Tools of the Laboratory

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Tools of the Laboratory
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The 5 Is of Culturing Microbes

• Inoculation
• Isolation
• Incubation
• Inspection
• Identification

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1. Inoculation

• Introduction of a sample into a container of
  sterile media
• Spread sample on solid media or introduce to
  liquid tube
• produces a culture of observable growth

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2. Isolation

• separating one species from another
• If individual bacterial cell separated from other
  cells and has space on a nutrient surface, it
  will grow into a mound of cells
• colony
• consists of one species

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Isolation

• Obtaining Pure Cultures
• Cultures composed of cells arising from a single
  progenitor
• Progenitor is termed a CFU
• Aseptic technique prevents contamination of
  sterile substances or objects
• Two common isolation techniques
• Streak plates
• Pour plates

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Insert figure 3.2 Isolation technique
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Isolation Techniques

• streak plate technique
• pour plate technique

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Media

• Way to provide nutrients in the laboratory
• Media can be classified according to three
  properties
• Physical state
• Chemical composition
• Functional type

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Physical States of Media

• Liquid
• Broth
• does not solidify
• Semisolid
• clot-like consistency
• contains solidifying agent (agar or gelatin)
• Solid
• firm surface for colony formation
• contains solidifying agent
• liquefiable and nonliquefiable

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Media

• Most commonly used solidifying agent is agar
• 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 and nutrients
• not digestible for most microbes

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Media Providing Nutrients in the Laboratory

• Most commonly used media
• nutrient broth
• liquid medium containing beef extract and peptone
• nutrient agar
• solid media containing beef extract, peptone and
  agar

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Media

• Selective media
• contains one or more agents that inhibit growth
  of some microbes
• encourage growth of the desired microbes
• Differential media
• allows growth of several types of microbes
• displays visible differences among desired and
  undesired microbes

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3. Incubation

• Incubation
• temperature-controlled chamber at appropriate
  temperature and atmosphere
• under conditions that allow growth
• microbe multiplies and produces macroscopically
  observable growth

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4. Inspection

• Inspection
• Observation
• macroscopic and microscopic
• pure culture
• grows only single known species of microorganisms
• mixed cultures
• hold two or more identified species or
  microorganisms
• contaminated culture
• once pure or mixed culture that has unwanted
  microbes growing

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5. Identification

• Identification
• macroscopic and microscopic appearance
• biochemical tests
• genetic characteristics
• immunological testing

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Characteristics of Bacterial Colonies
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Staining

• Positive staining
• surfaces of microbes are negatively charged and
  attract basic dyes
• Negative staining
• microbe repels dye, the dye stains the background

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Staining

• Simple stains
• one dye is used
• reveals shape, size, and arrangement

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Staining

• Differential stains
• use a primary stain and a counterstain to
  distinguish cell types or parts
• examples
• gram stain
• acid-fast stain
• endospore stain

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Staining

• Special stains
• reveal certain cell parts not revealed by
  conventional method
• capsule and flagellar stains

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Identification