Microbial Growth

Microbial Growth

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 indentical except for the occassional mutation.
Binary fission requires:
cell mass to increase
chromosome to replicate
cell wall to be synthesized
cell to divide into two cells

Exponential growth

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 semilogarithmic 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 transfered 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 resynthesized.
Damaged cells need to repair before they can grow
Transfered cells need to synthesize new enzymes required for growth in the poor medium.
When is a lag phase not necessary?
When active cells are transfered 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 coverslip 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.
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 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).

Environmental factors -

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.

Oxygen -
1. Aerobes require oxygen up to 21% as in air.
2. Microaerophilic bacteria require reduced levels of oxygen
3. strict or obligate anaerobes require the absence of oxygen.
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 using anaerobic respiration when necessary.
5. 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.

Raw materials

which microorganisms must get from the environment!!!!
Carbon sources
Classify bacteria based on carbon source:
heterotrophs which use organic molecules such as sugars, amino acids, fatty acids, organic acids, aromatic compounds, nitrogen bases, and countless other organic molecules, as their source of carbon.
autotrophs which use the inorganic molecule carbon dioxide as their source of carbon.
Bacteria are about 50% Carbon by weight.
Nitrogen
Bacteria are about 14% nitrogen by weight. Nitrogen found in amino acids, nucleotides, and other cell constituents.
Nitrogen in organic and inorganic forms. Example of organic is amino acids or nucleotides. Examples of inorganic form are NH4 or NO3.
A few species can take atmospheric nitrogen N2 (80%+ of air and inert) and convert it to ammonium and ultimately amino acids or nucleotides. These bacteria are referred to as nitrogen-fixing bacteria.
Phosphorous
Phosphorous found in nucleotides and phospholipids.
Phosphorous occurs in nature as organic or inorganic as phosphates forms.
Sulfur
Sulfur found in specific amino acids and vitamins.
Chemically transformed (oxidation states change) due to microorganisms activities. Sulfur occurs in nature as organic or inorganic forms. Bacteria use sulfate or sulfide forms.
Required in smaller amounts
Potassium
Required as a cofactor for a number of enzymes.
Magnesium
Required to stabilize ribosomes, cell membranes, nucleic acids and required by a number of enzymes.
Calcium
Stabilizes cell wall and important for heat stability of spores.
Sodium
Required by some but not all microorganisms. Usually seawater organisms have a requirement but freshwater or terrestrial organisms do not.
Iron
Required in small amounts but still considered a macro-nutrient.
Required for cytochromes and iron-sulfur proteins that are important for electron transport.
Found as insoluble inorganic form in nature. Bacteria produce siderophores to chelate insoluble iron and transport it to the cell. Siderophores produced by Escherichia coli or S. typhimurium are called enterobactins.
Micronutrients are found in your text. These are inorganic elements that are required in trace amounts by some or all bacteria for growth. Used primarily as enzyme cofactors.
Organic growth factors
Includes vitamins, amino acids, purines or pyrimidines. Most microorganisms can synthesize these but others require them be presynthesized.
Again microorganisms must either obtain these from the environment or be able to synthesize them for themselves. Culture medium that we use must provide the necessary raw products for growth if the cell cannot synthesize it.

Types of medium:

Agar is used to solidify medium. Agar melts in boiling water, remains liquid down to about 40oC where it solidifies. We hold it at 50oC in the lab. After agar solidifies it can be incubated at temperatures approaching 100 oC and not melt.
Complex - which is chemically undefined. made up of extracts from plant, yeast or meats or digests of proteins from these sources.
Defined - which is chemically defined.

Selective vs differential medium
Selective medium are designed to suppress the growth of unwanted species of bacteria and support the growth of desired species. Example - the dye brilliant green is added to medium because it inhibits gram positive bacteria and selects gram-negative bacteria.
Differential medium are used to differentiate between species of bacteria. Some property of the bacteria is exploited. Blood agar plates are used to differentiate species of bacteria that can lyse red blood cells.
Often medium are designed that are selective and differential. Mannitol salts agar is both selective and differential. This medium has 7.5% salt to select for salt tolerant species of bacteria and the medium has an indicator to differentiate between acid and non acid producing colonies from the production of acid from mannitol. Staphylococcus aureus is both salt tolerant and produces acid from mannitol.
Enrichment medium selects for small populations of a species of bacteria among larger populations. Suppose you are interested in a species of bacteria that can use trichloroethylene as a carbon and energy source. You can select your inoculum, e.g., a soil sample, place it in a liquid medium with TCE as the sole carbon and energy source. Let this incubate for several weeks and back transfer some of it to a new medium with TCE. Ultimately, after several backtransfers, you should have enriched for a TCE degrading bacterium that grows on the TCE.