Tools of the Laboratory

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Tools of the Laboratory

• This lecture will address the types of media used
  for studying microbes in the lab as well as there
  basic nutrient needs.

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• All organisms require certain physical or
  environmental conditions to grow. If those
  physical conditions are not met, the organisms
  growth will either be inhibited or it will die.
• Temperature is an important physical condition.
  There are several different groups of organisms
  that prefer different temperature ranges for
  growth. Microbes that cause disease generally
  prefer a growing temperature equal to body
  temperature (37 degrees C).
• Psychrophiles A group of cold-loving microbes
  that generally grow at temperatures of -10 to 20
  degrees C.
• Mesophiles A group of moderate-temperature-lovin
  g microbes that generally grow at temperatures of
  10 to 50 degrees C. Pathogenic microorganisms
  are in this group. Refrigeration severely
  retards the growth of most pathogenic bacteria
• Thermophiles A group of heat-loving microbes
  that generally grow at temperatures of 40 to 73
  degrees C.

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• pH is another important physical characteristic
  for growth. Remember that the pH scale is from
  0-14. A low pH acidic whereas a high pH basic
  and 7 is neutral.
• The normal growth range for bacteria is pH 6.5 to
  7.5. Notice that the bodys pH is also within
  that range. (Most bacteria cant grow in orange
  juice because it is too acidic. So why does
  orange juice spoil?)
• Acidophiles are a small group of bacteria that
  can grow in pH 4 conditions. These organisms
  are non-pathogenic.
• Molds and yeasts grow in pH 5-6.
• Have you figured out the answer to the orange
  juice question? It is usually molds and yeasts
  that spoil the orange juice with their metabolic
  by products.

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• Osmotic pressure is used all the time in the food
  industry to for preservation.
• It is the addition of large quantities of salts
  or sugars to foods that result in shrinkage of
  cells due to the loss of water.
• Plasma membrane pulls away from the cell wall
  which results in inhibition of cell growth.
• Ex. In food prep salted fish, honey, sweetened
  and condensed milk, pickles
• All of the items above are preserved because of a
  large amount of salt or sugar.
• Halophiles A group of organisms that can grow
  in high salt concentrations.

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• All organisms also need some basic nutrients to
  sustain growth.
• Carbon makes up proteins, lipids, and
  carbohydrates
• N, S, P makes amino acids, ATP, DNA, RNA
• Trace elements iron, copper, molybdenum, zinc
• Used by enzymes for proper functioning.
• Organic growth factors needed for growth that
  cannot be synthesized by own enzymes (ie.
  Vitamins in humans)
• Oxygen, ATP, water
• If any of these major nutrients are missing then
  the organism cannot function properly and
  eventually stops growing (forms endospore) or
  dies.

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How microbes feed

• Heterotroph A group of microbes that use
  organic carbon as their carbon source for
  metabolism.
• Autotroph A group of microbes that use
  inorganic carbon as their carbon source, such as
  carbon dioxide.
• Phototroph A group of microbes that use light
  as their energy source.
• Chemotroph A group of microbes that use
  chemical compounds for their energy source, such
  as glucose.
• Photoautotroph
• Chemoautroph
• Photoheterotroph
• Chemoheterotroph
• Saprobe A group of organisms that use decaying
  matter as its carbon and energy source.
• Parasite Requires a host to get its carbon and
  energy sources.

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Gas Requirements

• Aerobe A group of organisms that use oxygen for
  metabolism.
• Facultative Anaerobe A group of organisms that
  do not require oxygen for metabolism.
• Microaerophile A group of organisms that
  require small amounts of oxygen for metabolism.
• Strict Anaerobe A group of organisms that
  require an element other than oxygen for
  metabolism, such as N, S, CO2.

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Ecological Association Among Microorganisms

• Symbiosis 2 organisms live together in a close
  partnership. There are several different types
  of symbiotic relationships.
• Mutualism Both organisms benefit from the
  relationship. For example, E.coli found in our
  intestines aids in our digestion and it benefits
  from us because our intestines provide a nice,
  warm, nutrient rich environment for it to grow.
• Commensalism In this relationship one organism
  benefits and the other is unaffected. For
  example, microbes that feed off of the dead skin
  cells that we shed are benefited by us but we are
  unaffected by them.
• Parasitism In this relationship one organism
  benefits at the expense of the other. A tape
  worm is an example of this relationship. The
  tape worm attaches itself to the intestine of the
  host. The host provides a nice, warm, nutrient
  rich environment. The tape worm grows and grows.
  It can eventually cause malnutrition or
  intestinal blockages that are harmful to the
  host.

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• Synergism This type of relationship is one in
  which there is an added effect by the close
  relationship.
• An example is found in biofilms. Many of you
  found that many groups of organisms make up a
  biofilm and that when these organisms are in a
  close relationship like that, new genes are
  turned on that benefit the whole group. Those
  genes are not activated when the organisms are
  alone.
• Antagonism This type of relationship is a
  competitive relationship between organisms.
• For example, many organisms make personal
  antibiotics that kill off competitors for
  nutrients. Many of the antibiotics that we use
  today were originally derived from these natural
  antibiotics. Penicillin is one!

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Uses of Media

• Because we understand the basic principles of
  nutrition for living cells, we can use that
  information to study them in the lab.
• The media that we used in lab to grow
  microorganisms is designed according to the
  metabolic properties of microorganisms.
• There are three basic properties that are used
  when deciding which media to use for growth of
  microbes.
• 1. Physical state
• Liquidbroth in a test tube. (The nutrient broth
  that we use in lab is an example of this type of
  media.)
• Semisolid broth that has a little agar added to
  it so that it has a thicker consistency but is
  not solid. (The motility agar that we used in
  our last lab is an example of this type of
  media.)
• Solid broth that has enough agar added to it to
  make it solid. (The agar plates that we have
  been using in lab are examples of this type of
  media.)

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• 2. Chemical composition We can manipulate the
  media to select for the growth of one organism
  and inhibit the growth of another. We can also
  manipulate the media to show us metabolic
  differences between organisms.
• Complex media is the type of media we have used
  thus far in lab. (Remember we talked about
  chicken broth for microorganisms. It is derived
  from yeast, meat, or plant digests and contains
  all the nutrients that a microbe needs to grow.
  In this type of media the amount of nutrients
  varies from batch to batch and is unknown.
• Chemically defined media is type of media in
  which the exact chemical or nutrient composition
  is known. Each nutrient is carefully weighed and
  added one by one to the media. In order for this
  media to support bacterial growth an organisms
  nutrient requirements must be known and planned
  for.

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• 3. Functional type This category of media
  determines if it will select for the growth of
  one type of organism or if it will show metabolic
  differences between organisms.
• Selective media This type of media suppresses
  the growth of unwanted bacteria and encourages
  the growth of desired organism.
• For example, lets say that we have a test tube
  that contains both G and G- bacteria in it. You
  want to study the G. To help you isolate the G
  from the G- bacteria you can use a media that
  inhibits the growth of G- bacteria, leaving only
  the G.
• Differential Media
• Differential media makes it easy to distinguish
  different genus and species of organisms.
• Blood agar is an easy way to distinguish between
  some organisms. It is media that contains sheep
  blood. It is red and solid. You cant see
  through it because of the red blood cell content.
  Some organisms have the ability to break down
  the red blood cells, S. pyogenes. When the red
  blood cells are broken down the media turns
  orange and transparent. Some organisms cant
  break down the red blood cells so the media
  appears unchanged.

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• Enrichment Media
• Allows for growth of microbes that are not
  normally detected. Some microbes grow in very
  small numbers because their nutrient requirements
  are not optimal. This type of media allows you
  to add the specific nutrient required to select
  for that one type of bacteria and encourage its
  growth so that it can be isolated from the rest.
• For example, you take a soil sample and want to
  study the organisms found within the sample. One
  of the organisms requires phenol as carbon and
  energy source. That is an unusual growth
  requirement so it has to be added to encourage
  the growth of that particular organism. When it
  is added to the media it will select for the
  growth of the organism you want, while other
  organisms are unable to grow in the presence of
  phenol.

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• Some special culture techniques are used to
  culture hard to grow organisms
• Ie. Mycobacterium, the organism that is
  responsible for Leprosy and Tuberculosis, has a
  low temperature requirement for growth.
  (Slightly lower than body temperature.) To study
  it in the lab it is grown in armadillos or in the
  foot pads of mice and rats because the body
  temperature is ideal.
• Some organisms only grow inside cells, such as
  viruses. So special tissue cultures have to be
  grown in order to replicate the viruses.
• Some organisms still cannot be grown in the lab
  because their growth requirements are not
  understood well enough.

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Growth of Bacterial Cultures

• Bacterial Division Bacteria reproduce through
  binary fission. This produces 2 identical cells
  each time the parent cell divides. See Fig.7.13
  for a great explanation on this concept.
• The Generation Time of a microbe is the time
  required for a cell to divide and the population
  to double.
• For example, E-coli doubles once every 20 min.
  10 cells become 20 cells after 20 minutes and 20
  cells become 40 cells after 20 additional
  minuets, etc.
• The following is the equation used to calculate
  the final population of a bacterial culture
• nfni(2)g
• nf is the final population ni
  is the initial population
  
• g is the number of generations 2 represents
  the doubling due to binary fission
• Try calculating the following problem. (Ill go
  over it in lab on Friday.)
• A culture was inoculated with 10 E-coli cells.
  Then it was put in the incubator for 2 hours.
  What was the final population?
• Hint first calculate the number of generations
  by figuring out how many times E.coli can divide
  within 2 hours if it has a doubling time of 20
  minutes.

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Bacterial Growth Curve

• 4 basic phases of growth
• 1. Lag phase New growth medium is added or a
  new culture is made. This period is a time of
  delayed growth while the bacterial cells prepare
  to divide. DNA is replicating, the cell wall and
  membrane or expanding, etc.
• 2. Log phase (exponential growth phase)
  Cellular division is most active during this
  period. The generation time reaches a constant
  minimum.
• 3. Stationary phase A state of equilibrium
  where the number of cell deaths equals the number
  of cell divisions.
• 4. Death phase The number of cell deaths
  exceeds the number of new cells. The nutrients
  are becoming depleted or a change in the physical
  conditions are making conditions unfavorable.
• Early death phase is when sporulation begins.
• See Fig.7.15 for a great example of the shape of
  the growth curve.

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Preserving bacterial cultures

• Once we are able to grow our bacteria we want to
  be able to preserve them so we dont have to go
  through the same tedious process to isolate and
  grow them. A couple of the methods that are
  used are
• 1. Deep-freezing liquid bacterial culture is
  added to liquid glycerin to prevent breaking of
  membrane due to freezing. Then it is frozen at
  temp. ranging from 50 to 72 degrees C.
• Lyophiliztion (freeze drying) The bacterial
  culture is quickly frozen at temp. ranging from
  54 to 72 degrees C. Then the water is removed
  by a vacuum leaving a powder like residue. The
  powder contains the organisms.
• Ill post the homework for this lecture on
  Thursday morning. 9/14/06 It will be due on
  Monday.