Microbiology: What is a microbe? A microbe or microorganism is a member of a large, extremely divers, group of organisms that are lumped together on the basis of one property. The fact that, normally, they are so small that they cannot be seen without the use of a microscope. Microbe Range of size Nature Viruses 0.01-0.25 µm Cellular Bacteria 0.1-10 µm Prokaryote Fungi 2 µm- 1 meter Eukaryote protozoa 2- 1000 µm

Prokaryotes and Eukaryote Eukaryotes DNA free in cytoplasm. DNA is contained with a membrane bound nucleus Only one chromosome. Diploid two copies of each chromosome. Energy metabolism associated with cytoplasmic membrane. Mitochondria 70 s ribosome. 80 s ribosome. Peptidoglycan cell walls. Polysaccharide cell walls (cellulose or chitin).

The importance of microbiology: 1-The environment: microbes are responsible for the cycling of carbon, nitrogen and phosphorus. 2- Medicine: production of antibiotics. 3- Food: wine and bread making, cheese and milk products production. 4- Biotechnology: Some microorganisms have been used to synthesize many chemicals such as acetone and acetic acid. 5- Research: microbes have been used as modal organisms for the biochemical and medical investigations.

Microscopy: Total magnification = objective magnification* eyepiece A microscope is the most important piece of equipment used in clinical microbiology. Microscopy forms 70-90% of the work. Working principle of a microscope: A microscope is a magnifying instrument. The magnified image of the object (specimen) is first produced by a lens close to the object called the objective lens. A second lens near the eye called the eyepiece enlarges the primary image, converting it into one that can enter the pupil of the eye. Total magnification = objective magnification* eyepiece magnification (4, 10, 40, 100 X) * ( 10 X)

Resolution of Microscope: It is the smallest distance that separate two sources of light points reflected from two particles close together on object. Resolution = light wavelength / 2 Example: yellow light of a wavelength of 0.4 μm give a resolution of 0.2 μm. Types of microscopy: 1- The light microscope: (simple and compound). A- The student microscope B- Phase contrast microscope. C- Darkfield illumination microscope. 2- The Fluorescence microscope. 3- The Electron microscope.

A- The light microscope: Brightfield illumination microscope is mainly used to examine stained smear

B-Phase contrast microscope: This is needed to visualize transparent microorganisms suspended in a fluid. The phase microscope takes advantage of the fact that light waves passing through transparent slid or objects (cells) emerge in different phases (different times). A special optical system coverts difference in phase into difference in intensity. So that some structure appear darker than others.

Micrcoccus species examined by phase contrast microscope ( left) and Leptospira examined by Darkfield microscope (Right):

C-Dark field illumination microscope: This method is used for visualizing organisms suspended in fluid and can not be stained by Gram’s stain. Both the structure and the motility of the organisms can be seen. In this method the light enters the special condenser which has a central blacked-out area, so that light can not pass directly through it to inter the objective. Instead the light is reflected to pass through the outer rim of the condenser at a wide angle which illuminates the microorganism by a ring of light surrounding them.

2- The fluorescence microscope: In this method, microorganisms must be stained with a fluorescent dye such as rhodamine. A fluorescent lamp emits visible light which is filtered off using optical filters. (Ultraviolet (UV) wavelengths will not be filtered off). A fluorescent dye will absorb this UV-light and change it to visible (yellow or orange) light.

3-The electron microscope: Electron microscope is an instrument that magnify very small objects which can not be seen by light microscopy. Resolution of this microscope is higher than the light one. The energy source of this kind is the electron beam while light waves are used in the light microscope. Electromagnetic lenses are used instead of the glass lenses of light microscope. Visible light of 500 nm wavelength give a resolution of 250 nm. Electron beam of 0.001 nm wavelength give a resolution of 0.0005 nm. Viruses with a diameter of 0.01-0.2 micrometer can be easily seen.

a a

Light waves and Electron beam: