Microbial growth and its Basics
- Bacterial Cell: Structure and its Composition
- Bacterial Stress Response
- Microbial Physiology: Yields of Microbes
Binary fission requires:
- cell mass to increase
- chromosome to replicate
- cell wall to be synthesized
- cell to divide into two cells
- Increase in cell number and increase in cell population mass both usually occur in a measurably coordinated fashion, and thus are often used interchangeably, even synonymously (though obviously we must be speaking in terms of populations of cells for this to be true).
- Microbial populations tend to increase in number and in cell mass simultaneously.
- Note that, for bacteria, while the cell population and population mass typically increase with time (with growth), over the course of population growth individual cells actually cycle through increases and decreases in cell mass (i.e., growth, division, growth, division, growth . . .)
Bias towards Cell Number:
- When a microbiologist speaks of microbial growth it is usually increase in cell number that she is after.
- The reason for this bias is that a typical microbiologist is more interested in population characteristics than in the characteristics of individual cells, or both (since the characteristics of individual cells tend to be studied, by necessity, within the context of populations of cells).
- Consequently, there is a tendency for microbiologists to follow microbial growth as populations rather than following the growth of individual cells, and therefore microbiologists tend to be more interested in population sizes than the size (mass) of any individual cell. Furthermore, the typical measurement of microbial growth will be done over the span of more than one microbial generation.
- Structure of Cell Membrane: Basic Guide for Students
- Active transport: Cell Membrane Mechanism
- Cell Cycle
Increase in cell number in Microbial Growth:
- An increase in cell number is an immediate consequence of cell division.
- Because most bacteria grow by binary fission, doubling in cell number usually occurs at the same rate that individual cells grow and divide.
Increase in Cell Mass:
Doubling in size:
- Individual cells of many species double in size between divisions.
- Cell mass thus increases at the same rate as cell number.
- The implication of this is that while increase in cell number may be emphasized while considering microbial growth, increases (and decreases) in individual cell masses are also occurring, though these increases and decreases balance each other out such that the average cell size tends to remain constant under constant conditions.
- The increase in mass is a consequence of anabolism.
- For anabolism to occur a cell must be situated in an environment that supplies all necessary nutrients and which physically falls into a range in which growth can occur.
Prokaryotic cell division:
- The first steps of binary fission include cell elongation and DNA replication.
- The cell envelope then pinches inward, eventually meeting.
- cross wall is formed and ultimately two distinct cells are present, each essentially identical to the original parent cell.
Generation time [Doubling time]:
Prokaryotic cell division:
- A bacterial generation time is also know as its doubling time.
- Doubling time is the time it takes a bacterium to do one binary fission starting from having just divided.
- And ending at the point of having just completed the next division.
Generation times vary with organism and environment and can range from 20 minutes for a fast growing bacterium under ideal conditions, to hours and days for less than ideal conditions or for slowly growing bacteria.
The best temperature for bacterial growth is:
Most bacteria are mesophilic. Mesophilic bacteria grow best at 30-37°C. Optimum temperature for growth of common pathogenic bacteria is 37°C. Bacteria of a species will not grow but may remain alive at a maximum and a minimum temperature.
Standard bacterial (or) Microbial growth curve:
- The standard bacterial or Microbial growth curve describes various stages of growth a pure culture of bacteria will go through, beginning with the addition of cells to sterile media and ending with the death of all of the cells present.
- The phases of bacterial growth typically observed include:
- lag phase
- exponential (log, logarithmic) phase
- stationary phase
- death phase (exponential or logarithmic decline)
- In standard bacterial growth curves one keeps track of cell growth by some measure or estimation of cell number.
Exponential growth (phase):
Exponential growth is a function of binary fission since at each division there are two new cells. The time between divisions is called generation time or the doubling time since this is the time for the population to double. These can range from minutes to days depending on the species of bacteria.
Growth rate is the change in cell number or mass per unit time.
What do we mean by exponential growth?
A population doubles each generation is exponential growth. Graphically on arithmetic coordinates the graph takes the shape of a J – a curve with ever increasing slope – growth rate. Plotted on semi-logarithmic paper, where the Y axis is logarithmic (base 10) and the X axis is arithmetic (either generation or time), you get a straight line.
Generation times – N = No2n where No is the original number of cells and n is the number of generations. g, generation time, equals t/n, time divided by generation.
How do you calculate n?
N = No2n
log N = log No + nlog2
log N – log No = n log2
n = [log N – log No] / log2
n = [log N – log No] / 0.301
We also know that the slope of the semilog line equals 0.301 divided by the generation time.
Batch culture of bacteria:
Culturing bacteria in a Erlenmeyer flask where you simply inoculate it and let the bacteria grow.
There are 4 phases of growth in batch culture.
Lag phase – A newly inoculated culture usually does not begin growing immediately but rather after of period of no growth which is referred to as the lag phase.
Conditions that lead to a lag phase –
- inoculum which is in stationary phase inoculated into the same medium
- inoculum which is damaged but not killed inoculated into the same medium
- inoculum transferred from rich to poor medium
Why is there a lag phase?
- the cells are tooling up for growth.
- Stationary cells have probably depleted essential requirements and they need to be re-synthesized.
- Damaged cells need to repair before they can grow
- Transferred cells need to synthesize new enzymes required for growth in the poor medium.
When is a lag phase not necessary?
When active cells are transferred back to the same medium.
- Exponential or log phase – a consequence of each cell dividing to form two cells. Usually the phase with the greatest rate of increase in the population size. The rate is influenced by the environmental conditions such as temperature, aeration, and composition of medium.
- Stationary phase – realize that a bacterium – a single cell – with a generation time of 20 minutes would produce a population with the weight of 4000 times the earth after 48 hours. Wow A bacterium weighs about 10-12 gram.
What happens to stop this?
There are factors that limit population growth –
1. intraspecific competition for nutrients which are running out as the culture ages.
2. Build up of toxic metabolites
All of this leads to a stationary phase in which the growth rate of the population is zero.
Death phase – after stationary phase the cells may remain alive for a long period of time or begin to die off as in a death phase. The cells may begin to lyse as they die and other viable cells may grow on the remains of the lysed cells in what is called cryptic growth. Now remember that we are talking about a population – not a single cell.
How do we measure growth?
- Direct microscopic counts – use the microscope and a slide with a grid engraved on it. A cover-slip and placed over the grid which captures a known volume of liquid.
- Problems with direct microscopic counts
- dead cells are difficult to distinguish
- small cells are difficult to see
- method not suitable for dilute samples
- Viable counts – count only cells that are able to divide and form offspring. Referred to as plate counts or colony counts. Assumption each viable cell gives rise to a colony.
- spread plates and pour plates
Dilutions – to cover a cell density that ranges from 30 – 300 colony forming units per plate.
- Not all species of bacteria will form colonies on any particular medium.
- small colonies are not counted
- Despite problems, it is still widely used in ecology, food microbiology, medical microbiology, and dairy microbiology.
- Turbidity – cell suspensions look cloudy because each cell scatters light as it passes through a suspension of cells.
- Take advantage of the light scattering properties of a suspension using a spectrophotometer which measures unscattered light as it passes through.
- The scatter is proportional to cell number (density of cells) up to high density cultures because cells begin to cause rescatter the light back into the path of unscattered light. Therefore the optical density is not linear at high density suspensions.
- Need to develop a standard curve between OD and cell numbers (viable counts).
- Exponential growth is a physiological state marked by back-to-back division cycles such that the population doubles in number every generation time.
- Note that during exponential growth there is no change in average cell mass, though individuals cells are constantly changing in mass as they increase in mass, then divide thus rapidly decreasing in mass (while increasing in number).
The algebra of exponential growth:
- Note that during exponential growth the number of cells present at any given time is a multiplicative function of the number of cells present at a previous time.
- Under constant conditions the multiplicative increase in cell number consequently is constant for any given interval of the same duration.
- If a log phase culture goes from 2 cells to 4 cells during a 20 minute interval, then the culture will go from 4 cells to 8 cells during the next 20 minutes.
- If a log phase culture goes from 2 cells to 6 cells during a 60 minute interval, then the culture will go from 6 cells to 18 cells during the next 60 minutes.
- If during exponential phase there are 10 cells present at time 0, and 100 cells present at time 200, then at time 400 there will be 10,000 (100 * 100) cells present.
- A vegetative cell is one which is capable of actively growing.
- Contrast with endospore.
Lag in division:
- Upon a change in environment (especially from a rich environment to a poor environment), or when going from stationary phase to exponential phase, there is a lag before division resumes.
- For example, stationary phase Escherichia coli placed in an excess of sterile broth will go through a lag phase during which they increase in cell size but do not divide. They will divide only once they have reached the size of a cell which is about to divide during exponential growth under those conditions.
- During this time a culture is said to be in lag phase.
Increase in mass:
- During lag phase cells increase in mass but do not divide.
- In other words, there is no change in number, but an increase in mass.
The length of the lag phase is determined in part by characteristics of the bacterial species and in part by conditions in the media—both the medium from which the organisms are taken and the one to which they are transferred. Some species adapt to the new medium in an hour or two; others take several days. Organisms from old cultures, adapted to limited nutrients and large accumulated wastes, take longer to adjust to a new medium than do those transferred from a relatively fresh, nutrient-rich medium.” (p. 138, Black, 1996)
- Stationary phase is classically defined as a physiological point where the rate of cell division equals the rate of cell death, hence viable cell number remains constant.
- No cell division:
- Note that when cell division = 0 and cell death = 0, then the rate of cell division = rate of cell death.
- In other words, when cells stop dividing but have not yet started dying they are in stationary phase.
- A way to distinguish these possibilities is to compare viable count with total count.
- If both total counts and viable counts don’t change then you know that there is both no cell division and no cell death.
- If total count increases while viable counts remain constant, then you know that you are observing a true balance between ongoing cell division and cell death.
- Physiological adaptation to cell excess:
- Stationary phase usually occurs when cell concentration is so great and that some aspect of the environment is no longer able to serve the requirements of exponential growth.
- Stationary phase is a time of significant physiological change and particularly involves the physiological adaptation of cells to survival through periods of little growth.
- In single celled microorganisms cell death is the point at which re initiation of division is no longer possible.
- Qualified definition:
- Note that the concept of cell death is actually dependent on how one attempts to re-initiate growth.
- Particularly, there are ways to gently revive some microbes from physiological states that would result in permanent lack of growth in other growth environments.
- An analogous situation would be a person with an injury that is inevitably fatal in a third-world hospital, but readily treated in a first-world hospital.
- Example: seeds:
- Another analogy is with a plant seed. You can try to sprout it in all kinds of environments but not all will work out in the seed’s favor. You may end up killing the seed by allowing it to attempt to germinate in the wrong environment.
- The more degraded is the seed prior to planting, the greater the likelihood that germination will not successfully occur unless you take great care to make sure sprouting conditions are as close to ideal as you can make them.
Death phase [logarithmic decline, Exponential decline]:
- Death phase is a physiological point at which cell deaths exceed cell births.
- More specifically, viable count declines.
- “During the decline phase, many cells undergo involution—that is, they assume a variety of unusual shapes, which makes them difficult to identify.” (p. 140, Black, 1996)
Endospore [Spore, Sporulation, Sporogenesis, Activation and germination]:
Tough, dormant state:
- A very tough, dormant form of certain bacterial cell that is very resistant to desiccation, heat, and a variety of chemical and radiation treatments that are otherwise lethal to non-endospore bacterial cells.
- At least part of the toughness associated with a spore is found in its very tough outer layers, called a coat.
- Only some bacteria produce endospores.
- Endospores of some bacteria can last so long under proper conditions that various endospores found in such things as Egyptian mummies are likely the oldest living things.
Sporulation and sporogenesis:
- Sporulation and sporogenesis refer to the formation of endospores by vegetative (i.e., growing) cells.
- The endospore is actually the intracellular product of sporogenesis.
- A spore is an endospore which has been released from a cell, i.e., it exists is a free state.
- In bacteria the formation of a spore is not considered to be an act of reproduction. Indeed, the formation of the endospore is directed by the DNA that will ultimately be found in the spore, and the sister DNA found in the vegetative part of the cell ultimately is destroyed.
- The first step of germination, often requires some kind of coat traumatizing insult such as high temperature or low pH.
- The transformation from the endospore state to the vegetative state.
- The key thing to worry about with endospores is that they are capable of germinating despite harsh treatment, and thus can potentially produce actively replicating cells where there may have been none previously prevent.
- Of those bacteria on your list, the following are spore formers (note that all are gram-positives):
- Bacillus anthracis
- Bacillus subtilis
- Clostridium botulinum
- Clostridium perfringens
- Clostridium tetani
Temperature – as temperature increases, the growth rate increases until a point at which the growth rate declines.
- Minimum temperature – below growth does not occur may be due to the stiffening of the cytoplasmic membrane.
- optimum temperature where the growth rate is maximum
- Maximum temperature– above which growth does not occur which reflects when proteins may be denatured, nucleic acids and other cellular components are irreversibly damaged.
Classification of bacteria based on temperature optimum:
- psychrophiles – low temperature optima <15 C – may even be killed by brief warming or thawing.
- mesophiles – midrange temperature optima 25 – 40 C
- thermophiles – high temperature optima 40 – 80 C
- hyperthermophiles – very high temperature optima >80 C
- Psychrophiles : open ocean water is between 1 and 3 C. Artic and Antartic regions are cold.
Adaptation: membranes rich in unsaturated fatty acids
Thermophiles: hot springs all over the world, fermenting compost
Adaption: thermostable proteins with usually a few changes in the amino acid sequence when compared to a mesophile’s protein. saturated fatty acids in their membranes
Acidity and alkalinity: Most environments are between 5 and 9 and optima are between these values, around neutral pH of 7.
Acidophiles: live at low pH
Obligate acidophiles such as Thiobacillus. cytoplasmic membrane actually dissolves and the cell lysis at more neutral pH.
Alkaliphiles: live at high pH such as soda lakes and carbonate soils. Important to biotechnology since they have hydolytic proteases that function at alkaline pH and are used in household cleaners.
Water availability – bacteria need water as a solvent.
Water availability is expressed as water activity – how much water is available. Solutes and surfaces affect water activity – both decrease it. Water moves from high water activity values to lower values in the process of osmosis. Different bacteria have different tolerances towards low water activities. In fact, preservation process takes advantage of lower water activity which causes plasmolysis or pulling away of the membrane from the cell wall. This inhibits cell growth.
Halophiles require 1-6% for mild halophiles and 7-15% salt for moderate halophiles. Extreme halophiles require 15-30% salt.
- Aerobes require oxygen up to 21% as in air.
- Microaerophilic bacteria require reduced levels of oxygen
- strict or obligate anaerobes require the absence of oxygen.
- Facultative anaerobes can grow aerobically if oxygen is present and switch to fermentation or anaerobic respiration if oxygen is absent. E. coli is a facultative anaerobe that grows aerobically and using anaerobic respiration when necessary.
- Aerotolerant anaerobes don’t use oxygen for growth but tolerate its presence. Can grow on the surface of solid medium with out the special anaerobic conditions required for the strict anaerobes.
- Anaerobic culture conditions – add reducing reagents such as thioglycolate, bubble nitrogen gas through your solutions to remove oxygen after autoclaving, add a dye such as resazurin to indicate when oxygen is penetrating, use an anaerobic jar with an atmosphere containing hydrogen gas and carbon dioxide.
Why go to such great troubles for the strict anaerobes?
Because they contain lots of flavins which react with oxygen to produce toxic oxygen species that are very reactive.
- Oxygen species – singlet oxygen which the valence electrons become highly reactive and oxidize organic matter readily.
- Superoxide anion, hydrogen peroxide, and hydroxyl radical which are inadvertant byproducts during respiration. These can all damage cell macromolecules by oxidation processes.
- Measures to counter these toxic oxygens – catalase degrade hydrogen peroxide to oxygen and water.
- peroxidases – destroys hydrogen peroxides too but requires NADH. no oxygen evolved.
- super oxide dismutase produces hydrogen peroxide from super oxides. Aerobes and facultative aerobes generally contain catalase and super oxide dismutase.