Microbial Growth 

I. The Growth Curve

Closed system = Batch Culture– Closed culture vessel

One batch of culture medium Different from continuous culture (see below) Nutrients used up, culture eventually dies

Four stages of bacteria growth in batch culture

A period of apparent inactivity in which the cells are adapting to a new environment and preparing for reproductive growth, usually by synthesizing new cell components– ATP– Ribosomal proteins– rRNA– tRNA– Co-factors– Enzymes

Lag PhaseLag Phase

Varies in length depending upon the condition of the microorganisms and the nature of the medium

Assessment of medium – Receptors

DNA synthesized – initiation of cell division

Exponential phase (Log Phase)Exponential phase (Log Phase) Optimal growth rate and cell division dependent on

medium, O2, temperature, pH, genetic composition Regular, constant cell division (logarithmically) Smooth curve – division not synchronous Most useful phase for biochemical, physiological

and DNA replication studies– Biotechnology applications – competent cells – uptake of

plasmid DNA– Late log = optimal plasmid concentration

The population is most uniform in terms of chemical and physical properties during this period

Stationary PhaseStationary Phase

When the population reaches ~109/ml (106 for protozoan and algal cultures), cell division = cell death (stasis)

Nutrients become scarce O2 is depleted Toxic waste accumulates The number of viable microorganisms remains

constant either because metabolically active cells stop reproducing or because the reproductive rate is balanced by the rate of cell death

Death PhaseDeath Phase

Viable cell mass decreasesOften logarithmicCells not viable when inoculated into

fresh medium Cells have reached the carrying capacity of

their environment

The mathematics of growth-microbial growth can be described by certain mathematical terms:–Mean generation (doubling) time (g) is the time required for the population to double

–Mean growth rate constant is the number of generations per unit time, often expressed as generations per hour

Generation times vary markedly with the species of microorganism and environmental conditions –they can range from 10 minutes for a few bacteria to several days with some eukaryotic microorganisms

–Population size = 2n where n = the number of generations

II. Measurement of Microbial Growth

Counting cells directly (live and Counting cells directly (live and dead)dead)Petroff-Hausser Counting Chamber

– Slide with depressed etched grids (25 squares)– Covered with a coverslip– 25 squares (area) = 1mm2

– Depth = 0.02mm– Volume = 2 x 10-5 ml in 25 squares– Determination of cell numbers:

20 cells in one square x 25 squares/2 x 10-5 ml = 2.5 x 107 cells/ml

Electronic Counter– Coulter counter– Measures electrical resistance as cells pass

single file through a thin stream– RBC and WBC are counted– Less accurate with small cells

High interference Clumping

Counting only live cellsCounting only live cells

Plating techniques (spread plate, pour plate) using serial dilutions

Colony forming units (CFU) usually arise from one organism (but may be several if clumpy)

Membrane filtration assay– Membrane traps bacterial on the surface– Membrane transferred to an agar plate– Colonies grow counted– Can use selective media (e.g. Endo agar for coliform

counts in contaminated water supplies)

Measurement of cell massMeasurement of cell mass Cell mass increases as cell number increases Dry weight measurements

– Growth, concentration, wash, dried, weighed Spectrophotometric determination

– Light is scattered and is proportional to cell number– Linear relationship between absorbance and cell

density– Often written as % transmittance (as absorbance

increases, transmittance decreases)– Requires cultures to be ~107/ml and upwards (slight

turbidity)

III. Continuous Culture Techniques

Used to maintain cells in the exponential growth phase at a constant biomass concentration for extended periods of time

Conditions are met by continual provision of nutrients and removal of wastes = OPEN SYSTEM

Constant conditions are maintained

Chemostat– A continuous culture device that

maintains a constant growth rate by:supplying a medium containing a limited amount of an essential nutrient at a fixed rate

removing medium that contains microorganisms at the same rate

– As fresh media is added to the chamber, bacteria are removed

– Limiting nutrients control growth rates– Cell density depends on nutrient

concentration

Turbidostat– A continuous culture device that

regulates the flow rate of media through the vessel in order to maintain a predetermined turbidity or cell density

There is no limiting nutrient Absorbance is measured by a photocell

(optical sensing device) The number of cells in culture controls

the flow rate and the rate of growth of culture adjusts to this flow rate

Balanced and Unbalanced Growth

Balanced (exponential) growth occurs when all cellular components are synthesized at constant rates relative to one another

Unbalanced growth occurs when the rates of synthesis of some components change relative to the rates of synthesis of other components. –This usually occurs when the environmental conditions change

IV. Environmental Factors Affecting Microbial Growth

Solutes and Water ActivitySolutes and Water Activity

Osmotic concentrations affect microbes (e.g. plasmolysis in hypertonic solutions)

Water activity (aw) = measurement of availability of water in particular environments

Aw = Psolution/Pwater (P = vapor pressure)= inversely related to osmotic pressure

If the solution has a high osmotic pressure (high extracellular solute concentration), then its Aw = low

Energy is required by microbes to tolerate low aw because in order to keep water, solute concentration inside of cells must be kept high= Osmotolerance

S. aureus can tolerate up to 3M NaCl Archaebacteria halophiles tolerate 2.8-6.2M NaCl

(Great Salt Lake, Dead Sea)

– Avoidance of plasmolysis

pH is the negative logarithm of the hydrogen ion concentration

pH (Log scale of 0 – 14; each pH pH (Log scale of 0 – 14; each pH unit = 10x change)unit = 10x change)

–Acidophiles grow best between pH 0 and 5.5

–Neutrophiles grow best between pH 5.5 and 8.0

–Alkalophiles grow best between pH 8.5 and 11.5

– Extreme alkalophiles grow best at pH 10.0 or higher

– Despite wide variations in habitat pH, the internal pH of most microorganisms is maintained near neutrality either by proton/ion exchange or by internal buffering

– Sudden pH changes can inactivate enzymes and damage PMs

Reason for buffering culture medium, usually with a weak acid/conjugate base pair (e.g. KH2PO4/K2HPO4 – monobasic potassium/dibasic potassium)

Microorganisms are sensitive to temperature changes– Usually unicellular and

poikilothermic– Enzymes have temperature optima– If temperature is too high, proteins

denature, including enzymes, carriers and structural components

Temperature ranges are enormous (-20 to 100oC)

TemperatureTemperature

– Organisms exhibit distinct cardinal temperatures (minimal, maximal, and optimal growth temperatures)

– If an organism has a limited growth temperature range = stenothermal (e.g. N. gonorrhoeae)

– If an organism has a wide growth temperature range = eurythermal (E. faecalis)

–Psychrophiles can grow well at 0C, have optimal growth at 15C or lower, and usually will not grow above 20CArctic/Antarctic ocean Protein synthesis, enzymatic activity and transport systems have evolved to function at low temperatures

Cell walls contain high levels of unsaturated fatty acids (semi-fluid when cold)

– Psychrotrophs (facultative psychrophiles) can also grow at 0C, but have growth optima between 20C and 30C, and growth maxima at about 35C

Many are responsible for food spoilage in refrigerators

– Mesophiles have growth minima of 15 to 20C, optima of 20 to 45C, and maxima of about 45C or lower

Majority of human pathogens

–Thermophiles have growth minima around 45C, and optima of 55 to 65CHot springs, hot water pipes, compost heaps

Lipids in PM more saturated than mesophiles (higher melting points)

–Hyperthermophiles have growth minima around 55C and optima of 80 to 110CSea floor sulfur vents

Oxygen concentration– Obligate aerobes are completely

dependent on atmospheric O2 for growthOxygen is used as the terminal electron acceptor for electron transport in aerobic respiration

– Facultative anaerobes do not require O2 for growth, but do grow better in its presence

– Aerotolerant anaerobes ignore O2 and grow equally well whether it is present or not

–Obligate (strict) anaerobes do not tolerate O2 and die in its presence

–Microaerophiles are damaged by the normal atmospheric level of O2 (20%) but require lower levels (2 to 10%) for growth

Oxygen tolerance is determined by an organism’s ability to destroy toxic oxidizing products of oxygen reduction– Remember, because oxygen has two unpaired

outer orbital electrons, it accepts electrons readily

Toxic compounds– Superoxide radical:

O2 + e- O2•-

– Hydrogen peroxide: O2•- + e- + 2H+ H2O2

– Hydroxyl radical: H2O2 + e- + H+ H2O + OH•

These compounds are used deliberately by phagocytic WBC to break down intracellular microbes (respiratory burst)

Solution used by obligate aerobes and facultative anaerobes:– Produce enzymes that convert these toxic oxidizing

products to non-toxic compounds Superoxide dismutase

2O2•- + 2H+ O2 + H2O2

Catalase2H2O2 2H2O + O2

– Aerotolerant microbes have SOD; Obligate anaerobes lack SOD and catalase or have low concentrations

Laboratory considerations– Aerobic cultures

Shaken or sterile air introduced to medium

– Anaerobic cultures Remove oxygen

– Include reducing agents in medium (e.g. thioglycollate or cysteine

• Dissolved oxygen is destroyed

• Growth beneath surface

– Replace oxygen with nitrogen gas and CO2 gas

– Gas-Pak jar

• H2 + palladium catalyst + O2 H2O

– Bags or pouches CaCO3 CO2 rich atmosphere

Pressure–Barotolerant organisms are adversely affected by increased pressure, but not as severely as are nontolerant organisms

–Barophilic organisms require, or grow more rapidly in the presence of, increased pressure

Radiation–Ultraviolet radiation damages cells by causing the formation of thymine dimers in DNAPhotoreactivation repairs thymine dimers by direct splitting when the cells are exposed to blue light

Dark reactivation repairs thymine dimers by excision and replacement in the absence of light

– Ionizing radiation such as X rays or gamma rays are even more harmful to microorganisms than ultraviolet radiationLow levels produce mutations and may indirectly result in death

High levels are directly lethal by direct damage to cellular macromolecules or through the production of oxygen free radicals