BIOL121 Lecture 2 2023-24 PDF

Nutrition and culture of bacteria • What do they need for growth? • Nutrients must provide all elements that take part in the synthesis of cell material • Macroelements (Macronutients) - required in large amounts - present in all cells • Microelements (Micronutients, trace elements) - required in sm...

Nutrition and culture of bacteria • What do they need for growth? • Nutrients must provide all elements that take part in the synthesis of cell material • Macroelements (Macronutients) - required in large amounts - present in all cells • Microelements (Micronutients, trace elements) - required in small amounts - not required by all organisms Main nutritional elements for bacteria 10 Macro elements • • • • • • Carbon Oxygen Hydrogen Nitrogen Phosphorus Sulphur • • • • Potassium Calcium MagnesiumIron - • Proteins Components of • Nucleic acids • Carbohydrates • Lipids required for activity of some enzymes stabilises cell wall stabilises ribosomes, membranes, DNA part of enzymes and electron transport proteins Microelements for bacteria Microelements: components of various enzymes » Copper » Zinc » Nickel » Vanadium » Selenium » etc Types of bacterial media • How are these elements provided in the laboratory? • Culture media (singular, medium): nutrient solutions that provide all the elements required for growth • Chemically defined media: the exact chemical composition is known • Complex media: exact chemical composition not known made of digests of complex material such as milk protein, beef, soybeans, yeast etc. Bacterial growth on agar How do we grow them in the laboratory? • Solid culture media (s., medium): Nutrient agar plates Petri dish containing nutrient agar Aseptic technique working with bacteria From Brock Sterile disposable loops Growing bacteria in liquid media Liquid culture media: Erlenmeyer flasks Test tubes Bacterial multiplication Microbiology: growth = increase in cell numbers Most bacteria: cell divides into two new cells = binary fission When one cell divides in to two, one generation has occurred DNA replication Cell elongation Septum formation Cell separation Bacterial cell division: septum formation Bacterial generation time 1. Generation 2. Generation 3. Generation Time needed for a population to double =Doubling time = Generation time E. coli -20 mins at 37oC: others hours/days/weeks Bacterial growth may be exponential under ideal conditions Exponential growth Time needed for a population to double =Doubling time = Generation time Exponential growth: 5000 6 4000 5 Log (Number of cells) Number of cells growth with a constant doubling time 3000 2000 1000 5 Time (hours) 10 log 1000000 = log106 4 3 log 1000 = log 103 2 log 100 = log 102 1 log 10 = log 101 5 Time (hours) 10 Growth phases in a batch culture • Batch culture: culture is grown in a ‘closed system’ no additional nutrients added and no bacterial waste products removed during the culture period Log (Number of cells) Typical growth curve for bacterial population Time (hours) Time interval between inoculation and maximal division rate: Cells adjust to new environment Bacteria grow exponentially: • Constant doubling time • Growth rate is maximal Bacteria can no longer reproduce but are still alive (e.g., no nutrients left or growth inhibited by bacterial products Bacteria die How do you measure bacterial growth? Bacterial growth • Culturable bacteria: can be grown on media (liquid/solid) • Viable cells: some can be cultured but: – Viable but nonculturable bacteria refers as to bacteria that are in a state of very low metabolic activity and do not divide but are alive and have the ability to become culturable once resuscitated. – Or they just can’t grow on the conventional media (e.g. Legionella pneumophila) – Such bacteria can still cause disease (e.g. cholera) Counting bacterial by microscopy • Total Count: Non-specific dye that stains all bacteria – (culturable, viable and VBNC and in many cases dead cells) • Viable Count: uses fluorescent activity dyes – Counts all cells (culturable, viable and VBNC ) with activity (e.g. enzymatic, active membranes etc). • Culturable count: counts cells that can form colonies on solid media or increase turbidity in liquid media. Measuring bacterial growth microscopy (total cell count) DAPI/Acridine Orange/Live-Dead staining of bacteria Acridine Ornage DAPI Live-Dead stain Measuring bacterial growth using agar plates Culturable count (= plate count = colony count) Put sample on plate Spread sample Incubate Assumption: each culturable cell will grow and divide to yield one colony Mix sample with liquid agar Incubate Measuring bacterial growth by serial dilution 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml A serial dilution to obtain appropriate colony numbers: 9 ml broth 10-1 10-2 10-3 10-4 10-5 10-6 (20-200 cfu per plate) Total, viability, culture count: within same sample 1 Total, viability, culture count: within same sample 2 • Cholera is an acute, diarrheal illness caused by infection of the intestine with the toxigenic bacterium Vibrio cholerae serogroup O1 or O139. • An estimated 1.3 to 4 million people around the world get cholera each year and 21,000 to 143,000 people die from it (WHO) Monitoring Vibrio Cholerae • Culture: • Positive water not safe • Negative - Safe or not safe ? Safe or not safe? • Negative culture: not present –SAFE or • Negative culture: present- Viable but not culturablestill pathogenic- NOT SAFE Make water safe? Measuring bacterial growth turbidity Indirect measurement of bacterial growth A cell suspension looks cloudy (turbid): cells scatter light passing through the suspension Spectrophotometer for turbidity of bacterial growth photocell Effect of antimicrobial agents on growth Time Total cell count Bacteriolytic Log cell number Bacteriocidal Log cell number Log cell number Bacteriostatic Time Time Viable/culturable cell count Arrow indicates time at which growth inhibitory antimicrobial agent is added to the exponentially growing culture Metabolic classification of bacteria • Very important: carbon and energy sources • Carbon sources Heterotrophs: require organic molecules made by other organisms Autotrophs: CO2 is principal carbon source • Energy sources Phototrophs: use light as energy source (to produce ATP) Chemotrophs: oxidise organic or inorganic compounds PS. You do need to learn these terms! Environmental effects on bacterial growth Oxygen Temperature pH Osmolarity Oxygen and bacterial growth Oxygen high low Obligate aerobes Need O2 for growth Obligate anaerobes Cannot grow in presence of O2 Facultative anaerobes Can grow with and without O2 Aerotolerant anaerobes Do not need O2, but tolerate it Microaerophilic Need O2, but tolerate it only at low concentration Temperature and bacterial growth 1 Temperature and bacterial growth 2 Temperature and bacterial growth 3 • Psychrophiles - grow best below 15 °C - don’t grow above 20 °C - can grow below 0 °C Temperature and bacterial growth 4 Mesophiles - grow best between 20- 40 °C - many bacteria in our body are mesophiles (body temperature 37 °C) Thermophiles - grow best between 45- 80 °C - live in hot springs, compost heaps etc. Hyperthermophiles - grow best above 80 °C - live in hot springs Temperature and bacterial growth 5 Boulder Spring (Yellowstone National Park) is superheated (1-2 °C above boiling point) Temperature and bacterial growth 6 • Current record holder: Geogemma barossii • - grows between 80-121 °C • - survives at least 2 hours at 130 °C pH and bacterial growth Picrophilus oshimae pH 0.7 volcanic soils & waters gastric juices Acidophiles Grow best in acidic habitats Most bacteria acid soil pure water seawater very alkaline soil Alkaliphiles Grow best in alkaline habitats (soda lakes, carbonate soils) extremely alkaline soda lakes Natronobacterium gregoryi pH 12 Osmolarity and bacterial growth • Halophiles: grow in habitats with high salt concentration (sodium chloride) • Mild halophiles: grow with 1-6% NaCl • Moderate halophiles: grow with 7-15% NaCl • Extreme halophiles: grow with 15-30% NaCl High salt concentrations found in salt lakes – (e.g., Dead Sea) Do you know...? • How a hyperthermophile differs from a psychrophile? – >80oC vs 15oC or less • What is a facultative aerobe? – Not required but grows better with oxygen • A useful enzyme produced by thermophilic bacteria? – Taq polymerase, from Thermus aquaticus, used for PCR