History of Microbiology

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.

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

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

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 mm1 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 wide3- typical size of an animal cell would be from 10 - 100 µm

Microscope• Light microscope• Uses light

Bright field microscope

Light path

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.

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

• 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.

• 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

• 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).

• 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.

• 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.

• 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.

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

• 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.

• 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

• 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.