Dr Rita Oladele Dept of Med Micro &Para CMUL/LUTH Bacterial Growth Dr Rita Oladele Dept of Med Micro &Para CMUL/LUTH

AIMS Understand the concept of microbial growth Describe the steps involved in bacterial replication (binary fission) Differentiate between bacterial cell growth, bacterial replication and bacterial population growth Understand the calculation of population growth parameters Describe the bacterial population growth curve (batch culture) Describe what is happening to bacterial cells at each stage of the batch cycle growth cycle Describe commonly used methods for counting bacteria – direct microscopic count, viable count, turbidometric methods Differentiate batch culture from continuous culture

Bacterial growth Increase in number of bacterial cells rather than increase in size of individual bacteria Bacterial species only maintained if population continues to grow Growth depends on temperature, pH, osmotic pressure, oxygen, and nutrients

Microbial Growth contd • Growth= an increase in the number of cells, not an increase in size • Generation=growth by binary fission • Generation time=time it takes for a cell to divide and the population to double; most are 1-3 hours (E.coli: every 20 min.)

Bacterial cell replication • Bacterial cell is able to duplicate itself – asexualprocess – binary fission • Involves many chemical reactions – Energy transformation – Biosynthesis of small molecules (enzymes, cofactors) – Polymerisation of macromolecules from monomers • Assembly of macromolecules, formation of cellular structures (cell wall, cytoplasmic membrane ……….)

Bacterial division

Binary Division

Phases of Growth Lag phase Log or exponential Active growth Stationary Death = Growth rate Death Nutrients consumed pH too low (why?) Optimize curves in production

Bacterial growth curve

4/27/2017 Growth Curve – Chapter 6

Lag phase • When microbes inoculated into fresh medium they do not start to grow immediately (lag phase) • Length of lag phase variable – depends on history of the culture and growth conditions – exponentially growing culture inoculated into same media, -same growth conditions – no lag phase – old culture, same media & conditions – lag phase because cells need to replenish essential constituents to start growth & cell division cycle – Cells damaged (heat, radiation, toxic chemicals) lag phase as cells repair damage – Cells transferred from rich medium to poor culture medium, lag phase as cells have to synthesise more enzymes etc to enable synthesis of macromolecules not present in poor culture

Log or Exponential phase • Each cell divides to form 2 cells; 2 cells divide to form 4 cells …… • Rate of exponential growth influenced by environmental conditions(temperature,composition of culture medium) & genetic characteristics of organism

Stationary phase • In a batch culture exponential growth cannot occur indefinitely – Essential nutrients in medium used up and/or organism builds up to an inhibitory level. – Exponential growth ceases = stationary phase • In stationary phase – no net increase or decrease in cell numbers • Many cell functions continue – energy metabolism, biosynthesis • In some populations some slow growth may continue – some cells die and some grow – 2 processes balance out so no net change

Death phase • If incubation continues after stationary phase, cells may remain alive and continue to metabolise OR they may die = death phase • In some cases cell death is accompanied by lysis • Rate of cell death generally slower than that of exponential growth

Requirements for bacterial growth • Physical requirements – Temperature, pH, osmotic pressure, oxygen • Chemical requirements – Carbon, nitrogen, sulfur, and phosphorous – Trace elements

Optimum Growth Temperatures • Psychrophiles-optimum 15oC • Psychrotrophs- range from 20-30oC • Mesophiles- range from 25-40oC • Thermophiles-range from 50-60oC

pH – Most bacteria grow between pH 6.5 and 7.5 – Molds and yeasts grow between pH 5 and 6 – Acidophiles grow in acidic environments

Osmotic Pressure – Hypertonic environments, increase salt or sugar, cause plasmolysis; used to preserve foods – Extreme or obligate halophiles require high osmotic pressure – Facultative halophiles tolerate high osmotic

Requirements for bacterial growth • Physical requirements – Temperature, pH, osmotic pressure, oxygen • Chemical requirements – Carbon, nitrogen, sulfur, and phosphorous – Trace elements

Requirements for bacterial growth • Physical requirements – Temperature, pH, osmotic pressure, oxygen • Chemical requirements – Carbon, nitrogen, sulfur, and phosphorous – Trace elements

Requirements for bacterial growth • Physical requirements – Temperature, pH, osmotic pressure, oxygen • Chemical requirements – Carbon, nitrogen, sulfur, and phosphorous – Trace elements

The Requirements for Growth: Chemical • Nitrogen – In amino acids, proteins – Most bacteria decompose proteins – Some bacteria use NH4 + or NO3− – A few bacteria use N2 in nitrogen fixation • Sulfur – In amino acids, thiamine, biotin – Some bacteria use SO4 2− or H2S Phosphorus – In DNA, RNA, ATP, and membranes – PO43− is a source of phosphorus Carbon

Direct Measurement of microbial growth • Population growth is measured by: Direct microscopic count – Following changes in the number of cells – Weight of some component of cell mass eg protein – Total dry weight of

Viable count Only counts living cells • Viable cell = cell that is able to divide & form offspring • Usual method – determine the number of cells in the sample able to form colonies on suitable agar medium = platecount or colony count • Assumption: – Each viable cell can form one colony

Turbidity measurements of microbial growth

Continuous culture • Continuous cultures – flow system of constant volume where fresh medium is added continuously and spent culture medium removed continuously – at a constant rate. • Once the system is in equilibrium, the cell number and nutrient status remain constant steady state

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