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How do you study something that you cant see?

• You look at it under the microscope– But certain microbes (e.g. bacteria) do not have

too many identifying attributes

• You grow large populations of them (i.e. culture them).– You observe the behavior of the population under

various environmental conditions• On Solid media• In the presence of certain biochemicals• Based on the biochemical reactions they cause

Tools of the Laboratory:The Methods for Studying

Microorganisms

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compound light microscope

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Pathway of light

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Effect of wavelength on resolution

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Effect of magnification

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Oil immersion lens

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• magnification – ability to enlarge objects

• resolving power – ability to show detail

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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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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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Bacterial shapes and arrangements

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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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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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The 5 I’s 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. Inspection5. 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

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Isolation technique

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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 stains

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Carbohydrate fermentation media

• carbohydrate fermentation medium- contains 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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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

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• 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

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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