Microbiology: Tools of the Laboratory

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Microbiology Tools of the Laboratory
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Inoculation and Isolation

• Inoculation producing a culture
• Introduce a tiny sample (the inoculums) into a
  container of nutrient medium
• Isolation separating one species from another
• Separating a single bacterial cell from other
  cells and providing it space on a nutrient
  surface will allow that cell to grow in to a
  mound of cells (a colony).
• If formed from a single cell, the colony contains
  cells from just that species.

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Figure 3.2
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Streak Plate Method

• Streak plate method- small droplet of culture or
  sample spread over surface of the medium with an
  inoculating loop
• Uses a pattern that thins out the sample and
  separates the cells

Figure 3.3 a,b
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Loop Dilation Method

• Loop dilation, or pour plate, method- sample
  inoculated serially in to a series of liquid agar
  tues to dilute the number of cells in each
  successive tubes
• Tubes are then poured in to sterile Petri dishes
  and allowed to solidify

Figure 3.3 c,d
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Spread Plate Method

• Spread plate method- small volume of liquid,
  diluted sample pipette on to surface of the
  medium and spread around evenly by a sterile
  spreading tool

Figure 3.3 e,f
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Media Providing Nutrients in the Laboratory

• At least 500 different types
• Contained in test tubes, flasks, or Petri dishes
• Inoculated by loops, needles, pipettes, and swabs
• Sterile technique necessary
• Classification of media
• Physical state
• Chemical composition
• Functional type

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Classification of Media by Chemical Content

• Synthetic media- compositions are precisely
  chemically defined
• Complex (nonsynthetic) media- if even just one
  component is not chemically definable

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Classification of Media by Function

• General purpose media- to grow as broad a
  spectrum of microbes as possible
• Usually nonsynthetic
• Contain a mixture of nutrients to support a
  variety of microbes
• Examples nutrient agar and broth, brain-heart
  infusion, trypticase soy agar (TSA).

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Enriched Media

• Enriched media- contain complex organic
  substances (for example blood, serum, growth
  factors) to support the growth of fastidious
  bacteria. Examples blood agar, Thayer-Martin
  medium (chocolate agar)

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Figure 3.6
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Selective and Differential Media

• Selective media- contains one or more agents that
  inhibit the growth of certain microbes but not
  others. Example Mannitol salt agar (MSA),
  MacConkey agar, Hektoen enteric (HE) agar.
• Differential media- allow multiple types of
  microorganisms to grow but display visible
  differences among those microorganisms.
  MacConkey agar can be used as a differential
  medium as well.

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Figure 3.9
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Miscellaneous Media

• Reducing media- absorbs oxygen or slows its
  penetration in the medium used for growing
  anaerobes or for determining oxygen requirements
• Carbohydrate fermentation media- contain sugars
  that can be fermented and a pH indicator useful
  for identification of microorganisms
• Transport media- used to maintain and preserve
  specimens that need to be held for a period of
  time
• Assay media- used to test the effectiveness of
  antibiotics, disinfectants, antiseptics, etc.
• Enumeration media- used to count the numbers of
  organisms in a sample.

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Figure 3.10
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Incubation

• Incubation an inoculated sample is placed in an
  incubator to encourage growth.
• Usually in laboratories, between 20 and 40C.
• Can control atmospheric gases as well.
• Can visually recognize growth as cloudiness in
  liquid media and colonies on solid media.
• Pure culture- growth of only a single known
  species (also called axenic)
• Usually created by subculture
• Mixed culture- holds two or more identified
  species
• Contaminated culture- includes unwanted
  microorganisms of uncertain identity, or
  contaminants.

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Inspection and Identification

• Inspection and identification Using appearance
  as well as metabolism (biochemical tests) and
  sometimes genetic analysis or immunologic testing
  to identify the organisms in a culture.
• Cultures can be maintained using stock cultures
• Once cultures are no longer being used, they must
  be sterilized and destroyed properly.

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Figure 3.2 Microscopes and Magnification.
Unaided eye 200 ?m
Light microscope 200 nm 10 mm
Tick Actual size
Scanning electron microscope 10 nm 1 mm
Red blood cells
Transmission electron microscope 10 pm 100 ? m
E. coli bacteria
T-even bacteriophages (viruses)
Atomic force microscope 0.1 nm 10nm
DNA double helix
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Units of Measurement

• 3-1 List the metric units of measurement that are
  used for microorganisms.

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Units of Measurement

• 1 µm 106 m 103 mm
• 1 nm 109 m 106 mm
• 1000 nm 1 µm
• 0.001 µm 1 nm

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Light Microscopy

• The use of any kind of microscope that uses
  visible light to observe specimens
• Types of light microscopy
• Compound light microscopy
• Darkfield microscopy
• Phase-contrast microscopy
• Differential interference contrast microscopy
• Fluorescence microscopy
• Confocal microscopy

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Figure 3.1a The compound light microscope.
Ocular lens (eyepiece) Remagnifies the image
formed by the objective lens
Fine focusing knob
Coarse focusing knob
Body tube Transmits the image from the objective
lens to the ocular lens
Arm
Objective lenses Primary lenses that magnify the
specimen
Stage Holds the microscope slide in position
Condenser Focuses light through specimen
Diaphragm Controls the amount of light entering
the condenser
Principal parts and functions
Illuminator Light source
Base
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Compound Light Microscopy

• In a compound microscope, the image from the
  objective lens is magnified again by the ocular
  lens
• Total magnification objective lens ? ocular lens

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Compound Light Microscopy

• Resolution is the ability of the lenses to
  distinguish two points
• A microscope with a resolving power of 0.4 nm
  can distinguish between two points 0.4 nm
• Shorter wavelengths of light provide greater
  resolution

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Figure 3.3 Refraction in the compound microscope
using an oil immersion objective lens.
Oil immersion objective lens
Unrefracted light
Without immersion oil most light is refracted and
lost
Immersion oil
Air
Glass slide
Condenser lenses
Condenser
Iris diaphragm
Light source
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Brightfield Illumination

• Dark objects are visible against a bright
  background
• Light reflected off the specimen does not enter
  the objective lens

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Darkfield Illumination

• Light objects are visible against a dark
  background
• Light reflected off the specimen enters the
  objective lens

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Phase-Contrast Microscopy

• Accentuates diffraction of the light that passes
  through a specimen

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Fluorescence Microscopy

• Uses UV light
• Fluorescent substances absorb UV light and emit
  visible light
• Cells may be stained with fluorescent dyes
  (fluorochromes)

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Figure 3.6b The principle of immunofluorescence.
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Electron Microscopy

• Uses electrons instead of light
• The shorter wavelength of electrons gives greater
  resolution

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Transmission Electron Microscopy (TEM)

• Ultrathin sections of specimens
• Light passes through specimen, then an
  electromagnetic lens, to a screen or film
• Specimens may be stained with heavy-metal salts

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Transmission Electron Microscopy (TEM)

• 10,000100,000? resolution 2.5 nm

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Scanning Electron Microscopy (SEM)

• An electron gun produces a beam of electrons that
  scans the surface of a whole specimen
• Secondary electrons emitted from the specimen
  produce the image

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Scanning Electron Microscopy (SEM)

• 1,00010,000? resolution 20 nm

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Figure 3.22
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Preparing Smears for Staining

• Staining coloring the microbe with a dye that
  emphasizes certain structures
• Smear a thin film of a solution of microbes on
  a slide
• A smear is usually fixed to attach the microbes
  to the slide and to kill the microbes

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Preparing Smears for Staining

• Live or unstained cells have little contrast with
  the surrounding medium. Researchers do make
  discoveries about cell behavior by observing live
  specimens.

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Simple Stains

• Simple stain use of a single basic dye
• A mordant may be used to hold the stain or coat
  the specimen to enlarge it

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Differential Stains

• Used to distinguish between bacteria
• Gram stain
• Acid-fast stain

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Gram Stain

• Classifies bacteria into gram-positive or
  gram-negative
• Gram-positive bacteria tend to be killed by
  penicillin and detergents
• Gram-negative bacteria are more resistant to
  antibiotics

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Gram Stain
Color of Gram-Positive Cells Color of Gram-Negative Cells
Primary Stain Crystal Violet Purple Purple
Mordant Iodine Purple Purple
Decolorizing Agent Alcohol-Acetone Purple Colorless
Counterstain Safranin Purple Red
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Figure 3.12b Gram staining.
Rod (gram-negative)
Cocci (gram-positive)
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Special Stains

• Used to distinguish parts of cells
• Capsule stain
• Endospore stain
• Flagella stain

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Endospore Staining

• Primary stain malachite green, usually with heat
• Decolorize cells water
• Counterstain safranin

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Figure 3.14b Special staining.
Endospore
Endospore staining