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Tools of the Laboratory:

The Methods for Studying Microorganisms

The 5 Is of culturing microbes


1. Inoculation introduction of a sample into a container of media 2. Incubation under conditions that allow growth 3. Isolation separating one species from another 4. Inspection 5. Identification
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Isolation
If an individual bacterial cell is separated from other cells & has space on a nutrient surface, it will grow into a mound of cells- a colony A colony consists of one species

Isolation technique

Media providing nutrients in the laboratory


Most commonly used:
nutrient broth liquid medium containing beef extract & peptone nutrient agar solid media containing beef extract, peptone & agar

agar is a complex polysaccharide isolated from red algae


solid at room temp, liquefies at boiling (100oC), does not resolidify until it cools to 42oC provides framework to hold moisture & nutrients not digestible for most microbes
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Types of media
synthetic contains pure organic & inorganic compounds in an exact chemical formula complex or nonsynthetic contains at least one ingredient that is not chemically definable general purpose media- grows a broad range of microbes, usually nonsynthetic enriched media- contains complex organic substances such as blood, serum, hemoglobin or special growth factors required by fastidious microbes
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Enriched media

selective media- contains one or more agents that inhibit growth of some microbes and encourage growth of the desired microbes differential media allows growth of several types of microbes and displays visible differences among desired and undesired microbes
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selective & differential media

Selective media

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Differential media

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Miscellaneous media
reducing medium contains a substance that absorbs oxygen or slows penetration of oxygen into medium; used for growing anaerobic bacteria carbohydrate fermentation mediumcontains sugars that can be fermented, converted to acids, and a pH indicator to show the reaction; basis for identifying bacteria and fungi
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Carbohydrate fermentation media

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magnification ability to enlarge objects resolving power ability to show detail

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compound light microscope

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Pathway of light

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Oil immersion lens

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Types of light microscopes


Bright-field most widely used, specimen is darker than surrounding field Dark-field brightly illuminated specimens surrounded by dark field Phase-contrast transforms subtle changes in light waves passing through the specimen into differences in light intensity, best for observing intracellular structures
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3 views of a cell

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Fluorescence Microscope
Modified compound microscope with an ultraviolet radiation source and a filter that protects the viewers eye Uses dyes that emit visible light when bombarded with shorter uv rays. Useful in diagnosing infections

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Electron microscopy
Forms an image with a beam of electrons that can be made to travel in wavelike patterns when accelerated to high speeds. Electron waves are 100,000X shorter than the waves of visible light. Electrons have tremendous power to resolve minute structures because resolving power is a function of wavelength. Magnification between 5,000X and 1,000,000X
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2 types of electron microscopes


Transmission electron microscopes (TEM) transmits electrons through the specimen; darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts Scanning electron microscopes (SEM) provides detailed three-dimensional view. SEM bombards surface of a whole, metal-coated specimen with electrons while scanning back and forth over it. 24

Transmission Electron Micrograph

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Scanning Electron Micrograph

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Specimen preparation
wet mounts & hanging drop mounts allow examination of characteristics of live cells: motility, shape, & arrangement fixed mounts are made by drying & heating a film of specimen. This smear is stained using dyes to permit visualization of cells or cell parts.
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Staining
cationic dyes - basic, with positive charges on the chromophore anionic dyes - acidic, with negative charges on the chromophore surfaces of microbes are negatively charged and attract basic dyes positive staining. negative staining microbe repels dye & it stains the background
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Staining
simple stains one dye is used differential stains use a primary stain and a counterstain to distinguish cell types or parts. examples: Gram stain, acid-fast stain and endospore stain special stains: capsule and flagellar stains

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Types of stains

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