Download Microbial nutrition, ecology and growth

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.
• All organisms require certain physical or
environmental conditions to grow. If those
physical conditions are not met, the organism’s
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-loving
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.
• 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 body’s pH is also within that range.
(Most bacteria can’t 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.
• 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.
• 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.
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.
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–
–
–
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Photoautotroph
Chemoautroph
Photoheterotroph
Chemoheterotroph
Saprobe: A group of organisms that use decaying matter as it’s carbon and
energy source.
– Parasite: Requires a host to get it’s carbon and energy sources.
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.
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.
• 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!
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
• Liquid:broth 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.)
– 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 organism’s
nutrient requirements must be known and planned for.
– 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, let’s 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 Gbacteria, 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 can’t 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
can’t break down the red blood cells so the media appears
unchanged.
• 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 it’s 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.
• 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.
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:
nf=ni(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. (I’ll 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.
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.
Preserving bacterial cultures
Once we are able to grow our bacteria we want to be able to
preserve them so we don’t 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.
• I’ll post the homework for this lecture on
Thursday morning. 9/14/06 It will be due on
Monday.