Microbial or Bacterial Growth

Microbial growth refers to the reproduction and proliferation of microorganisms, including bacteria, fungi, and viruses. It is a basic part of microorganisms’ biology and is important for their survival and evolution.

Microbial growth occurs in a variety of environments, including soil, water, and the human body, and is influenced by a range of factors, including temperature, pH, and the availability of nutrients. Microorganisms can adapt to a wide range of conditions and can grow in many different places, even in extreme ones like high salt levels or low temperatures.

The study of microbial growth is important for a variety of applications, including the production of food and beverages, the development of drugs and other products, and the understanding of disease outbreaks. It is also important for studying the role of microorganisms in the environment, such as how they affect the health of ecosystems and how nutrients cycle through the environment.

Microbial growth may be described as occurring in different ways under different circumstances. Microbial growth is usually studied as a population, not an individual. Individual cells divide in a process called binary fission, where two daughter cells arise from a single cell. The daughter cells are identical except for the occasional mutation.

Overall, microbial growth is a key part of how microorganisms live and has important effects on a wide range of fields and applications. 

Binary fission requires

  • cell mass to increase
  • chromosome to replicate
  • cell wall to be synthesised
  • cell to divide into two cells

increase in both population size and population mass

  • Increases in cell number and cell population mass typically occur in a measurably coordinated manner, and thus are frequently used interchangeably, if not synonymously (though we must obviously be speaking in terms of cell populations for this to be true).
  • Microbial populations tend to increase in number and 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, and so on).

Bias towards cell number

  • When a microbiologist speaks of microbial growth, it is usually an increase in cell number that she is after.
  • This is because a typical microbiologist is more interested in the characteristics of populations of cells than in the characteristics of individual cells, or both, since the characteristics of individual cells usually have to be studied in 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 an individual cell. Furthermore, the typical measurement of microbial growth will be done over the span of more than one microbial generation.

increase in cell number in microbial growth

  1. An increase in cell number is an immediate consequence of cell division.
  2. Most bacteria grow by a process called binary fission. This means that the number of cells usually doubles at the same rate that each cell grows and divides.

Increase in Cell Mass

  1. Doubling in size

    1. Individual cells of many species double in size between divisions.
    2. Cell mass thus increases at the same rate as the cell number.
    3. The implication of this is that while an increase in cell number may be emphasised when 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.
  2. Anabolic process:

    • 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 that physically falls into a range in which growth can occur.

Binary fission

Prokaryotic cell division

Binary fission is the process by which most prokaryotes replicate. During binary fission, a single cell usually splits into two more or less identical daughter cells. Each of these daughter cells has at least one copy of the DNA from the parent cell. Binary fission is the basic feature of microbial growth.

Stepwise process

  1. The first steps of binary fission include cell elongation and DNA replication.
  2. The cell envelope then pinches inward, eventually meeting.
  3. The cross wall is formed, and ultimately two distinct cells are present, each essentially identical to the original parent cell.

Time of generation [time of doubling]

Bacterial cell division

  1. Bacterial generation time is also known as its doubling time.
  2. Doubling time is the time it takes a bacterium to do one binary fission, starting from having just divided.
  3. and ending at the point of having just completed the next division.

Generation times vary from 20 minutes for a fast-growing bacterium in perfect conditions to hours or days for bacteria that grow slowly or in less than perfect conditions.

The best temperature for bacterial growth is

Most bacteria are mesophilic. Mesophilic bacteria grow best at 30-37 °C. The optimum temperature for the growth of common pathogenic bacteria is 37 °C. Bacteria of a certain species won’t grow, but they may still be able to live at very high or very low temperatures.

Standard bacterial or  microbial growth curve

  1. The standard bacterial or microbial growth curve describes various stages of growth that 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.
  2. The phases of bacterial growth typically observed include:
    • lag phase
    • phase exponential (logarithmic, logarithmic)
    • stationary phase
    • death phase (exponential or logarithmic decline)
  3. In standard bacterial growth curves, cell growth is tracked using 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 known as generation time or doubling time because this is when the population doubles. These can range from minutes to days, depending on the species of bacteria.

The change in cell number or mass per unit time is referred to as the growth rate.

What do we mean by “exponential growth”?

Exponential growth refers to a population that doubles every generation. Graphically, on arithmetic coordinates, the graph takes the shape of a J-shaped curve with an ever-increasing slope and 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 No + nlog2 = log N
n log2 = log N – log No
n = log2 / [log N – log No].
[log N – log No] / 0.301 = n
We also know that the slope of the semilog line equals 0.301 divided by the generation time.

The batch culture of bacteria

Culturing bacteria in an Erlenmeyer flask, where you simply inoculate it and let the bacteria grow,

There are four phases of growth in batch culture.

1. Lag phase

Most cultures don’t start growing right away after being inoculated. Instead, there is a period of no growth, called the “lag phase.”
Conditions that lead to a lag phase

  • inoculum, which is in the stationary phase, is inoculated into the same medium.
  • inoculum, which is damaged but not killed, is inoculated into the same medium.
  • Transfer of inoculum from rich to poor medium

Why is there a lag phase?

  • The cells are gearing up for growth.
  • Stationary cells have probably depleted essential requirements, and they need to be resynthesised.
  • Damaged cells need to be repaired before they can grow.
  • Transferred cells need to synthesise 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.

2. Exponential or log-phase

a consequence of each cell dividing to form two cells. Most of the time, this is the phase when the population grows the fastest. The rate is affected by things like the temperature, the amount of oxygen in the air, and the makeup of the medium.

3. Stationary phase

It realizes that a bacterium—a single cell—with a generation time of 20 minutes would produce a population with a weight 4000 times that of the earth after 48 hours. Wow, a bacterium weighs about 10–12 grammes.

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.

4. Death phase

After the stationary phase, the cells may remain alive for a long period of time or begin to die off, as in the 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 counting: 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 is 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 are needed to cover a cell density that ranges from 30 to 300 colony-forming units per plate.
  • Problems:
    • 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 scatter the light back into the path of unscattered light. Therefore, the optical density is not linear in high-density suspensions.
    • There is a need to develop a standard curve between OD and cell numbers (viable counts).

Back-to-back divisions

  • Exponential growth is a physiological state marked by back-to-back division cycles such that the population doubles in number every generation.
  • There is no change in average cell mass during exponential growth, but individual cells are constantly changing in mass as they increase in mass, then divide, 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 rate at which the number of cells increases by multiplying is the same for any given interval of the same length.

  • 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 the 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.

Vegetative cell

  1. A vegetative cell is one that is capable of actively growing.
  2. In contrast to endospore.

Lag phase:

  1. Lag in the division

    • Upon a change in the 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 that is about to divide during exponential growth under those conditions.
    • During this time, a culture is said to be in the “lag phase.”
  2. Increase in mass:

    1. During the lagged phase, cells increase in mass but do not divide.
    2. 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 the 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:

  1. The stationary phase is classically defined as a physiological point where the rate of cell division equals the rate of cell death, hence the viable cell number remains constant.
  2. No cell division:
    • Note that when cell division = 0 and cell death = 0, then the rate of cell division equals the rate of cell death.
    • In other words, when cells stop dividing but have not yet started dying, they are in the stationary phase.
  3. A way to distinguish between these possibilities is to compare the viable count with the 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 the 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.
  4. Physiological adaptation to cell excess:
    • Stationary phase usually occurs when the cell concentration is so high that some aspect of the environment can no longer meet the needs of exponential growth.
    • The stationary phase is a time of significant physiological change and particularly involves the physiological adaptation of cells to survival through periods of little growth.

Cell death:

  1. In single-celled microorganisms, cell death is the point at which re-initiation of the division is no longer possible.
  2. Qualified definition:
    • Note that the concept of cell death is actually dependent on how one attempts to re-initiate growth.
    • In particular, there are ways to gently revive some microbes from physiological states that would result in a permanent lack of growth in other growth environments.
  3. An analogous situation would be a person with an injury that is inevitably fatal in a third-world hospital but readily treatable in a first-world hospital.
  4. 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 the seed is prior to planting, the greater the likelihood that germination will not occur successfully 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]:

  1. he “death phase” is a physiological point at which cell deaths exceed cell births.
  2. More specifically, the viable count declines.
  3. “During the decline phase, many cells undergo involution—that is, they assume a variety of unusual shapes, which makes them difficult to identify.”

Endospore [spore, sporulation, sporogenesis, activation, and germination]

  1. Tough, dormant state:

    • a very tough, dormant form of certain bacterial cells 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 survive for so long under the right conditions that endospores found in Egyptian mummies are likely the oldest living things.
  1. 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 that has been released from a cell, i.e…, it exists in 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 is ultimately destroyed.
  2. The first stage of germination frequently necessitates a traumatising insult, such as high temperature or low pH.
  3. The transformation from the endospore state to the vegetative state
  4. 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.
  5. Of those bacteria on your list, the following are spore-forming organisms (note that all are gram-positive):
    • Bacillus anthracis
    • Bacillus subtilis
    • Clostridium botulinum
    • Clostridium perfringens
    • Clostridium tetani

Environmental factors

Temperature: As the temperature increases, the growth rate increases until a point at which it declines.

  • Minimum temperature: if growth does not occur below this temperature, it may be due to the stiffening of the cytoplasmic membrane.
  • the optimum temperature where the growth rate is maximum
  • Maximum temperature—above which growth does not occur—reflects when proteins may be denatured, nucleic acids are damaged, and other cellular components are irreversibly damaged.

1. Classification of bacteria based on temperature optimum

  • Brief warming or thawing can even kill psychrophiles (low-temperature optima of 15 C).
  • Mesophiles: optimal midrange temperatures are 25-40 C.
  • Thermophiles: high-temperature optima: 40–80 C
  • Hyperthermophiles: very high-temperature optimum temperatures > 80 C
  • Psychrophiles: open ocean water is between 1 and 3 C. The Arctic and Antarctic regions are cold.

Adaptation: membranes rich in unsaturated fatty acids
Thermophiles: hot springs all over the world, fermenting compost
Adaptation: Compared to mesophile proteins, the order of amino acids in thermostable proteins doesn’t change much. saturated fatty acids in their membranes

Acidity and alkalinity: Most environments are between 5 and 9, and optima are between these values, around a neutral pH of 7.
Acidophiles are organisms that thrive in environments with low pH.
Thiobacillus obligate acidophiles At a more neutral pH, the cytoplasmic membrane dissolves and cell lysis occurs.
Alkaliphiles live in high-pH environments such as soda lakes and carbonate soils.

They are important to biotechnology because they have proteases that break down proteins and work at an alkaline pH. These proteases are also used in household cleaners.

Water availability: bacteria need water as a solvent.
Water availability is expressed as “water activity,” or how much water is available. Soluble 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, the preservation process takes advantage of lower water activity, which causes plasmolysis, or the pulling away of the membrane from the cell wall. This inhibits cell growth.
Halophiles require 1-6% salt for mild halophiles and 7-15% salt for moderate halophiles. Extreme halophiles require 15–30% salt.

2. Oxygen

  1. Aerobes require up to 21% of the oxygen in the air.
  2. Microaerophilic bacteria require reduced levels of oxygen.
  3. The absence of oxygen is required by strict or obligate anaerobes.
  4. 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 uses anaerobic respiration when necessary.
  5. Aerotolerant anaerobes don’t use oxygen for growth but tolerate its presence. can grow on the surface of the solid medium without the special anaerobic conditions required for the strict anaerobes.
  6. 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, and use an anaerobic jar with an atmosphere containing hydrogen gas and carbon dioxide.

Why go to such lengths 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, in which the valence electrons become highly reactive and oxidise organic matter readily.
  • superoxide anion, hydrogen peroxide, and hydroxyl radical, which are inadvertent by-products during respiration. All of these can damage cell macromolecules through oxidation processes.
  • To combat these toxic oxygens, catalase converts hydrogen peroxide to oxygen and water.
  • peroxidases—destroy hydrogen peroxides too but require NADH. No oxygen evolved.
  • Superoxide dismutase produces hydrogen peroxide from superoxide. Aerobes and facultative aerobes generally contain catalase and superoxide dismutase.

Final words on Microbial growth

Microbial growth refers to the reproduction and proliferation of microorganisms, including bacteria, fungi, and viruses. It is a basic part of microorganisms’ biology and is important for their survival and evolution.

The study of microbial growth is important for a variety of applications, including the production of food and beverages, the development of drugs and other products, and the understanding of disease outbreaks. It is also important for studying the role of microorganisms in the environment, such as how they affect the health of ecosystems and how nutrients cycle through the environment.

There are several factors that can influence microbial growth, including the availability of nutrients, temperature, pH, and the presence of other microorganisms. For example, microorganisms require a source of energy and specific nutrients to grow, such as carbohydrates, proteins, and lipids. Most microorganisms also have a preferred temperature range in which they can grow and thrive. pH can also affect the growth of microorganisms.

Different methods can be used to study microbial growth, such as culturing, which involves growing microorganisms in a controlled environment, and microscopy, which lets scientists look at microorganisms at the level of their cells.

Overall, studying how microorganisms grow is important for learning about their biology and has important implications for a wide range of fields and applications.