Chapter 9- Microbial growth Flashcards
In prokaryotes, reproduction is always
Asexual. Genetic reproduction can occur in the form of horizontal gene transfer.
What form is bacterial DNA usually in?
Most bacteria have a single circular chromosome
Binary fission (3)
The most common mechanism of cell replication in bacteria.
1. The cell grows and increases its number of cellular components
2. DNA replication begins at the origin of replication
3. The center of the enlarged cell constricts until two daughter cells are formed
Origin of replication
The region of the bacterium’s circular chromosome that is attached to the inner cell membrane. This is where DNA replication begins during binary fission. Replication continues in opposite directions along the chromosome until it reaches the terminus
How do the offspring resemble the original cell after binary fission?
The offspring are clones of the original cell. They receive a complete copy of the parental genome and a division of the cytoplasm through cytokinesis
FtsZ
A protein that directs the process of cytokinesis and cell division in bacteria. It assembles into a Z ring on the cytoplasmic membrane. The Z ring is anchored by FtsZ binding proteins and provides a plane for cell division
FtsZ
A protein that directs the process of cytokinesis and cell division in bacteria. It assembles into a Z ring on the cytoplasmic membrane
Divisome
During binary fission, additional proteins are added to the Z ring to form this structure. The divisome will eventually produce a peptidoglycan cell wall wall and will build a septum that divides the two daughter cells
Division septum
Separate the two daughter cells during binary fission. This is where the cell’s cell wall and outer membranes are remodeled to finish the division process
Generation time
In prokaryotes, this is also called the doubling time- the time it takes for the population to double through one round of binary fission. Different species of bacteria have different generation times
Growth curve
The growth pattern of microorganisms plotted as a logarithm of bacterial cells (y axis) and time (x) axis
Culture density
The number of cells per unit volume. In a closed environment, this is also a measure of the number of cells in the population
Phases of the growth curve (4)
- Lag phase- no increase in the number of living bacterial cells
- Log phase- exponential increase in the number of living bacterial cells- positive slope
- Stationary phase- plateau in number of living bacterial cells, the rate of cell division is equal to the rate of cell death
- Decline phase- exponential decrease in the number of living bacterial cells
Inoculum
The small number of cells that are initially added to a fresh culture medium
Culture medium
A nutritional broth that supports growth
Lag phase
The part of the growth curve where cells are gearing up for the next phase of growth. The cells grow larger and are metabolically active so that they can synthesize necessary proteins for growth in that medium
Logarithmic (log) growth phase
The part of the growth curve where cells are actively dividing by binary fission- their number increases exponentially. Generation time under specific growth conditionals is genetically determined. Cells in this phase have constant growth and uniform metabolic activity. This is the stage where bacteria are most susceptible to disinfectants and antibiotics that affect protein, DNA, and cell wall synthesis
Intrinsic growth rate
The genetically predetermined generation time of bacteria in specific conditions
Stationary phase
A plateau in the number of living bacterial cells in the growth curve. At this point, the rate of cell division is equal to the rate of cell death, so the total population of living cells is mostly stagnant. This occurs because as the number of cells increase, waste products accumulate, oxygen is depleted, and nutrients are used up- this creates unfavorable living conditions and cells start to die. During this phase, cells switch to a survival mode of metabolism. Many cells undergo sporulation if they are capable of producing endospores. Cells synthesize secondary metabolites, including endospores, in this phase. Virulence factors are also synthesized
What determines a culture’s carrying capacity?
The carrying capacity/maximum culture density depends on the types of microorganisms in the culture and the specific conditions of the culture. However, carrying capacity is constant for a given organism grown under the same conditions
How do cells’ metabolism change during the stationary phase?
Metabolism switches to survival mode. As the growth of the cells slows down, so does the synthesis of peptidoglycans, proteins, and nucleic acids. This means that stationary cultures are less susceptible to antibiotics that disrupt these processes
Virulence factors
Products that contribute to a microbe’s ability to survive, reproduce, and cause disease in a host organism. S. aureus bacteria produce enzymes that can break down human tissue and cellular debris, which allows bacteria to spread to new tissue where nutrients are more plentiful. These products are typically synthesized during the stationary phase
Death phase
The phase of the growth curve where cells die in greater numbers, and the number of dying cells exceeds the number of dividing cells. This occurs because the culture medium begins to accumulate toxic waste and nutrients are exhausted. Some cells lyse and release nutrients, which allows some of the other cells to survive and form endospores.
Persisters
Cells that are characterized by a slow metabolic rate. These cells are associated with chronic infections that do not respond to antibiotic treatment, such as tuberculosis
Chemostat
A culture vessel that is used to maintain a continuous culture where nutrients are supplied at a constant rate. Bacterial suspension is removed at the same rate at which nutrients flow in to maintain an optimal growth environment
Why is it important to estimate the number of bacterial cells in a sample?
The number of bacteria in a clinical sample can indicate the extent of an infection. Estimates of bacterial counts in drinking water, food, medication, and cosmetics are used to detect contamination and prevent the spread of disease
Direct cell count
Refers to counting the cells in a liquid culture or colonies on a plate. It is a direct method of estimating how many organisms are present in a sample
Direct microscopic cell count
Involves transferring a known volume of a culture to a calibrated slide (Petroff-Hausser chamber) and counting the cells under a light microscope. It involves a counting chamber which is etched into squares of different sizes. A sample of the culture is added to a chamber. The concentration of cells in the original sample can be estimated by counting individual cells in a certain number of squares, and determining the volume of the sample observed. Cells in several small squares are counted and averaged in order to get an accurate measurement.
Petroff-Hausser chamber
The calibrated slide used in a direct microscopic cell count. It is similar to a hemocytometer used to count red blood cells.
Pros and cons of the direct microscopic cell count method
Pros- it is a cheap, easy to use, and fast method
Cons- the counting chamber does not work well with dilute cultures. Also, it can be difficult to differentiate between living and dead cells with this method
Fluorescence staining
Used to distinguish between living and dead bacteria. These stains bind to nucleic acids, but the primary and secondary stains differ in their ability to cross the cytoplasmic membrane. The primary stain is fluorescent green and can penetrate intact cytoplasmic membranes, and stains living and dead cells. The secondary stain is red and can stains dead cells, because it only stains a cell if the cytoplasmic membrane is damaged.
Coulter counter
An electronic cell counting device that detects and counts the changes in electrical resistance in saline solution. Cells are drawn through a class tube, and an electrode is located inside and outside the tube. The electrodes measure the brief change in resistance between them as the cells pass through. Each resistance change represents a cell. This method is fast and accurate within a range of concentrations. However, if the culture is too concentrated, multiple cells could pass through at one time and skew the results. It also can’t differentiate between living and dead cells
Viable
Living- refers to live cells in this context
In which situations is it necessary to estimate the number of living cells in a sample?
Counts of live cells are needed to determine the extent of an infection, the effectiveness of antimicrobials, or the contamination of food and water. In these cases, direct cell counts aren’t helpful
Viable plate count
Produces an estimated count of viable cells, based on the principle that viable cells replicate and give rise to visible colonies when incubated. The results are expressed as colony-forming units per millimeter. Microbiologists use plates with 30-300 colonies to get the most reliable numbers.
What are the limitations of a viable plate count?
More than one cells can occupy one spot, which is why the results have to be expressed as colony-forming units per millimeter. Some samples of bacteria grow in clusters or chains that are difficult to disperse, and a colony may represent multiple cells. Also, some cells can’t form colonies on solid media. These limitations mean that viable plate counts are low estimates of the actual number of cells
2 approaches to a viable plate count
- Pour plate
- Spread plate
Serial dilution
The first step of a viable plate count, before a pour plate or a spread plate. It is used in order to obtain plates that have between 30-300 colonies. Serial dilutions are usually done in multiples of 10 to simplify the process, but the number of dilutions is determined based on culture density. The culture is diluted in sterile broth, and the dilution continued until the culture is a specific concentration. The sample is plated on solid medium using either the pour plate method or the spread plate method, and the plates are incubated until colonies appear. 2-3 plates are used and the number of colonies on each plate are averaged.
CFU/mL
Stands for colony forming units per millimeter
Pour plate method (4 steps)
- The bacterial sample is mixed with warm agar (45-50 degrees)
- The sample is poured onto a sterile plate
- Sample is swirled to mix and allowed to solidify
- The plate is incubated until bacterial colonies grow
Spread plate method (3 steps)
- A .1 mL sample is poured onto solid medium
- Spread sample evenly over the surface
- The plate is incubated until bacterial colonies grow on the surface of the medium
Membrane filtration technique
Known volumes of a sample are vacuum-filtered aseptically though a membrane with a pore size small enough to trap microorganisms. The membrane is transferred to a Petri plate containing a growth medium, and the colonies are counted after incubation. Cell density is calculated by dividing the cell count by the volume of filtered liquid. This method is used for a very dilute sample that doesn’t contain enough organisms to use either of the plate count methods
Most probable number (MPN) method
A statistical procedure for estimating the number of viable microorganisms in a very dilute sample. It evaluates detectable growth by observing changes in turbidity or color due to metabolic activity. It can be used for water and food samples
Turbidity
The cloudiness of a sample of bacteria in a liquid suspension. A spectrophotometer is the instrument used to measure turbidity. This is an indirect cell count method
How does a spectrophotometer work?
A light beam is transmitted through a bacterial suspension, and a detector measures the amount of light that is passing through the sample. The amount of light that reaches the detector is converted into a percent transmission or a logarithmic value called absorbance/optical density. As the numbers of bacteria in a suspension increase, the turbidity also increases and causes less light to reach the detector. The decrease in light passing through the sample and reaching the detector is associated with a decrease in percent transmission and increase in absorbance measured by the spectrophotometer