The document provides an overview of microscopic organisms and the tools used to observe them, focusing on the metric units for measuring sizes of microbes and the characteristics of various types of microscopes. Key concepts include the use of micrometers and nanometers for microbial sizes, comparing simple and compound microscopes, and the functionality of advanced microscopy techniques like electron and atomic force microscopes. It emphasizes the importance of resolving power in microscopy and describes specific applications such as darkfield and phase-contrast microscopy.

1. Chapter II:
Viewing the
Microbial
World
REGIE L. MAGALLANES, LPT, M.ED
Microbiology and Parasitology -
Instructor

2. At the end of the unit, the student
should be able to:
1. State the metric units used to express the
sizes of bacteria, protozoa and viruses.
2. Compare and contrast the various types of
microscopes.

3. Microbes are so tiny. But how small
are they? Most of the time, some
microscopes are required to view
them; thus, microbes are considered
to be microscopic. We will learn the
various types of microscopes in this
lesson.

4. The metric system units of length
will also be discussed because
they are used to express the sizes
of microbes and the resolving
power of microscopes.

5. At the end of the unit, the student
should be able to:
1. State the metric units used to
express the sizes of bacteria,
protozoa and viruses.
2. Compare and contrast the
various types of microscopes.

6. USING THE METRIC
SYSTEM TO EXPRESS
THE SIZES OF
MICROBES

7. In microbiology,
metric units
(primarily
micrometers and
nanometer) are
used to express
the sizes of
microbes.

8. A meter can be divided into 10 (101
)
equally spaced units called
decimeters; or 100 (102
) equally
spaced units called centimeters; 1,000
(103
) equally spaced units called
millimeters, or 1 million (106
) equally
spaced units called micrometers; or 1
billion (109
) equally spaced units called

9. It should be noted that the old terms
micron (μ) and millimicron (mμ) have
been replaced by the terms
micrometer (μm) and nanometer (nm),
respectively. An angstrom (Å) is 0.1 nm.

10. Using this scale,
human red blood
cells are
approximately
about 7 μm in
diameter.

11. The sizes of bacteria
and protozoa are
usually expressed in
terms of micrometers.
For example, a typical
spherical bacterium
(coccus; pl., cocci) is
approximately 1 μm in
diameter.

12. A typical rod-shaped
bacterium (bacillus; pl.,
bacilli) is about 1 μm wide
3 3 μm long, although
some bacilli are shorter,
and some form very long
filaments. The sizes of
viruses are expressed in
terms of nanometers.

13. Most of the viruses that cause human
disease range in size from about 10 to
300 nm, although some (e.g., Ebola
virus, a cause of hemorrhagic fever)
can be as long as 1,000 nm (1 μm).
Some very large protozoa reach a
length of 2,000 μm (2 mm).

14. In the microbiology
laboratory, the sizes
of cellular microbes
are measured using
an ocular
micrometer, a tiny
ruler within the
eyepiece (ocular) of
the compound light
microscope.

15. Before it can be
utilized to measure
objects, however,
the ocular
micrometer must
first be calibrated,
using a measuring
device called a
stage micrometer.

17. Calibration must be done for each of
the objective lenses to determine the
distance between the marks on the
ocular micrometer.

18. The ocular micrometer can then be
used to measure lengths and widths of
microbes and other objects on the
specimen slide

19. MICROSCOPES

20. A microscope is
an optical
instrument that
is used to
observe tiny
organisms that
cannot be seen
with the unaided

21. Each optical instrument has a limit
as to what can be seen using that
instrument.

22. This limit is referred to as the
resolving power or resolution of
the instrument.

23. TYPES
OF
MICROSCOPES

24. Simple Microscopes
A simple microscope contains only
one magnifying lens.

25. Simple Microscopes
During the late 1600s, Anton van
Leeuwenhoek, used simple
microscopes to observe many tiny
objects, including bacteria and
protozoa. His simple microscopes had
a maximum magnifying power of
about x100 (300 times).

27. Compound Microscopes
A compound microscope
contains more than one
magnifying lens which usually
magnifies objects about 1,000
times.

28. Compound
Microscopes
Photographs taken
through the lens
system of compound
microscopes are
called
photomicrographs.

29. Compound Microscopes
It is the wavelength of visible light
(approximately 0.45 μm) that
limits the size of objects that can
be
seen using the compound light
microscope.

30. Compound Microscopes
When using the light microscope,
objects cannot be seen if they are
smaller than about 0.225 μm.

31. Compound
Microscopes
The compound light
microscopes usually
contain two magnifying
lens systems. Within the
eyepiece is the ocular lens
which has a magnifying
power of x10.

33. Compound Microscopes
The other magnifying lens system
is found in the objective which is
situated immediately above the
object to be viewed.

34. Compound Microscopes
The four objectives used in most
laboratory compound light
microscopes are X4, X10, X40, and
X100 objectives.

35. Compound Microscopes
The higher the magnification, the
more light that is needed. Hence,
as magnification is increased, the
amount of light striking the
specimen to be studied must also
be increased.

36. Compound Microscopes
Image clarity depends on the
microscope’s resolving power or
resolution which is the ability of
the
lens system to distinguish
between two adjacent objects.

37. Compound
Microscopes
When objects are
observed against a
bright background,
that microscope is
referred to as the
brightfield
microscope.

38. Compound
Microscopes
In the laboratory,
darkfield
microscopy is
used to diagnose
syphilis where the
causative agent is
the spiral-shaped
bacterium,
Treponema

40. Compound Microscopes
Phase-contrast microscopes can be used to
view unstained living microorganisms.
Because the
light refracted by living cells is different from
the light refracted by the surrounding
medium, contrast is
therefore increased, and the specimens are
clearly viewed.

42. Compound Microscopes
Fluorescence microscopes,
however, contain a built-in
ultraviolet light source which emit
longer
wavelength light, causing them to
glow against dark background.

44. Electron Microscopes
Electron microscopes utilize an electron beam as
a source of illumination and magnets to focus the
beam. Because the wavelength of electrons
traveling in a vacuum is much shorter than the
wavelength of visible light, electron microscopes
have a much greater resolving power than
compound microscopes.

45. Electron Microscopes
There are two types of electron
microscopes: transmission electron
microscopes (TEMs) and
scanning electron microscopes (SEMs).

46. Electron Microscopes
A transmission electron microscope
(TEM) has a tall column at the top of which
an electron gun
fires a beam of electrons downward.
Viruses can be observed using a TEM. A
TEM reveals the
internal structure of cells.

48. Electron Microscopes
A scanning electron microscope (SEM), on the
other hand, has a shorter column, where the
specimen is placed at the bottom of the column.
Electrons that bounce off the surface of the specimen
are captured by detectors, and an image of the
specimen appears on a monitor. Hence, SEMs are
used to observe the outer surfaces of specimens.

50. Atomic Force
Microscopes
Atomic Force
Microscopes (AFMs)
provide a true three-
dimensional surface
profile.

51. Atomic Force Microscopes
AFMs have silicon or silicon nitride having
a sharp tip or probe at its end which is
used to scan the specimen surface.

52. Atomic Force Microscopes
When the tip is brought in
proximity to a sample surface,
forces between the tip and the
sample lead to a deflection of the
cantilever.

53. Atomic Force Microscopes
The deflection is measured using
a laser spot reflected from the top
surface of the cantilever into an
array of photodiodes creating an
image on a monitor screen.

54. Let’s
Recap!

55. A compound microscope contains
more than one magnifying lens
which usually magnifies objects
about 1,000 times.

56. When objects are observed
against a bright background, that
microscope is referred to as the
brightfield microscope.

57. Phase-contrast microscopes can be
used to view unstained living
microorganisms. Because the light
refracted by living cells is different
from the light refracted by the
surrounding medium, contrast is
therefore increased, and the
specimens are clearly viewed.

58. Fluorescence microscopes,
however, contain a built-in
ultraviolet light source which emit
longer
wavelength light, causing them to
glow against dark background.

59. A transmission electron microscope
(TEM) has a tall column at the top of
which an electron gun fires
a beam of electrons downward.
Viruses can be observed using a
TEM.

60. An scanning electron microscope
(SEM), on the other hand, has a
shorter column, where the
specimen is placed at the bottom
of the column. SEMs are used to
observe the outer surfaces of
specimens.

61. Atomic Force Microscopes (AFMs)
provide a true three-dimensional
surface profile. AFMs have
silicon or silicon nitride having a
sharp tip or probe at its end which
is used to scan the specimen
surface.