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 and the X axis is arithmetic, 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 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).
Continuous culture
In a batch culture, culture conditions are dynamic since there are a fixed amount of nutrients and the number of cells is changing. Continuous culture has been developed to maintained constant conditions. Continuous cultures are flow through cultures where there is input and output and the conditions reach an equilibrium where the cell number and nutrient concentration remains constant at steady state.
Chemostat - common continuous culture devise where the concentration of nutrients and dilution rate are controlled. The dilution rate controls the growth rate of the bacteria and the nutrient concentration controls the growth yield of the culture (see figure 5.11)
At low dilution rates the cells are starving and dying and the culture may washout. At higher dilution rates the cells may wash out also if the dilution rate is greater than their growth rate.
Cell density is controlled by nutrient concentration in the input. Keeping the dilution rate constant and increasing the nutrient concentration results in a larger population with the same growth.
Environmental factors -
Temperature - as temperature increases, the growth rate increases until a point at which the population dies.
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 45 - 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
Biotechnology
Taq DNA polymerase from Thermus aquaticus.
Acidity and alkalinity
Most environments are between 5 and 9 and optima are between these values.
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.
Halophiles require 1-6% for mild halophiles and 7-15% salt for moderate halophiles. Extreme halophiles require 15-30% salt.
Osmophiles grow in sugary solutions and xerophiles grow in dry environments.
As the water activity or water potential drops, bacteria will lose water to their environment unless they stop it. Compatible solutes are solutes produced by the bacteria to counteract the effects of more negative water potentials. Compatible solutes include amino acids, sugars, and alcohols such as glycerol.
Oxygen -
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.
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.
super oxide dismutase produces hydrogen peroxide from super oxides.
Aerobes and facultative aerobes generally contain catalase and super oxide dismutase.
