Microbial Growth and Physiology Lecture Notes PDF

MICR20010 Lecture 5 Growth and Physiology Dr. Jennifer Mitchell Microbiology School of Biomolecular and Biomedical Science Lecture 4 • Prokaryotic cell morphology • Bacterial cell structure • The gram stain – Gram stain mechanism • Bacterial shapes – Different morphological shapes • Bacterial ce...

MICR20010 Lecture 5 Growth and Physiology Dr. Jennifer Mitchell Microbiology School of Biomolecular and Biomedical Science Lecture 4 • Prokaryotic cell morphology • Bacterial cell structure • The gram stain – Gram stain mechanism • Bacterial shapes – Different morphological shapes • Bacterial cell structure – G+ve G-ve Archaea – – – – Cell membrane Cell wall Outer membrane Cell appendages Learning Outcomes • Microbial Growth and Physiology • Growth of Bacteria – Bacteria Divide by Binary Fission – Growth of Bacteria on Solid Medium – Growth of Bacteria in Liquid Medium • • • • • Growth Phases of liquid Bacterial Culture Measurements of Bacterial Growth Direct Measurements of Bacterial Growth: Indirect Measurements of Bacterial Growth: Growth Requirements Microbial growth and physiology In the laboratory Liquid broths and Nutrient Agar plates GROWTH OF BACTERIA: • ASEPTIC TECHNIQUE • STERILE GROWTH MEDIA • BOIL – KILL ALL CELLS 100⁰C / 30 MIN • AUTOCLAVE – KILL ALL CELLS & SPORES 120 ⁰ C / 30 MIN • DRY HEAT – 150 ⁰ C / 120 MIN Bacteria divide by Binary Fission • Binary Fission • Chromosome divides to produce two identical copies • These copies segregate to opposite ends of the cell • Cell wall is laid down the middle of the cell to ultimately produce two new cells which are identical Binary Fission Bacterial growth is Exponential 1->2->4->8->16->32->64->128->256->512 etc • Bacterial growth proceeds exponentially • Generation times (time for bacterial mass to double) can be as fast as 20 minutes • Contributes to the remarkable adaptability of bacteria • Growth in a hostile environment can create a selective pressure for mutant cells which can persist. • One mutant cell which can survive will rapidly grow and take over. GROWTH OF BACTERIA ON SOLID MEDIUM • • • • AGAR: MELTS AT 100⁰C, SOLIDIFIES AT 40⁰C STERILIZED BY AUTOCLAVING BACTERIA GROW AS COLONIES SINGLE COLONY PURIFICATION GROWTH IN LIQUID MEDIUM • • • • • • COTTON WOOL BUNG GAS EXCHANGE KEEP CONTENTS STERILE INCUBATE STANDING OR AGITATED TURBID CULTURE ~ 109 CELLS/ML Growth Phases of a Bacterial Culture 1. Lag Phase – Adaptation 2. Logarithmic Phase – Cells multiply at the maximum rate 3. Stationary Phase – Lack of nutrients and build up of toxic metabolic intermediates means multiplication is balanced by cell death 4. Phase of decline Generation Times of Bacteria Bacterium Medium Generation Time (minutes) Escherichia coli Glucose-salts 17 Bacillus megaterium Sucrose-salts 25 Milk 26 Lactose broth 48 Staphylococcus aureus Heart infusion broth 27-30 Lactobacillus acidophilus Milk 66-87 Rhizobium japonicum Mannitol-salts-yeast extract 344-461 Mycobacterium tuberculosis Synthetic 792-932 Treponema pallidum Rabbit testes 1980 Streptococcus lactis Streptococcus lactis Measurements of bacterial growth • Direct measurements of bacterial growth: I. Total cell count. Using microscope and counting chamber II. Total viable count. Cells in culture are diluted and spread on nutrient agar plates. • Only viable cells will reproduce to give rise to a colony. Direct measurements of bacterial growth: • Counting chamber Direct measurements of bacterial growth: • The area and volume under each square is known. • Can determine the number of cells in sample volume. Total Viable Count Serial 10-fold dilutions Total Viable Count: Spread plate method and pour plate method Indirect measurements of bacterial growth • Turbidity • (Cloudiness) • Measures live and dead cells How a spectrophotometer measures turbidity (cloudiness) Chemostat culture 1. Cell density controlled by nutrient conc. 2. Growth rate controlled by flow rate of nutrient Growth requires • Energy, • The building blocks required for the construction of cellular machinery • Appropriate environmental conditions Growth Requirements • Nutrient Requirements – Water – Carbon (carbohydrate) – Nitrogen (protein) – Inorganic salts Iron - siderophores – Oxidation of organic compounds – (carbohydrates, lipids, proteins) Temperature 20⁰C- 110 ⁰ C !! pH 4.0 - 9.0 Atmosphere O₂ / No O₂ Energy • Derived from the enzymatic breakdown of organic substrates (carbohydrates, lipids or proteins) in a process called Catabolism • Energy generated from catabolism is used to synthesise cellular constituents in a process called Anabolism • Catabolism + Anabolism = Metabolism Bacterial growth in diverse environments • In addition to carbohydrates, lipids and proteins bacteria can also derive energy from plastic, rubber and toxic compounds like phenol. • Important implications for decontamination of environmental pollution Exxon Valdez Oil Spill in Alaska: Engineered bacteria that “eat” hydrocarbons” were fertilised onto beaches contaminated with oil. Known as bioremediation, this method was successful on several beaches where the oil was not too thick. Auxotrophs • The ability of individual bacterial species to produce their own cellular components will dictate its nutritional requirements • E.g. some species can synthesis all essential amino acids whereas others need amino acids to be added to their growth media (auxotrophs). Oxygen • Obligate aerobes – grow only in presence of O2. • Obligate anaerobes – grow only in absence of O2, killed by O2 • Facultative anaerobes – grow in presence & absence of O2 Temperature • Psychrophile - cold loving bacteria (10-20⁰C) • Mesophile (20-40 ⁰ C) – human body temperature – pathogens – opportunists • Thermophile - heat loving (>60 ⁰ C) pH • Many bacteria grow best at neutral pH • Some can survive/grow – acid – alkali Extremophiles Antartica Hot geyser at Yellowstone National Park Further Reading • Brock, Biology of Microorganisms, Madigan, Martinko and Parker 10th Ed. • Chapter 5 “Nutrition, Laboratory Culture and Metabolism of Microorganisms” • Chapter 6 “Microbial Growth”