Tools of the Laboratory:

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Tools of the Laboratory

• The Methods for Studying Microorganisms
• Chapter 3

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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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• Inoculation Incubation
• Prior to inoculation the specimen must be
  obtained. How and where it is obtained is
  extremely important to the process. It is is
  obtained improperly or from the wrong site, the
  experiment or analysis can fail. For instance,
  if you did a throat swab for someone with a
  diarrheal infection, you would probably not
  isolate the causative agent. Unfortunately, a
  rectal swab or a stool sample would be dictated
  in this case. Likewise, if the specimen is not
  kept alive, the experiment will fail. For this
  reason, specimens are often taken and immediately
  placed in rich media or on ice. It is also
  necessary to protect them from outside
  contamination prior to inoculation so they are
  placed in sterile tubes/containers immediately
  after they are collected.

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The media that is used for inoculation is also
important because many bacteria have very
explicit growth factor requirements and will not
grow in anything that does not have this growth
factor. In some cases we know these and use
special media or special hosts. For instance,
viruses are often inoculated into embryonated
eggs. When we dont know the growth requirements
of an organism (or dont know the organism) we
use growth media that is very rich, containing
many different growth factors. In some cases
this wont even work because the concentration of
salt of some other substance in the rich medium,
inhibits growth. The fact is that there are more
than 4,000 individual bacterial species in a gram
of soil (rich soil from the Midwest). We can
only grow in culture about 1 of these because
the rest have media/growth requirements that we
cannot duplicate. They are termed unculturable
organisms.
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The characteristics of incubation are also very
important. For instance, bacteria have optimum
growth temperatures. Bacteria isolated from
humans tend to grow best at 37C, whereas
bacteria isolated from the soil tend to grow
better at 25 C. Why do you think this is? In
fact, some soil organisms die when incubated at
37 C. Oxygen requirements are also important
during incubation. Some bacteria can only grow
in air (presence of oxygen). They are termed
strict aerobes. Other bacteria can only grow
in the absence of oxygen. They are termed
strict anaerobes. Other bacteria can grow in
either the presence or absence of oxygen. They
are facultative anaerobes (sometimes called
facultative aerobes). So before you incubate, you
must know something about the organism. The
collection site can tell you a lot.
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• at what temperature would you incubate an
  organism if you collected it from a deep-sea
  thermal vent?
• what aerobic conditions would you use if you
  isolated an organism from someones face?
• how about if you isolated an organisms from the
  mud found at the bottom of the salt river?

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Isolation
Why? In order to study a species you must have
it isolated (in pure culture) before you can
attribute specific traits. If you had the Mormon
tabernacle choir sing to you, could you tell who
were the best singers? Not likely.
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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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The same type of isolation can be done using the
pour plate dilution method, or by simply making
dilutions of a sample and plating them out
(spread plate 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 (like
  Jello)
• provides framework to hold moisture nutrients
• not digestible for most microbes

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Types of media

• liquid tubes, flasks, fermentors. Nutrient
  broth (peptone and beef extract)
• semisolid fluid thioglycolate. Has a low
  percentage of solidifying agent in it so it is
  viscous, but not solid. Usually tubes.
• solid converted to liquid. Nutrient agar.
  Usually plates, slants, or stabs.
• solid cannot be liquefied. potato slices, rice
  grains, etc.

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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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An example of synthetic medium
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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
It is important to note that selective media is
usually not totally selective, but also takes
into account the normal flora of the collection
site.
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Examples of selective media
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Differential media
TSI slant no growth, growth but no acid, acid
only in bottom, acid througout, acid and H2S
production Chromagar
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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 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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When analyzing characteristics of bacteria, it is
usually very important that you have a pure
culture. Otherwise, you will not be able to
attribute your results to a single species.
Fortunately, bacterial colonies can be quite
diverse in their appearance and the establishment
of a pure culture can be determined by
macroscopic observation. To insure that a pure
culture is obtained, new plates are made by
inoculating from a single colony.
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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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Effect of wavelength on resolution
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The basis for the resolution of the electron
microscope D ?/2 x N.A. The numerical
aperture of a light microscope and an electron
microscope are virtually the same. However,
electron waves are 100,000X shorter than the
waves of visible light. So what does that do to
the equation? If resolution of light microscope
is 0.2 micrometers, then resolution of electron
microscope is about 0.000002 micrometers, or
0.002 nm or 2 picometers. (I personally think
that this is pushing it a bit. globular proteins
are about 5 nm in width (average) so you can
actually see molecules)
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Oil immersion lens
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Effect of magnification
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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.

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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