How do you study something that you cant see

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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

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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 Is of culturing microbes

• Inoculation introduction of a sample into a
  container of media
• Incubation under conditions that allow growth
• Isolation separating one species from another
• Inspection
• 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