Download Chapter 6 Microbial Growth

Chapter 6—Microbial Growth
Note: I will not lecture on the material up to Roman Numeral V (below), but you are responsible for knowing
the material covered in these sections.
I.
Microbial growth = increase in number of cells, not cell size.
a. Physical Requirements:
i. Temperature. Fig. 1.
1. Minimum growth temperature = lowest temperature at which a species will grow.
2. Optimum growth temperature = temperature at which a species will grow best.
3. Maximum growth temperature = highest temperature at which growth is possible.
4. Psychrophiles are cold loving microbes that can grow at 0°C but grow optimally
around 15°C.
a. Found deep in the ocean or in polar regions.
5. Psychrotrophs are also cold loving microbes.
a. Grow at 0°C but optimally at 20-30°C.
b. Cause food spoilage because they are common and grow well in
refrigerators.
6. Mesophiles are moderate temperature loving microbes.
a. Grow optimally between 25-40°C.
b. Most common types of microbes. Many organisms that grow inside
animals fall into this group.
c. Includes many common spoilage and disease organisms.
i. When large amounts of food are to be refrigerated, the time
necessary for adequate cooling can allow bacteria to grow to high
numbers. Figs. 2 and 3.
7. Thermophiles are heat loving microbes.
a. Many grow optimally between 50-60°C.
8. Archaean hyperthermophiles (extreme thermophiles) grow optimally at
temperatures up to 80°C or greater.
a. Found in geothermal hot springs and vents.
b. Deep ocean hydrothermal vents have bacterial populations that can
survive temperatures of about 110°C; [pressure vs. boiling point].
ii. pH:
1. Most bacteria grow between pH 6.5 and 7.5.
a. In the lab, bacteria often produce acids that can inhibit their own growth
with increasing [H+].
2. Molds and yeasts grow between pH 5 and 6.
3. Acidophiles grow in acidic environments.
iii. Osmotic Pressure. Fig. 4.
1. When a microbe is suspended in a hypertonic solution, water moves out of the
cell via osmosis.
a. This causes plasmolysis = shrinkage of the cell’s cytoplasm.
i. This can permanently damage the plasma membrane.
b. Growth is inhibited as the plasma membrane pulls away from the cell
wall.
c. Salted meats, honey, and sweetened condensed milk are preserved via this
mechanism.
i. Extreme and obligate halophiles require high salt concentrations in
order to grow.
ii. Facultative halophiles do not require high salt concentrations, but
can grow in salt concentrations up to 2%, which inhibits the
growth of many microbes.
2. When a microbe is suspended in a hypotonic solution, water enters the cell via
osmosis. If the microbe has a relatively weak cell wall, it may lyse (osmotic
lysis).
b. Chemical Requirements:
i. Carbon:
1. Used to make structural organic molecules and energy rich molecules.
2. Heterotrophs use organic carbon sources.
3. Autotrophs use CO2.
ii. Nitrogen:
1. Found in amino acids, proteins, DNA, RNA, ATP.
2. Most bacteria decompose proteins.
3. Some bacteria use ammonium ions (NH4+) or nitrate ions (NO3) as nitrogen
sources.
4. A few bacteria use atmospheric N2 in nitrogen fixation.
a. Anabaena spp., Rhizobium spp.
iii. Sulfur:
1. Found in the R group of some amino acids and the vitamins thiamine and biotin.
2. Also necessary for synthesis of DNA & RNA.
3. Some bacteria use sulfate ions (SO42) or H2S as their sources of sulfur.
iv. Phosphorus:
1. Found in DNA, RNA, ATP, and phospholipids.
2. Phosphate ion (PO43) is a source of phosphorus.
v. Trace Elements:
1. Inorganic elements required in small amounts.
2. Usually needed to serve as enzyme cofactors.
3. Iron, copper, molybdenum, zinc, etc.
vi. Oxygen (O2): Table 6.1.
1. Obligate aerobes require oxygen to live, and use molecular oxygen as the final
electron acceptor in the ETC of the aerobic pathway.
a. Microaerophiles are aerobic, but grow only when oxygen concentrations
are lower than that found in air.
2. Facultative anaerobes can use oxygen for aerobic respiration when it is present,
but are able to grow via fermentation or anaerobic respiration when oxygen is not
available.
3. Obligate anaerobes generally are not able to grow in the presence of molecular
oxygen, but do use oxygen atoms already present in cellular molecules.
4. Aerotolerant anaerobes cannot use oxygen for metabolism, but tolerate it fairly
well.
5. Toxic Forms of Oxygen:
a. Singlet oxygen: 1O2- boosted to a higher energy state.
i. Extremely reactive. Present in phagocytic white blood cells.
b. Superoxide free radicals: O2
i. Note: The symbol “” refers to an unpaired electron. Only free
radicals have an unpaired electron.
ii. Highly unstable; formed during normal aerobic cellular respiration
when oxygen is used as the final electron acceptor.
iii. Steal electrons from neighboring molecules, turning them into
radicals.
II.
III.
iv. Neutralized by superoxide dismutase to produce H2O2.
c. Peroxide anion: O22
i. Two peroxide anions combine with 2 protons to form O2 and H2O2.
H2O2 is then neutralized by catalase to form water and O2.
d. Hydroxyl radical (OH).
i. Note: This is not the same thing as OH.
ii. Produced by ionizing radiation and when H2O2 reacts with certain
metal ions.
iii. Highly reactive.
vii. Organic Growth Factors:
1. Organic compounds obtained from the environment that the organism is unable to
synthesize itself.
2. Vitamins, certain amino and fatty acids, purines, pyrimidines.
Biofilms.
a. Complex communities of microorganisms living together.
i. Can communicate with one-another to coordinate activities, similar to the way cells in a
multicellular organism communicate.
ii. Are often difficult to destroy, and are commonly found on catheters.
Culture Media.
a. Culture Medium: Nutrients prepared for microbial growth.
b. Sterile: No living microbes.
c. Inoculum: Introduction of microbes into the medium.
d. Culture: Microbes growing in/on culture medium.
e. Agar:
i. Developed by Hess, 1882.
ii. Complex polysaccharide.
iii. Used as solidifying agent for culture media in Petri plates, slants, and deeps.
iv. Generally not metabolized by microbes.
v. Liquefies at 100°C.
vi. Solidifies ~40°C.
f. Chemically Defined Media: Exact chemical composition is known.
i. Provides a source of energy, carbon, nitrogen, sulfur, phosphorous, and growth factors.
ii. Organisms that require many growth factors are termed fastidious organisms.
iii. Normally used for the growth of autotrophic bacteria (use CO2 as the carbon source).
g. Complex Media:
i. Extracts and digests of yeasts, meat, or plants that provide vitamins and organic growth
factors.
ii. Peptone is the primary energy, carbon, nitrogen, and sulfur source.
iii. Used to grow heterotrophic bacteria and fungi (which require an organic carbon source).
iv. Liquid form is called a nutrient broth.
v. Solid form is called a nutrient agar.
h. Anaerobic Growth Media and Methods:
i. Reducing media.
1. Contain chemicals (such as sodium thioglycolate) that combine with O2.
2. Heated to drive off O2.
ii. Anaerobic jar. [DO NOT COVER]
iii. Anaerobic chamber. [DO NOT COVER]
iv. Capnophiles require high CO2 concentrations for growth.
1. Candle jar. [DO NOT COVER]
2. CO2 packet. [DO NOT COVER]
i. Selective Media.
IV.
V.
VI.
i. Halts or inhibits the growth of a particular group of microorganisms, while generally not
affecting the growth of another group of microorganisms.
ii. 7% NaCl.
j. Differential Media.
i. Provides a visual difference between groups of organisms that is not related to how well
the organisms grow on the medium.
ii. Blood Agar. Fig. 9.
k. Eosin Methylene Blue (EMB).
i. Selective against G+ bacteria, and differential on the basis of lactose fermentation.
l. Enrichment Media.
i. Encourages growth of desired microbe but not others, so it is also a selective medium.
ii. Assume a soil sample contains a few phenol-degrading bacteria and thousands of other
bacteria.
iii. Inoculate phenol-containing broth culture medium with the soil and incubate.
1. Phenol is the only source of carbon and energy.
iv. Transfer 1 ml to another flask of the phenol medium and incubate.
v. Transfer 1 ml to another flask of the phenol medium and incubate.
vi. Only phenol-metabolizing bacteria will be growing.
m. Table 6.5 summarizes the major types of culture media.
Obtaining Pure Cultures.
a. A pure culture contains only one species or strain of microorganism.
b. A colony is a population of cells arising from a single cell or spore or from a group of attached
cells.
i. A colony is often called a colony-forming unit (CFU).
c. Streak Plate Method. Fig. 11.
i. Also known as streaking for isolation.
ii. Works well when the organism to be isolated is present in large numbers relative to the
total microbial population. Otherwise, the organism must be grown in an enrichment
medium first.
Preserving Bacterial Cultures.
a. Short-term storage is accomplished by refrigeration.
b. Long term storage:
i. Deep-freezing: a pure culture in a suspending liquid which is then quick frozen at -50° to
-95°C.
1. Preserves the culture for several years.
ii. Lyophilization (freeze-drying): Frozen (-54° to -72°C) and dehydrated in a vacuum
(sublimation).
1. Preserves the culture for many years.
Growth of Bacterial Cultures.
a. Bacterial Division.
i. Binary fission. Fig. 12.
ii. Budding.
1. Cell forms a small outgrowth that enlarges until it is nearly as large as the parent
cell. It then separates.
b. Generation Time.
i. Time required for a cell to divide.
ii. Can be as fast as 20 minutes. Most bacteria have a generation time of 1-3 hours. Some
require more than 24 hours.
c. Logarithmic Representation of Bacterial Populations. Figs. 13 and 14.
d. Phases of Growth. Fig. 15.
i. When an inoculation is made and microbial numbers are counted at regular intervals, a
bacterial growth curve can be generated.
ii. Lag Phase:
1. Initially, the number of cells changes very little because they immediately do not
reproduce in a new medium.
2. Can last from an hour to several days.
3. Cells are metabolically active synthesizing new molecules, but are not dividing.
iii. Log Phase = Exponential Growth Phase:
1. As cells begin to divide, generation time reaches a constant minimum and
exponential growth of the population occurs.
2. Cells are maximally metabolically active during this time.
iv. Stationary Phase:
1. Growth rate slows, and microbial death rate approaches the rate of new microbe
formation via binary fission.
2. Exhaustion of nutrients, accumulation of waste products, and changes in pH may
all play a role in halting the exponential growth phase, which causes this
stationary phase.
v. Death Phase:
1. Number of cells dying exceeds the number of new cells being formed.
(Logarithmic decline phase = exponential decline phase).
2. The population dies off until only a small number of cells survive, or the
population dies out entirely.
e. Direct Measurements of Microbial Growth.
i. Plate Counts: Fig. 16.
1. Perform serial dilutions of a sample.
2. Inoculate Petri plates from serial dilutions.
3. After incubation, count colonies on plates that have 25-250 colonies (CFUs).
4. The count is used to estimate the number of bacteria in the original sample.
5. Plate counts are done using either the pour plate method or the spread plate
method. Fig. 17.
ii. Filtration. Fig. 18.
1. Used when bacterial numbers are low within a liquid or gaseous medium.
2. Filter the liquid, then transfer the filter to a Petri dish containing a pad saturated in
liquid nutrient medium.
3. Colonies will grow on the filter’s surface.
4. Often used to detect bacteria in water bodies/supplies.
iii. Multiple tube most probable number (MPN) test. Fig. 19.
1. Count positive tubes and compare to statistical MPN table.
iv. Direct Microscopic Count. Fig. 20.
1. A measured volume of liquid is placed on a defined area of a microscope slide,
and the bacteria present are counted.
a. Not good for counting motile bacteria.
b. Non-motile dead bacterial cells are as likely to be counted as live ones.
c. A high concentration of cells is required in order for them to be counted.
d. No incubation time is required to make a count.
f. Estimating Bacterial Numbers by Indirect Methods.
i. Turbidity. Fig. 21.
ii. Metabolic activity.
1. Assumes that a bacterial metabolic product accumulates in direct proportion to the
number of bacteria present.
iii. Dry weight.
1. Used for filamentous bacteria and molds.
2. The organism is filtered from the growth medium/sample, then dehydrated and
weighed.