The document provides a history of key discoveries in microbiology from the 1500s to the late 1800s. It discusses how Hieronymus Fracastorius first discussed contagious infection in 1546. In 1835, Bassi showed a disease in silkworms was caused by a fungus, the first recognized microorganism causing animal disease. In 1857, Pasteur proposed the germ theory of disease. Koch later demonstrated the role of bacteria in specific diseases and developed techniques to isolate and culture microbes. The document also describes microscopy techniques such as brightfield, darkfield, and calculating magnification and resolution.

1. History of Microbiology

2. Proof that microbes cause disease
1546: Hieronymus Fracastorius (Girolamo Fracastoro) wrote "On Contagion", the 1st
known discussion of the phenomenon of contagious infection.
1835: Agostino Bassi de Lodi showed that a disease affecting silkworms was caused by
a fungus - the first microorganism to be recognized as a contagious agent of animal
disease.
1847: Ignaz Semmelweiss (1818-1865), a Hungarian physician- decided that doctors in
Vienna hospitals were spreading childbed fever while delivering babies. He started
forcing doctors under his supervision to wash their hands before touching patients.
1857: Louis Pasteur proposed the “Germ theory of disease”.
• - Ancients believed that disease was the result of a divine punishment.
Pasteur fought to convince surgeons that germs existed and carried diseases, and
dirty instruments and hands spread germs and therefore disease. Pasteur's
pasteurization process killed germs and prevented the spread of disease.
1867: Joseph Lister (1827-1912) introduced antiseptics in surgery. By spraying carbolic
acid on surgical instruments, wounds and dressings, he reduced surgical mortality due
to bacterial infection considerably.
1876: Robert Koch (1843-1910). German bacteriologist was the first to cultivate
anthrax bacteria outside the body using blood serum at body temperature.

3. Robert Koch
demonstrated the first
direct role of a bacterium
in disease
• "Koch's postulates" (1884), the critical test for the
involvement of a microorganism in a disease:
1. The agent must be present in every case of the disease.
2. The agent must be isolated and cultured in vitro.
3. The disease must be reproduced when a pure culture of
the agent is inoculated into a susceptible host.
4. The agent must be recoverable from the
experimentally-infected host.
• This eventually led to:
• Development of pure culture techniques
• Stains, agar, culture media, petri dishes
Koch's postulates

4. Preparing smears for staining
• Staining- coloring microbe with a dye to emphasize
certain structure
• Smear- A thin film of a microbe solution on a slide, a
smear is usually fixed to attach microbes to the slide
and kill microbes

5. How do we view microorganisms?
• Units of measurement
When talking about cells and microscopic organisms, you
would be measuring using MICROMETRE (abbreviated: µ
--micron ) or stated as: µm (micrometer).
1 µm = 1 x 10-6 meters/ 1 x 10-3 mm
1 mm= 1 x 103 nanometers/ 1 x 103 µm
To give you the idea of how small a micro metre is,
1- a human hair is about 100 µm, wide,
2- a red blood cell would be around 8 µm wide
3- typical size of an animal cell would be from 10 - 100 µm

6. Microscope
• Light microscope
• Uses light

7. Bright field microscope
Light path

8. Microscopy
• The objective lens forms an enlarged real image
within the microscope, and the eyepiece lens
further magnifies this primary image.
• When one looks into a microscope, the enlarged
specimen image, called the virtual image, appears
to lie just beyond the stage about 25 cm away.
• The total magnification is calculated by
multiplying the objective and eyepiece
magnifications together. For example, if a 45
objective is used with a 10 eyepiece, the overall
magnification of the specimen will be 450 X.

9. Microscopy
• Resolution is the ability of a lens to separate or distinguish
between small objects that are close together.
• The minimum distance (d) between two objects that reveals
them as separate entities is given by the Abbe equation, in
which lambda (λ) is the wavelength of light used to illuminate
the specimen and n sin θ is the numerical aperture (NA).
d= 0.5 λ/n sin θ

10. • 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.

11. • The curved upper part of the arm holds the body
assembly, to which a nosepiece and one or more
eyepieces or oculars are attached.
• More advanced microscopes have eyepieces for
both eyes and are called binocular microscopes.
• The nosepiece holds three to five objectives with
lenses of differing magnifying power and can be
rotated to position any objective beneath the
body assembly

12. • When one looks into a microscope, the enlarged
specimen image is a virtual image.
• As d becomes smaller, the resolution increases,
and finer detail can be discerned in a specimen.
• The greatest resolution is obtained with light of
the shortest wavelength, light at the blue end of
the visible spectrum (in the range of 450 to 500
nm).

13. • Theta is defined as 1/2 the angle of the cone of light entering an objective.
• Light that strikes the microorganism after passing through a condenser is cone-
shaped.
• When this cone has a narrow angle and tapers to a sharp point, it does not spread
out much after leaving the slide and therefore does not adequately separate
images of closely packed objects.
• The resolution is low.
• If the cone of light has a very wide angle and spreads out rapidly after passing
through a specimen, closely packed objects appear widely separated and are
resolved.
• The angle of the cone of light that can enter a lens depends on the refractive index
(n) of the medium in which the lens works, as well as upon the objective itself.
• The refractive index for air is 1.00.
• Since sin θ cannot be greater than 1 (the maximum θ is 90° and sin 90° is 1.00),
no lens working in air can have a numerical aperture greater than 1.00.

14. • To raise the numerical aperture above 1.00, and therefore achieve
higher resolution, is to increase the refractive index with immersion
oil, a colorless liquid with the same refractive index as glass.
• If air is replaced with immersion oil, many light rays that did not
enter the objective due to reflection and refraction at the surfaces
of the objective lens and slide will now do so.
• An increase in numerical aperture and resolution results.
• The working distance of an objective is the distance between the
front surface of the lens and the surface of the cover glass (if one
is used) or the specimen when it is in sharp focus.

15. • Resolution can be improved with a substage
condenser, a large light-gathering lens used to
project a wide cone of light through the slide
and into the objective lens, thus increasing the
numerical aperture.

16. The Dark-Field Microscope
• Living, 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

17. • 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.

18. • Light enters the microscope for illumination
of the sample.
• A specially sized disc, the patch stop blocks
some light from the light source, leaving an
outer ring of illumination.
• A wide phase annulus can also be
reasonably substituted at low magnification.
• The condenser lens focuses the light
towards the sample.
• The light enters the sample.
• Most is directly transmitted, while some is
scattered from the sample.
• The scattered light enters the objective lens,
while the directly transmitted light simply
misses the lens and is not collected due to
a direct illumination block .
• Only the scattered light goes on to produce
the image, while the directly transmitted
light is omitted

19. • Dark field microscopy is a very simple yet effective
technique and well suited for uses involving live
and unstained biological samples, such as a smear from a
tissue culture or individual, water-borne, single-celled
organisms.
• Considering the simplicity of the setup, the quality of
images obtained from this technique is impressive.
• The main limitation of dark field microscopy is the low light
levels seen in the final image.
• This means the sample must be very strongly illuminated,
which can cause damage to the sample.
• Dark field microscopy techniques are almost entirely free
of artifacts, due to the nature of the process.