M I C RO B I O L O G Y A N D PA R A S I T O L O G Y

Joshua V. Manalo, RMT, MSPH(cand)

MICROSCOPY

Reading assignment:
1. Aberration 7. Köhler Illumination
2. Contrast 8. Mechanical tube length
3. Depth of field 9. Numerical aperture
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4. Depth of focus 10. Refractive index
5. Immersion fluid 11. Resolution
6. Working distance 12. Parfocal
SIMPLE MICROSCOPE
- A simple microscope contains a single bi-convex magnifying lens which is
thicker in the center than at the periphery.

- It produces a magnified image that is in the same orientation as the original

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object.

- Uses: dissection, examination of bacterial colonies, and interpretation of

agglutination reactions.

Examples: jeweler‘s loupes, photographic slide viewers, and simple magnifying or

reading glasses
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 COMPOUND MICROSCOPE
The first compound microscopes were constructed around 1590 by Dutch
spectacle makers Zaccharias Janssen and Hans Janssen.

The Janssen microscope consisted of an object lens (objective) that was placed
close to the specimen and the eye, or an ocular lens that was placed close to the 5
eye. The lenses were separated by a body tube.

- The objective lens projects a magnified image into the body tube and the
eyepiece magnifies the projected image
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THE LIGHT MICROSCOPE
- The light microscope is an instrument for visualizing fine detail of an
object. It does this by creating a magnified image through the use of a
series of glass lenses, which first focus a beam of light onto or through an
object, and convex objective lenses to enlarge the image formed.
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Types of light microscope

1.
2.
3.
4.
BRIGHT FIELD MICROSCOPE
- An ordinary type of microscope
- It forms a dark image against a brighter background

DARK-FIELD MICROSCOPE 8

- It uses darkfield condenser that blocks light that would enter

- It directs the light to hot the specimen at an oblique angle
• All the other light that passes through the specimen will miss the
objective, thus making the background a dark field.
• Organisms will appear extremely bright against a dark field
PHASE CONTRAST MICROSCOPE
- Converts slight differences in refractive index and cell density into
easily detected variations in light intensity
- The phase differences are seen through the microscope as
different degrees of brightness
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- Staining is not part of phase-contrast microscopy
FLUORESCENCE MICROSCOPE
- When some molecules absorb radiant energy, they become
excited and release much of the
trapped energy as light.
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Fluorochromes – stains used in this technique
• Fluoresce brightly upon exposure to light of a specific
wavelength
• The microscope forms an image of the fluorochrome-labeled
microorganisms
ELECTRON MICROSCOPE
- is one of the most advanced and important types of microscopes
- highest magnifying capacity.
- electrons are used to illuminate the tiniest particles.
- more powerful tool than light microscopes
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TRANSMISSION ELECTRON MICROSCOPE (TEM)

- ultrathin slices of microorganisms or viruses are placed on a wire grid
- Stain: gold or palladium
- The densely coated parts of the specimen deflect the electron beam,
and both dark and light\ areas show up on the image.
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PRESENTATION TITLE 20XX

The Scanning Electron Microscope (SEM)
- The more contemporary form electron microscope.
- this microscope gives lower magnification than the TEM
- permits three-dimensional views of microorganisms and other
objects.

The Scanning tunneling Microscope (STM) 13

- shows three-dimensional images of a sample

- the structure of a surface is studied using a stylus that scans the
surface at a fixed distance from it.
- electrons tunnel between the surface and the stylus, producing an
electrical signal.
- stylus tip is formed by one single atom
Microbiology
- Study of organisms and agents too small to be seen by the unaided eye
- Study of microorganisms

Branches of Microbiology 14
ü Bacteriology
ü Mycology
ü Virology
ü Phycology
ü Protozoology
BIOLOGICAL WEAPONS
Biological warfare may be defined as
intentional use of doses to harm or kill an
adversary military forces, population, food or
livestock and includes any living, or nonliving 15
organisms or its bioactive substance (toxin).

• British distributed blankets to Indians in 1763.

• British had detonated an experimental anthrax bomb in Gruinard Island
• 750 people fell ill to food poisoning in Oregaon because of spread of salmonella
BIOLOGICAL WEAPONS
Bacillus anthracis (causing pneumonia
with a mortality rate 95%, if untreated),

Smallpox (contagious with high mortality

rate),
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Yersinia pestis (plague causing bacteria),

Francisella tularensis (tularemia),

Ebola Marburg viruses (hemorrhage fever)

Clostridium botulinum (botulinism)

HISTORY AND THE GERM THEORY OF DISEASE

Roman Philosopher Lucretius (98 – 55 BC) and Girolamo

Fracastoro (1478 – 1553)
- Suggested that diseases were caused by ―invisible living creatures

Anton van Leeuwenhoek (1632 – 1723) 17

- He was the first person to observe and describe microorganisms

accurately
- He described the microorganisms from the pond water as ―animalcules
- He used his self-made single lens microscope with 50 – 300x
magnification
SPONTANEOUS GENERATION
People (including scientists) believed that simple living organisms
could come into being by spontaneous generation. This was the idea
that non-living objects can give rise to living organisms. It was 18
common "knowledge" that simple organisms like worms, beetles,
frogs, and salamanders could come from dust, mud, etc., and food left
out, quickly "swarmed" with life.
Francesco Redi (1626 – 1697)
- In 1668, even before Leeuwenhoek‘s discovery of the microscope, he
demonstrated that maggots do not arise spontaneously from decaying
meat.
- His results were a serious blow to the long-held belief that large forms
of life could arise from non-life. 19

John Needham (1748)

- He observed that boiled mutton broth (tightly sealed) eventually
became cloudy with microorganisms
- He proposed that organic matter possessed a ―vital force that could
give rise to life.
Lazzaro Spallanzani (1729 – 1799)
- He observed that no growth took place as long as the flasks remained
sealed.
- He proposed that air carried microorganisms to the culture medium
and that external air might be essential for the growth of animals
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present already in the medium

Rudolf Virchow (1858)

- He challenged spontaneous generation with the concept of biogenesis
Louis Pasteur (1822 – 1895)
- He resolved the issue of spontaneous generation
- He stated that microorganisms are indeed present in the air and can
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contaminate seemingly sterile solutions, however the air itself does not
create microbes
- He showed that microorganisms can be present in non-
living matter
- He stated that microbial life can be present in non-living
matter
- He stated that microbial life can be destroyed by heat (basis 22

of the aseptic technique

- He provided evidence that microorganisms cannot originate
from mystical forces present in nonliving materials
John Tyndall (1820 – 1893)
- He showed that dust carry germs which contaminates sterile broth
- tyndallization– form of sterilization for three consecutive days

The Germ Theory of Disease 23

The germ theory of disease is the currently accepted scientific

theory for many diseases. It states that microorganisms known as
pathogens or "germs" can lead to disease.
Joseph Lister (1827 – 1912)
- He developed the antiseptic system of surgery
- He demonstrated the use of phenol for treating surgical wound and
also sprayed phenol over
the surgical area
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Robert Koch (1843 – 1910)

- He discovered Bacillus Anthracis – causes Anthrax (1876 – 1877)
- He was the first to culture bacteria on boiled potatoes, gelatin and
used meat extracts and protein digests for cultivation
- He developed culture media for observing growth of bacteria isolated
from the human body.
Koch’s Postulate
Ø The microorganism must be present in every case of the disease
but absent from healthy organisms
Ø The suspected microorganism must be isolated and grown in a
pure culture
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Ø The same disease must result when the isolated microorganism
is inoculated into a healthy host

The same organism must be isolated again from the diseased host
Fannie Eilshemius Hesse – suggested the use of agar as a solidifying agent
Richard Petri – developed the petri dish (plate)
Martinus Beijerinck and Sergie Winogradsky – developed the enrichment-
culture technique and the use of selective media
TAXONOMY
Bacterial taxonomy is concerned with the naming of bacterial
organisms and with organizing these names according to various
criteria. Overall, classification involves the recognition of similarities
and relationships as a basis for the arrangement of the bacteria into 26
taxonomic groups or taxa. The basic taxon is the species.
Identification also involves the recognition of a bacterium as a
member of one of the established taxa, appropriately named, by the
comparison of a number of characters with those in the description.
3 PARTS OF TAXONOMY

- Classification
• Arrangement of organisms into groups or taxa (sing. taxum)
• Based on mutual similarity or relatedness
- Nomenclature 27

• Branch of taxonomy concerned with assignment of names to

taxonomic groups in agreement with published rules
- Identification
• Process of determining that a particular isolate belong to a re-
organized taxon
BACTERIAL CLASSIFICATION

Kingdoms

2-kingdom classification by Aristotle (384 – 322 BC)

- Plants
-Animals 28

3-kingdom classification by Ernst Haeckel (1735)

- Plants
- Animals
- Protista
4-kingdom classification by Herbert Copeland (1938)
- Plants – includes fungi
- Animals
- Protista – amoebas, diatoms, some unicellular and multicellular
eukaryotes
- Monera/Prokaryotes – bacteria 29

5-kingdom classification by Robert Whittaker (1957)

- Plants
- Animals
- Protista – algae and protozoa
- Monera/Prokaryotae – bacteria and archaeans
- Fungi
Binomial System – by Carl Von Linne/ Carolus Linnaeus
- Latinized, italicized name
- Generic name
• First part
• Capitalized
• May be changed
- Specific epithet 30
• Second part
• Stable
• The oldest epithet for a particular organism takes precedence and
must be used
- Names may be shortened by abbreviating the genus name with a
single capital letter
ü e.g. E. coli