BACTERIAL GROWTH Dr. Faten Mostafa Professor of Medical Microbiology & Immunology, Faculty of Medicine, Ain Shams University 6/26/2019 Dr. faten mostafa

At the end of this chapter you should be able to: Define bacterial growth, and generation time. Describe the process of bacterial replication. Demonstrate the phases of microbial growth and describe their relation to generation time. Discuss clinical significance of growth curve. 6/26/2019 Dr. faten mostafa

At the end of this chapter you should be able to: Compare and contrast between autotrophic and heterotrophic bacteria regarding nutritional requirements. Differentiate bacteria according to their gaseous requirements. Describe physical conditions necessary for bacterial growth 6/26/2019 Dr. faten mostafa

Short 20 minutes long. 18 hours E.coli. Mycobacteria Growth involves both an increase in Generation Time (doubling time) Short 20 minutes E.coli. long. 18 hours Mycobacteria 6/26/2019 Dr. faten mostafa

Doubling time (generation time) time required for a population of bacteria cells to double • varied from one species to another - doubling time depends on nutritional and genetic factors eg. Escherichia coli doubles in ~20 min 6/26/2019 Dr. faten mostafa

DNA DNA replication Cell separation Septum formation Microbial growth Binary fission: asexual division of one cell into two DNA DNA replication Septum formation DNA is attached to membrane during separation Cell separation 6/26/2019 Dr. faten mostafa

Bacterial Reproduction 4-protoplasm becomes divided into 2 equal parts by inward growth of CM & CW 1-Simple binary fission 2-double stranded DNA (bacterial chromosome) separate 3-acts as a template for another copy. 5-parent cell separates into 2 daughter identical cells. 6/26/2019 Dr. faten mostafa

1 2 4 8 16 Exponential increase in the number of cells Microbial growth an increase in the number of cells 1 2 4 8 16 Exponential increase in the number of cells 6/26/2019 Dr. faten mostafa

BACTERIAL GROWTH In Vitro Facultative intracellular living cells artificial culture media obligate intracellular 6/26/2019 Dr. faten mostafa

both IC outside host can be cultivated on artificial media outside host can not cultivated Treponema pallidum (of syphilis) and Mycobacterium leprae (of leprosy) cannot grow in vitro 6/26/2019 Dr. faten mostafa

Give reasons chlamydia and Rickettsia can grow only within living cells . They are obligate intracellular parasites. is that they lack the ability to produce sufficient ATP and must use ATP produced by the host cells. • 6/26/2019 Dr. faten mostafa

Bacterial Growth Curve If No of bacterial cells present in any culture is measured &No is plotted in relation to time (growth period), the resulting figure is called Bacterial Growth Curve. It is formed of 4 phases Exponential Phase (Log phase) Stationary Phase Decline or Death Phase Lag Phase 6/26/2019 Dr. faten mostafa

Bacterial Growth Curve 6/26/2019 Dr. faten mostafa

Growth phases Lag Exponential (Log) Stationary Death log # of viable cells Slower growth rate Time Dr. faten mostafa 6/26/2019

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log # of viable Lag cells Time Lag phase cells adapt to their new environment They start to synthesize enzymes and molecules required for bacteria growth No cell division length of this phase depends on the type of the organism, size of the inoculum and type of growth medium. one hour up to several days depending log # of viable cells Lag Time Dr. faten mostafa 6/26/2019

Lag phase an increase in bacterial mass per unit of volume, but no increase in cell count. 6/26/2019 Dr. faten mostafa

log # of viable cells Time Exponential (log) phase cell number increases logarithmically most active phase . cells dividing at their maximum rate of division phase continues until the nutrient of the medium is exhausted or toxic metabolites accumulate. Bacteria are highly susceptible to antibiotics at this phase Exponential (log) phase ascending straight line. ● log # of viable cells Time Dr. faten mostafa 6/26/2019

log # of viable cells Time Stationary Stationary phase . nutrient in the medium are exhausted and toxic products accumulate, the rate of growth decrease. The number of dying cells equal to the number of newly formed cells. The number of viable bacteria remains constant. Process of sporulation in spore forming bacteria start in this phase Stationary log # of viable cells 6/26/2019 Dr. faten mostafa Time

log # of viable cells Time Death Death phase exhaustion of nutrients, accumulation of waste products and harmful changes in pH continue; the rate of division becomes less and less, and the death rate increases until it reaches a steady level.   NO of dying bacteria exceeds the number of newly formed bacteria. NOof viable living bacteria is decreasing until the population dies out entirely. Death log # of viable cells Time 6/26/2019 Dr. faten mostafa

Clinical significance of bacterial Growth Curve natural infections and diseases Lag Phase incubation period. Exponential &stationary Phases the clinical signs and symptoms of disease. Decline Phase convalescence period &recovery from a disease. 6/26/2019 Dr. faten mostafa

Growth Requirements Chemical nutritional gaseous Physical temperature OP 6/26/2019 Dr. faten mostafa

Nutritional requirements Minerals Carbon & Nitrogen According to carbon requirement, bacteria are subdivided Growth factors utilize simple inorganic . CO2,Ho2, and inorganic salts they synthesize organic substances proteins, carbohydrates Sulfur, Phosphorus, Potassium, Magnesium Calcium. Heterotrophic Autotrophic yeast extract, blood, B complex vitamins, amino acids, purines pyrimidines. Require organic source of carbon. free living, non-pathogenic derived from living host are called parasite 6/26/2019 Dr. faten mostafa

Heterotrophic pathogenic or commensals according to nitrogen requirement nonexacting bacteria that utilize inorganic source of nitrogen exacting bacteria that utilize organic source of nitrogen. growing only in special artificial cultures containing specific growth factors Heterotrophic bacteria include both pathogenic and commensal 6/26/2019 Dr. faten mostafa

N.B. Fastidious bacteria are those bacteria with complex nutritional needs. 6/26/2019 Dr. faten mostafa

Growth Requirements O2 CO2 Gaseous (chemical ) Requirements Physical Nutritional requirements Carbon & Nitrogen Growth factors O2 CO2 Temperature Inorganic ions pH 6/26/2019 Dr. faten mostafa

Gaseous Requirements Oxygen carbon dioxide. low O2 tension Microaerophilic CO­2 present in air (0.05%) is sufficient for growth higher con of CO2 (5-10%) for growth,. low O2 tension Obligate aerobes Presence of O2 Capnophilic Bactria Obligate anaerobes absence of O2 pathogenic Neisseria Brucella abortus. Facultative anaerobes Aerobic &anaerobic most of pathogenic bacteria Dr. faten mostafa 6/26/2019

Gaseous Requirements 6/26/2019 Dr. faten mostafa

. Effect of Oxygen Some organisms require O2 Some organisms are killed by O2 Some are not affected by O2 Classes based on oxygen requirement 1. obligate aerobes 2. microaerophiles 3. facultative anaerobes 4. strict (obligate) anaerobes 6/26/2019 Dr. faten mostafa

require ambient concentration of O2 (21%) 1. aerobes: require ambient concentration of O2 (21%) 2. microaerophiles: • grow only at low [O2] • require O2 for aerobic respiration, but have some enzymes that are inactivated by O2 3. facultative anaerobes: • can grow aerobically or anaerobically • can use either O2 or another substance as the terminal electron acceptor 4- obligate anaerobes. These bacteria grow only in absence of O2 6/26/2019 Dr. faten mostafa

H2O2 + e- + H+  H2O + OH• hydroxyl radical OH• + e- + H+  H2O Aerobes obtain their energy by a series of oxidation-reduction reaction in which the hydrogen acceptor is atmospheric O2 (aerobic respiration). O2 + e-  O2- superoxide O2- + e- + 2H+  H2O2 peroxide H2O2 + e- + H+  H2O + OH• hydroxyl radical OH• + e- + H+  H2O Overall: O2 + 4 e- + 4 H+  2 H2O 6/26/2019 Dr. faten mostafa

Toxic forms of oxygen Toxic, reactive oxygen species oxidize cellular macromolecules (e.g. DNA, proteins, and lipids) O2- superoxide H2O2 peroxide OH• hydroxyl radical most damaging 6/26/2019 Dr. faten mostafa

SOD 2 H2O2  2 H2O + O2 O2- H2O2 + NADH + H+  2 H2O + NAD+ 2. Adaptations to toxic oxygen: detoxifying enzymes ability of cells to grow in the presence of O2 depends on the presence of enzymes that eliminate these O2 toxic products, e.g. catalase enzyme which destroys H2O2 and superoxide dismutase which destroys superoxide O- radicals. a. Catalase 2 H2O2  2 H2O + O2 b. Peroxidase H2O2 + NADH + H+  2 H2O + NAD+ c. Superoxide dismutase (SOD) O2- + O2-  2 H2O2 + O2 O2- to catalase reaction SOD 6/26/2019 Dr. faten mostafa

SOD appears to be required for the survival of aerobes Superoxide dismutase (SOD) SOD appears to be required for the survival of aerobes O2- anaerobe •. GR .Anaerobic bacteria lack these enzymes 6/26/2019 Dr. faten mostafa

Facultative and anaerobic bacteria ferment, but aerobes, which can grow only in the presence of oxygen, do not. Aerobes,, produce metabolites that enter the Krebs cycle by processes other than fermentation, such as the deamination of amino acids 6/26/2019 Dr. faten mostafa

but others die in the presence of oxygen (anaerobes). Some bacteria can grow in the presence of oxygen (aerobes and facultatives), but others die in the presence of oxygen (anaerobes). The use of oxygen by bacteria generates toxic products such as superoxide and hydrogen peroxide. Aerobes and facultatives have enzymes, such as superoxide dismutase and catalase, that detoxify these products, but anaerobes do not and are killed in the presence of oxygen. 6/26/2019 Dr. faten mostafa

Microaerophiles need oxygen because they cannot ferment or respire anaerobically. they are poisoned by high concentrations of oxygen. 6/26/2019 Dr. faten mostafa

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Physical Requirements Temperature Hydrogen ion concentration (pH): Psychrophilic: range (5 - 25C) (cold loving microbes ) 15 0C most pathogenic bacteria is 6.5 and 7.5 alkaline pH Vibrio cholerae Mesophilic: range (25- 45C) moderate temp. loving microbes) 37 0C acidic pH Lactobacillus Thermophilic: range (45 –75C) (heat loving microbes) 60 0C 6/26/2019 Dr. faten mostafa

Non pathogenic bacteria may be psychrophilic or thermophilic Most pathogenic bacteria are mesophilic, optimum temperature of growth at 37 0C. Non pathogenic bacteria may be psychrophilic or thermophilic 6/26/2019 Dr. faten mostafa

Physical Requirements Temperature except except Lower temp Most Higher temp e.g ,Yerssinia pestis causing plaque grow at 27 C Why ???? 1- Campylobacter and Helicobacter, both at 42 C 2- Geobacillus stearothermophilus at 120 C Most bacteria grow at optimum temp 37 C ,Why ?/?? 6/26/2019 Dr. faten mostafa

Osmotic Pressure  Growth of most microorganisms proceeds best when the osmotic pressure is optimum. i.e. when the salt concentration of microbial cytoplasm is the same as the external environment ( 1% NaCl; isotonic). Microorganisms that live in marine environments can tolerate high salt concentrations.  halophilic.    6/26/2019 Dr. faten mostafa