63. 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

64. Acid-fast Stain
13

65. 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

66. Special Stains
13

67. References
Images:
1. http://www.scienceheritage.com/wp-
content/uploads/wpsc/productimages/grandm.jpg
2. http://micro.magnet.fsu.edu/primer/anatomy/introduction.html
3. http://www.recyclind.it/dati/pagine/immagine_201210192337.jpg
4. https://s10.lite.msu.edu/res/msu/botonl/b_online/library/biology107/terry/images/
micro/resolvpower.gif
5. http://www.olympusmicro.com/primer/techniques/polarized/images/polmicroalign
mentfigure1.jpg
6. http://www.microscopemaster.com/images/cm501microscopediagram.jpg
7. http://www.microscope-microscope.org/images/BWScope.jpg
8. http://www.scienceprofonline.com/images/science-image-
library/microbiology/equipment/microscope-objective-lenses-SIL.jpg
9. http://classroom.sdmesa.edu/eschmid/Lecture2-Microbio.htm
10. https://classconnection.s3.amazonaws.com/553/flashcards/661553/jpg/picture1132
8380497708.jpg
11. http://microbialworld.blog.com/files/2011/09/P.png
12. http://php.med.unsw.edu.au/cellbiology/images/0/0d/Phase_contrast_microscopy.j
pg

68. 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

69. Thank You