# Really a very high class magnifying glass!

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Really a very high class magnifying glass!
The first person to observe and describe microorganisms accurately was Antony van Leeuwenhoek. Eventually led to the cell theory which states that cells are the fundamental units of life and carry out all the basic functions of living things. Really a very high class magnifying glass!

There are 1000 mm in a meter. 1000 μm in a mm. 1000 nm in a μm.
Fig. 3-2 Micrometer = μm nanometer = nm There are 1000 mm in a meter μm in a mm nm in a μm.

The properties of light that effect our ability to visualize objects include wavelength and resolution. Wavelength = lambda λ = the distance between two adjacent crests or two adjacent troughs of any wave. Fig. 3-3

The sun produces a continuous spectrum of electromagnetic radiation with waves of various lengths.
Fig. 3-4 White light is the combination of all colors of visible light. Black is the absence of visible light. The shorter the wavelength used, the grater the resolution that can be attained.

Resolution refers to the ability to see two items as separate and discrete units rather than as a fuzzy, overlapped single image. Fig. 3-5 Light must pass between two objects for them to appear to be separate things. As this distance gets smaller we need greater resolving power to see two objects

The effect of wavelength on resolution
The effect of wavelength on resolution. The smaller the wavelength, the more clearly we see between the legs of the E and the greater the resolving power we have. The resolving power is restricted by the wavelength of visible light and the numerical aperture (written on the ocular of your micaroscopes) of the lens being used. Fig. 3-6

Various interactions of light with objects it strikes:
1. Reflection - light bounces back, giving the object color. Example: green light reflects off the leaves of plants. 2. Transmission - light passes through the object 3. Absorption - light rays are taken up, used, by the object. Example: all wavelengths except green are taken up by the leaves and their energy is used in photosynthesis Example: a black object reflects no light, it absorbs all wavelengths and gains energy as heat faster than a white object Fig. 3-7

Various interactions of light with objects it strikes:
3. Absorption - light rays are taken up, used, by the object. Example: all wavelengths except green are taken up by the leaves and their energy is used in photosynthesis. When absorbed light rays are changed into longer wavelengths and reemitted, the object is fluorescent - many dyes used in microbiology are fluorescent. 4. Refraction - light rays speed up or slow down as they pass from one medium to another. This causes the light to bend at a particular angle - the angle of refrection.. [Pencil in water] Fig. 3-7

Microscopists use immersion oil between the slide and the lense since they have the same refractive index and light will not bend and blur the image. Staining a specimen increases differences in the indices of refraction, making it easier to observe details. See box on page 55 - A life of Crime [glass rod in immersion oil] Fig. 3-9

Various interactions of light with objects it strikes:
1. Reflection 2. Transmission 3. Absorption 4. Refraction 5. Defraction 5. Defraction - light waves bend around any opening that they travel through, like waves of water do. Defraction causes blurred images and, along with the wavelength of visible light, limits the total magnification capacity of oil immersion light microscope to 1000X.. See checklist on page 56 Fig. 3-10

Compound microscope = a microscope with more than one lens
Light microscopy = the use of any kind of microscope that uses visible light Compound microscope = a microscope with more than one lens An objective in our compound light microscopes is really a series of lenses. Fig. 3-11

Total magnification = ocular power X objective power.
Light enters from a source in the base, goes through a condenser, which converges the light beams so they pass through the specimen. The diaphram controls the amount of light - the higher the magnification the more light is needed. The objective lens magnifies the image, the ocular lens further magnifies the image and the mechanical stage allows precise movement of the slide. Total magnification = ocular power X objective power. Parfocal = focus in low power needs only minor adjustment when objective lens is changed. Fig. 3-12

Brightfield microscopy vs Darkfield
The dark-fild condenser deflects light rays so they reflect off the specimen The condenser of the brightfield microscope concentrates and transmits light directly through the specimen Fig. 3-13

Brightfield Darkfield

Differential Interference Contrast
Phase-Contrast Observing live, unstained cells Differential Interference Contrast Produces higher resolution

Differential Interference Contrast
Brightfield Dark-filed Phase-contrast Differential Interference Contrast

Fluorescence Microscopy: ultraviolet light is used to excite molecules that emit (release) light of a longer wavelength (visible light). Some cells fluoresce naturally. Most are “stained” with a fluorescent dye (fluorochromes). These dyes may specifically stain certain molecules, like nucleic acids or they may be attached to antibody molecules which then bind specifically to certain molecules on the specimen. Antibodies are made by the immune system. They are very specific for one molecule. Ex. Antibodies against syphilis will bind only to syphilis spirochetes. Diagnosis can be made in minutes.

Transmission Electron Microscope Scanning Electron Microscope
Fig. 3-19 The light microscope’s resolving power is limited by the wavelength of light and is 0.2 micrometers (μm). Electron microscope uses a beam of electrons instead of a beam of light. Electrons must travel through a vacuum or they will be scattered by the molecules in air. We can not see an image made by electrons. Instead an electron micrograph is created. With an electron microscope we can see molecules and even individual atoms. Two kinds include: Transmission Electron Microscope Scanning Electron Microscope

Light microscope Electron Microscope

Scanning Electron Microscope
specimen is embedded in a block of plastic and sliced very thin so the electrons can penetrate give a better view of the internal structure can resolve objects as close as 1nm, magnifying up to 500,000X due to short wavelength of electrons electron micrograph can be enlarged to obtain an image magnified 20 million times Transmission Electron Microscope used to create images of the surfaces of specimens

Transmission Electron
Scanning Electron See Checklist on page 63, See Table 3.2

Specimen preparation for light microscope
Wet mounts = a drop of medium containing the microbes is placed on a slide Hanging drop is a wet mount used with dark-field. Fig a Smears = a loopful of medium is spread onto a glass slide, allowed to air dry, and fixed to the slide. This kills the microbes. Not easy - too thick or too thin, stirred too much, not dry enough when fixed, etc. Heat fixation kills the organisms, sticks them to the slide so they wont wash off, and allows the microbe to take up stain better.

Principles of staining
A stain is a molecule that can bind to a cellular structure and give it color, making the microorganism easier to see. In some cases the different staining techniques help to differentiate between different organisms. Cationic or basic dyes such as methylene blue, crystal violet, etc are attracted to negatively charged cell components like the cell membrane Anionic or acidic dyes are attracted to any positively charged cell structures

Staining techniques See Table 3.3
Simple stain uses a single stain and reveals basic cell shapes and cell arrangements Differential staining techniques use two or more dyes to distinguish between two kinds of organisms or two different parts of an organism. Gram stain, acid fast stain Special stains can identify specialized structures. Flagellar stain

Fig. 3-28 The Gram Stain

Four groups of organisms can be distinguished with the Gram Stain.
Gram-positive organisms retain the crystal violet/iodine complex due to the structure of their cell walls (chapter 4) Gram-negative organisms do not retain this complex due to the structure of their cell walls (chapter 4) Gram-nonreactive organisms which do not stain or stain poorly Gram-variable organisms which stain unevenly. often due to age - organisms from cultures over 48 hours old

Ziehl-Neelsen acid-fast stain is used to detect tuberculosis- and leprosy-causing organisms, micobacterium. Most bacteria will lose the red carbolfuchsin stain when decolorized, only “acid fast” bacteria retain the dye due to lipid components in their walls (chapter 4). Used with a counter stain.

Negative staining is used when an organism or a structure does not take up stain easily, as with the capsule (chapter 4). A stain is used to stain the background (pink). A capsule is a layer of polysaccharide material that surrounds a bacteria, acting as a barrier to host defense mechanisms. A dye can be used to stain the microbe itself (blue).