Microbiology Chapter 5 - Growth
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an organism that grows best at low pH; typically below pH 5.5
an organism that can use O2 in respiration; some require O2
Define: Aerotolerant anaerobe
a microorganism that can tolerate O2 and grow in its presence even though they cannot use it
O2 is not required and grow no better with it
an organism that has a growth pH optimum of 8 or higher
an organism that cannot use O2 in respiration and whose growth is typically inhibited by O2
Define: Antimicrobial agent
a chemical compound that kills or inhibits the growth of microorganisms
Define: Antiseptic (germicide)
a chemical agent that kills or inhibits growth of microorganisms and is sufficiently nontoxic to be applied to living tissues
a sealed heating device that destroys microorganisms with temperature and steam under pressure
Define: Bacteriocidal agent
an agent that kills bacteria Bacteriostatic agent an agent that inhibits bacterial growth
Define: Batch culture
a closed-system microbial culture of fixed volume
Define: Binary fission
cell division following enlargement of a cell to twice its minimum size
an attached polysaccharide matrix containing bacterial cells
Define: Cardinal temperatures
the minimum, maximum, and optimum growth temperatures for a given organism
a device that allows for the continuous culture of microorganisms with independent control of both growth rate and cell number
Define: Compatible solute
a molecule that is accumulated in the cytoplasm of a cell for adjustment of water activity but that does not inhibit biochemical processes
Define: Death (as it pertains to microbiology)
The irreversible loss of ability to reproduce
treatment of a surface or object to make it safe to handle
an antimicrobial agent used only on inanimate objects
rendering a surface or object free of all pathogen microorganisms
a complex of proteins that directs cell division processes in prokaryotes
Define: Exponential growth
growth of a microbial population in which cell numbers double within a specific time interval
Define: Extreme halophile
a microorganism that requires very large amounts of NaCl, usually greater than 10% and in some cases near to saturation, for growth
with respect to O2, an organism that can grow in either its presence or absence
Define: Facultative Aerobes
aerobes that, under appropriate nutrient and culture conditions, can grow under either aerobic or anaerobic conditions (they can grow throughout medium). O2 is not required but they grow better with it.
a protein that forms a ring along the mid-cell division plane to initiate cell division
Define: Fungicidal agent
an agent that kills fungi Fungistatic agent an agent that inhibits fungal growth
Define: Generation time
the time required for a population of microbial cells to double
Define: Germicide (antiseptic)
a chemical agent that kills or inhibits growth of microorganisms and is sufficiently nontoxic to be applied to living tissues
the orderly increase of all the major chemical constituents of an organism
a microorganism that requires NaCl for growth
a microorganism that does not require NaCl for growth but can grow in the presence of NaCl, in some cases, substantial levels of NaCl
Define: HEPA filter
a high-efficiency particulate air filter that removes particles, including microorganisms, from intake or exhaust air flow
an organism that grows best at temperatures between 15 and 40°C
a prokaryote that has a growth temperature optimum of 80°C or greater
aerobes that can use O2 only when it is present at levels reduced from that in air, usually because of their limited capacity to respire or because they contain some oxygen-sensitive molecule
Define: Minimum inhibitory concentration (MIC)
the minimum concentration of a substance necessary to prevent microbial growth
an organism that grows best at neutral pH, between pH 5.5 and 8
Define: Obligate anaerobe
an organism that cannot grow in the presence of O2
Define: Obligate aerobes
aerobes that require O2 for growth
an organism that grows best in the presence of high levels of solute, typically a sugar
the heat treatment of milk or other liquids to reduce its total number of microorganisms
the negative logarithm of the hydrogen ion (H+) concentration of a solution
Define: Plate count
a method of counting viable cells; the number of colonies on a plate is used as a measure of cell numbers
Give an example
an organism with a growth temperature optimum of 15°C or lower, a maximum growth temperature below 20°C, and a minimum below 0°C
Snow algae, ocean water (3°C)
Capable of growing at 0°C, but optimum of 20-40°C; more widely distributed
an agent that reduces microorganisms to a safe level, but may not eliminate them
Define: Sterilant (sterilizer, sporicide)
a chemical agent that destroys all forms of microbial life
the killing or removal of all living organisms and viruses, including endospores
an organism whose growth temperature optimum lies between 45 and 80°C
formation of peptide cross- links between muramic acid residues in peptidoglycan synthesis
capable of reproducing
Define: Viable count
a measurement of the concentration of live cells in a population
Define: Viricidal agent
an agent that stops viral replication and activity
Define: Viristatic agent
an agent that inhibits viral replication
Define: Water activity (aw)
the ratio of the vapor pressure of air in equilibrium with a solution to the vapor pressure of pure water
an organism that is able to live, or that lives best, in very dry environments
True or False: Growth always implies increase total mass
Increase in total mass is not necessarily growth since this may be due to accumulation of cellular reserve material
Knowing growth "normally" results in cellular multiplication, is there a specific exception (organism) to this?
Growth normally results in cellular multiplication except in the special case of coenocytic (multinucleated) organisms
In a multicellular organism, cellular multiplication results in an increase in _______________. In unicellular organisms, it leads to an increase in _____________.
In a multicellular organism, cellular multiplication results in an increase in size of the individual. In unicellular organisms, it leads to an increase in the number of individuals
List and briefly describe the processes by which Growth relies on. Which of these factors is the most important?
- Metabolism: a large number of chemical reactions
- Fueling: some transformation of energy
- Biosynthesis: synthesis of small molecules (building blocks, coenzymes, vitamins, etc)
- Polymerization: reactions that make the macromolecules
is the most important, as the products that occur in this phase are synthesized and accumulated in the cell they are then assembled into cell structures and the cell grows in size and eventually divides
What is the most common form of unicellular microbial reproduction?
Transverse/Binary Fission (with the term “binary” expressing the fact that two cells have arisen from one cell)
What results from invagination of the cell membrane and cell wall?
A Septum results from invagination of the cell membrane and cell wall
What is invagination?
Invagination is the inward growth of the cytoplasmic membrane and cell wall from opposing directions
What proteins are essential for cell division, and are present in all Bacteria?
Fts proteins (filamentous temperature sensitive) are essential for cell division, and they are present in all Bacteria
True of False: Fts proteins are present in most, but not all, Bacteria
Fts Proteins are present in all bacteria
What could a mutation in the genes that code for Fts proteins cause?
Mutations in genes encoding for Fts proteins could cause the cell to not divide normally (Such as long filamentous cells that fail to divide)
Fts proteins interact in the cell to form ______
Fts proteins interact in the cell to form a division apparatus called the divisome
How does the formation of the FtsZ ring begin?
Formation begins with attachment of molecules of FtsZ in a ring around the center of the cell (10,000 proteins needed to polymerize) which attracts other divisome proteins
Briefly describe the following proteins...
- ZipA: an anchor that connects the FtsZ ring to the cytoplasmic membrane and stabilizes it
- FtsA: a protein related to actin; also helps to connect the TfsZ ring to the cytoplasmic membrane and recruit other divisome proteins
- FtsI: proteins needed for peptidoglycan synthesis; activity blocked by penicillin (is one of several penicillin-binding proteins)
Fts protein complex [graphic]
DNA replication and cell-division events [graphic]
The ________ defines the cell-division plane
The FtsZ ring defines the cell-division plane
Does DNA replicate before or after the FtsZ ring forms? Why or why not?
DNA replicates before the FtsZ ring forms because the ring forms in the space between the duplicated nucleoids; before the nucleoids segregate, they effectively block formation of the FtsZ ring.
What ensures that the FtsZ ring form only at the cell center?
Min proteins (MinC, MinD, and MinE)
How far into cell division cycle does the divisome form?
About 3/4 of the way through the cell division cycle
Describe the final few steps of cell division
FtsK and several other proteins help the two copies of the chromosomes to be pulled apart. As the cell constricts, the FtsZ ring depolymerizes, triggering inward growth of wall materials to form the septum and seal off one daughter cell from the other
What source of energy allows for the activity of FtsZ?
GTP hydrolysis provides energy for FtsZ’s enzymatic activity
Describe the role of MinC and MinD proteins
MinC and MinD proteins help dictate the center of the cell by inhibiting FtsZ ring formation outside the cell’s center (prevents cell division at the poles by being the most abundant protein at the cell poles)
MinD oscilates and helps MinC find the cytoplasmic membrane
The ________ is responsible for regulating synthesis of new cell membrane and wall material
The Divisome is responsible for regulating synthesis of new cell membrane and wall material
Describe what MreB does
The cytoskeleton protein MreB is an actin
analog that winds as a coil through the long axis of the rod shaped cells, making contact with cell membrane at sites of synthesis
In vibrio shaped bacteria, ________ is found along with MreB. This protein is similar to ______.
In vibrio shaped bacteria, crescentin is found along with MreB, this protein is similar to keratin
What presumably defines a bacterial cell's shape? How does it do this? What occurs when it is not present?
The MreB cytoskeleton presumably defines cell shape by recruiting other proteins that function in cell wall growth to group into a specific pattern. Inactivation of the gene encoding MreB in rod-shaped bacteria causes the cells to become coccus-shaped. Moreover, most naturally coccoid bacteria lack the MreB gene and thus do not make MreB. This indicates that the “default” morphology for a bacterium is most likely the sphere.
What role does Bactoprenol play in the biosynthesis of peptidoglycan?
- The lipid carrier molecule called bactoprenol plays a major role in the insertion of peptidoglycan precursors. Bactoprenol is a hydrophobic C55 alcohol that bonds to a N-acetylglucosamine/N-acetylmuramic acid/pentapeptide peptidoglycan precursor. Bactoprenol transports peptidoglycan precursors across the cytoplasmic membrane by rendering them sufficiently hydrophobic to pass through the membrane interior.
- Bactoprenol also interacts with enzymes called transglycosylases that insert cell wall precursors into the growing point of the cell wall and catalyze (make) the formation of new glycosidic bonds.
What occurs prior to insertion of cell wall precursors?
- Prior to the insertion of cell wall precursors, small gaps in the existing peptidoglycan are made by enzymes called autolysins, enzymes that function to hydrolyze (cut) the preexisting B-1,4-Glycosidic bonds that connect N-acetylglucosamine and N-acetylmuramic acid in the peptidoglycan backbone. Insertion of of peptidoglycan precursors occurs simultaneously
- New cell wall material is then added across the gaps
What is the final step in cell wall synthesis? What Fts protein catalyzes this step in E. coli and other cells?
- The final step in cell wall synthesis is transpeptidation. Transpeptidation forms the peptide cross-links between muramic acid residues in adjacent glycan chains.
- FtsI catalyzes the transpeptidase reaction in E. coli and other cells
What would occur to the cell in the absence of crosslinks and new cell wall synthesis?
In the absence of crosslinks and new cell wall synthesis, continued activity of autolysins weakens cell wall causing osmotic lysis (autolysis)
Peptidoglycan synthesis [graphic]
Briefly describe how certain bacterial cells are penicillin sensitive? What does penicillin cause in these cells?
Cells are penicillin sensitive because penicillin binds to FtsI and other penicillin binding proteins, thus no cross-linking occurs with new material.
A type of division which results in unequal distribution of cellular material between newly synthesized bud and the original cell
What ways can yeast cells divide?
Yeats cells can divide by budding or by binary fission
What can be quantitatively measured to determine growth in bacterial cells?
During growth, both the total cell number and mass double, both of which can be quantitatively measured and are usually equivalent due to non-synchronous growth
How is growth usually expressed?
Usually expressed as: (#cells/mL) or (mg cells/mL)
Cell mass is _________ whereas cell division is _________
Cell mass is continuous whereas cell division is discontinuous
_______ _______ is the pattern of population increase, where there is a doubling in the number of cells present
Exponential growth is the pattern of population increase, where there is a doubling in the number of cells present
Cells increase in size without dividing until the ______ _______ is reached
Cells increase in size without dividing until the generation time is reached
Measures made in what type of culture are equivalent to a single cell?
Measures made in Synchronous Culture are equivalent to a single cell
What does the Bacterial Growth Curve reflect? What does it not represent?
- The Bacterial Growth Curve reflects one selected portion of a normal growth curve, namely the Logarithmic/Exponential Phase.
- The Bacterial Growth Curve does not represent the normal pattern of bacterial growth
Cells divide at regular intervals known as ______
Cells divide at regular intervals known as generation time (g)
Although exponential growth is characteristic of microbial populations, it does not normally _______.
Although exponential growth is characteristic of microbial populations, it does not normally continue for long periods of time
Briefly describe the 4 phases of Bacterial Growth (think the growth curve) and relate it to K.
*(K = specific growth rate)
- Lag Phase: does not always occur in the growth of a bacterial population and when it does the duration can vary considerably (k = 0)
- Exponential or Log Phase: cannot continue indefinitely, limited (k = positive, reaches a max)
- Stationary Phase: no increase in cell number or cell mass (k = 0)
- Death Phase: cells die exponentially (k = negative)
What occurs each time a cell divides and produces two cells?
Each time a cell divides and produces two cells a generation (or doubling) occurs
Time required for a doubling to occur is the __________
g = ?
t = ?
n = ?
*Thus g = ?
Time required for a doubling to occur is the duration of growth (t)
- g = generation time
- t = duration of growth
- n = # of generations
- *Thus g = t/n
- Example problem: 1 cell multiples into 4 cells in 60 minutes. 1 -> 2 -> 4, so the # is doubling (ie # generations), which means n = 2.
- G = t/n, thus g = 60 min/2 doublings = 30 min/doubling = 30 min/gen
When a microbial population is inoculated into a fresh medium, growth usually does not take place immediately but only after a period of time called _______ does growth begin again.
When a microbial population is inoculated into a fresh medium, growth usually does not take place immediately; only after a period of time called the lag phase does growth begin again.
Why does the Lag Phase occur?
The lag phase occurs because for growth to occur in a particular culture medium, the cells must have a complete complement of enzymes for the synthesis of the essential metabolites
What metabolic subdivision occur in lag phase?
- Metabolism's subdivisions I, II, III & IV occur in lag.
- All phases are at maximal rate and cells multiply according to g
- As IV occurs, the cell gets larger and prepares to divide
When can a lag phase occur? Why?
- When you inoculate cells from stationary or death phase into a fresh medium (Cells are deficient from previous growth)
- When you inoculate cells from exponential phase into a fresh medium of different chemical composition (Cells growing on glucose are then placed in lactose)
- When you inoculate cells from a rich to a poor culture (aka medium shift) (Cells growing in complex medium are moved to defined medium)
When does a lag phase not occur?
When you inoculate cells growing exponentially into a fresh medium of the same chemical composition
Define: Biphasic Growth
Biphasic growth: two different carbon and energy sources (Ex: glucose and lactose)
IE: Able to ferment galactose and fructose
Give a description of an example of Biphasic Growth
- Stationary culture cells are inoculated into a medium containing both glucose and lactose. Growth first occurs on the most rapidly metabolized carbon source (glucose), and then lag occurs before growth on lactose.
- 2 extra enzymes needed for this specific case, Lactase permease to bring in the lactose and, B-galactosidase to break lactose down to galactose and glucose
The pattern of population increase where the number of cells doubles during each unit of time period is referred to as ________ _______
The pattern of population increase where the number of cells doubles during each unit of time period is referred to as exponential growth
Growth rate is maximal when?
- Cells are the healthiest
- Prokaryotes grow faster than eukaryotes
- Smaller cells grow faster than larger cells
What are the two reasons that cause Stationary Phase to occur?
Stationary phase occurs because either:
- 1) An essential nutrient of the medium is used up
- 2) Waste by-product of the organism builds up to an inhibitory level
In stationary phase there is no _______ in cell number, but many cell functions can continue
In stationary phase there is no net increase or decrease in cell number, but many cell functions can continue
Define: Cryptic growth
Some cells die while some cells are doubling
The Death Phase occurs when? How does the Death Phase compare to the exponential growth rate?
When the cells are no longer able to find the nutrients needed, thus the cells can die
The death phase is also exponential, but is slower than the growth exponential rate
True of False: The terms lag phase, exponential phase, stationary phase and death phase apply to individual cells
The terms lag phase, exponential phase, stationary phase and death phase do not apply to individual cells, and instead only to populations of cells
What does a Spectrophotometer measure? Nepholometer?
- Spectrophotometer: Measures light passing through the solution (Unscattered & unabsorbed)
- Nepholometer: Measures scattered light
Why are Biofilms an issue?
Biofilms are an issue because antibiotics may not penetrate and kill all the bacteria present
What is the Petroff-Hausser Counting Chamber?
A special slide and cover slip which contains a known volume.
The number of bacteria per milliliter (ml) = number of cells counted X dilution (if used) x 50,000
50,000 = 50 (cell depth is 1/50) X 1000 (1000 cubic mm = 1 milliliter)
Ex: 300 cells X 50,000 = 1.5 x 107 cells/ml
How does the Direct Microscopic Count procedure occur?
A small portion (0.01 to 0.02 mL) of a bacterial suspension is smeared on a glass slide in a prescribed area (usually 1 cm2).
The film is stained and the # of microorganisms per microscopic field (MF) is recorded
- You want to have 1-2 microscopic organisms per field and to count 50 microscopic fields
- If more than 1-2 microscopic organisms are present, fewer fields may be counted
Viable Plate Count [graphic]
Describe how the Direct Microscopic Count Calculations are made
The number of microscopic organisms/mL in a suspension= (Avg# of MO's/field)x(Microscopic factor)x(Dilution factor)
Microscopic Factor (MF) = Area of the film (Af
)/Area of the microscopic field (Amf
- Note: Af should be LARGER than Amf
- Amf is how much you can see in the microscope
What are the rates of growth and total amount of growth are governed by?
Rates of growth and total amount of growth are governed by the physical and chemical environment
List the various physical and chemical environmental factors that affect rates of growth and total amount of growth...
- Physical Environment: Temperature, Hydrostatic pressure, Osmotic Pressure, Surface tension, Visible radiation, UV radiation, Gravity, Adsorption phenomena, Viscosity
- Chemical Environment: Water activity (aw), Water structure, pH, Inorganic nutrients quantity and quality, Gasses quality and quantity, Organic nutrients quantity and quality, Hormones, growth regulations, metabolic control substances, Poisons, inhibitors and nutrient analogs, Oxidation-reduction potential
Temperature effects on growth comparisons [graphic]
What corresponds with a marked change in the environment (either physical or chemical)?
Any marked change in the environment produces a corresponding change in the morphological and/or physiological characteristics of the organism
Describe what makes up the Cardinal Temperatures
- For every organism there is a minimum temperature below which no growth occurs, an optimum temperature at which growth is most rapid, and a maximum temperature above which growth is not possible
- The optimum temperature is always nearer the maximum than the minimum
These three temperatures are called the cardinal temperatures
What adaptions in protein structure allow psychrophillic organisms to thrive in their environments?
Adaptations include changes in protein structure:
- 1) More α-helix, less B-sheets
- 2) More polar, less hydrophobic amino acids
- 3) More unsaturated and shorter fatty acids
If pH changes one pH unit (from 2 to 3), how much has [H+] changed?
If pH changes one pH unit (from 2 to 3), then [H+] changes 10 fold.
Thus for a pH change from 2 -> 4, the [H+] changes 10 x for each pH unit change or 10 x 10 = 100 fold
aw = ?
= Psoln. / Pwater
- Psoln. is the water vapor pressure above the solution
- Pwater is the water vapor pressure of pure water
aw is always less than or equal to _____
aw is always less than or equal to one (< or = 1)
Most bacteria fall within what range of pH?
Most bacteria fall within range pH 4-9
Most bacteria grow above aw = ?
Most bacteria grow above aw = 0.95
Bacteria growing in an environment where the inside of the cell has a higher solute concentration than the outside if the cell is said to be in _________.
Bacteria growing in an environment where the inside of the cell has a higher solute concentration than the outside of the cell is said to be in positive water balance
Organisms growing in a low water activity environment must drive water in by increasing the solute concentration inside through what two methods?
Organisms growing in a low water activity environment must drive water in by increasing the solute concentration inside through:
- 1) Pumping in solutes from the outside
- 2) Making compatible solutes (Solutes used inside the cell for adjustment of cytoplasmic water activity; Highly water soluble; Must be non-inhibitory; This is all genetically determined)
Staph uses the amino acid _______
Staph uses the amino acid proline
Thioglycolate Tubes contain which dye?
Thioglycolate Tubes contain the dye resazurin
which is pink
when oxidized, and colorless when reduced
_________ is used in halophilic bacteria and cyanobacteria
Glycine betaine is used in halophilic bacteria and cyanobacteria
What is a major form of toxic oxygen to the cell is? How is it toxic? How can some organisms prevent harm from it?
A major form of toxic oxygen to the cell is singlet oxygen (1O2)
Electrons on its outer shell become highly reactive and can carry out spontaneous and undesirable oxidation
Organisms which often encounter singlet oxygen contain pigments known as carotenoids which convert it to nontoxic form
Apart from singlet oxygen, what are some other forms of toxic oxygen?
Other forms of toxic oxygen include:
- 1) Superoxide anion (O2-)
- 2) Hydrogen peroxide (H2O2) – least reactive
- 3) Hydroxyl radical (OH*) – most reactive
What are all toxic oxygen's by-products of?
All are by-products of the reduction of O2
Even if a cell cannot respire O2, through what other means may it catalyze O2?
Even if a cell cannot respire O2, it contains enzymes such as quinones which catalyze the reduction from O2 to O2-
Obligate and facultative aerobes typically have which two enzymes?
Obligate and facultative aerobes typically have both catalase and superoxide dismutase (SOD)
Aerotolerant anaerobes typically lack which enzyme, but have which other enzyme?
Aerotolerant anaerobes typically lack catalase but have superoxide dismutase (SOD)
Obligate anaerobes typically lack which two enzymes?
Obligate anaerobes typically lack both catalase and superoxide dismutase (SOD)
Enzymes that destroy toxic oxygen species [graphic]
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