Transcript Lab 2
Chapter 6 Microbial Growth Lab 2 Goals and Objectives : Lecture: Chapter 6 (Microbial Growth) Learn aseptic technique and pure culture isolation • Exercise 9: Aseptic Technique • Exercise 10: Pure Culture Technique • Turn in cultures from home for incubation • Finish microscope worksheet if necessary • Microbiology is the study of life and organisms that are too small to be seen with the naked eye. • Microbial growth - an increase in the number of cells in a population , not increase in size. Reproduction of Prokaryotes - Binary fission Bacterial growth in culture • Bacteria multiply by binary fission • The population grows in geometric progression • • • • • • • • • • • • • • 12 2 4 3 8 4 16 5 32 6 64 7 128 8 256 9 518 10 1036 11 2064 12 4128 13 8256 ……… -Lag phase: initial period of little to no cell division as bacteria acclimate to new media -Log phase: period of exponential growth with a constant generation time -Stationary phase: cell growth is equal to cell death -Death phase: cell death exceeds cell growth Number of generation Generation time • Generation time - the time required for a cell to divide, to undergo one round of binary fission – If 100 cells growing for 5 hours produced 1,720,320 cells: • E. coli has a generation time of 20 min • Common bacterial generation times range 1-3 hrs Growing microorganisms 1. Culture Medium: Nutrients prepared for microbial growth • Sterile: No living microbes 2. Inoculation: Introduction of microbes into medium Inoculum: The material used in an inoculation 3. Culture: Microbes growing in/on culture medium Culture Media • Nutrient broth- liquid form • Nutrient agar- solid form – Agar - complex polysaccharide • Used as solidifying agent for culture media in Petri plates, slants, and deeps • Generally not metabolized by microbes • Liquefies at 100°C • Solidifies ~40°C Culture Media • Complex Media: Extracts and digests of yeasts, meat, or plants • Chemically Defined Media: Exact chemical composition is known • Differential Media: – Make it easy to distinguish colonies of different microbes. • Enrichment Media Encourages growth of desired microbe • Reducing media for anaerobic culture, Contain chemicals (thioglycollate or oxyrase) that combine O2 Heated to drive off O2 Figure 6.9b Quantifying Microbial Growth • Direct Measurements – – – – Plate Counts Filtration Most Probable Number (MPN) Direct Microscopic Count • Indirect Estimations – Turbidity – Metabolic Activity – Dry Weight Microbial Growth • The requirements for microbial growth • Physical – Temperature – pH – Osmotic pressure • Chemical • • • • • • • Oxygen Carbon Nitrogen Sulfur Phosphorus Trace elements Organic factors Physical Requirements -Temperature – Minimum growth temperature • the lowest temperature at which the species will grow – Optimum growth temperature • species grow best – Maximum growth temperature • the highest temperature at which growth is possible. Physical Requirements - 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 Physical Requirements - Osmotic Pressure • Osmotic pressure is the hydrostatic pressure produced by a difference in concentration between solutions on the two sides of a surface such as a semipermeable membrane. • An isotonic environment for a cell is created when the solution outside of the cell is isotonic (having equal accent )with the cytoplasm of the cell. • Hypotonic environment - the solution outside of the cell is hypotonic in comparison to the cytoplasm of the cell. – cause cell lyses for some cells and nothing to bacteria, because of cell wall . • Hypertonic environments - hypertonic solution has more dissolved solute than the cytoplasm of the cell ( increase salt or sugar) – cause plasmolysis ( shrink the cells ) – Facultative halophiles tolerate high osmotic pressure – Extreme or obligate halophiles require high osmotic pressure Figure 4.18 - Overview Chemical Requirements • Carbon – Structural organic molecules, energy source – Autotrophs use CO2 – Chemoheterotrophs use organic carbon sources • • • 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 – Most bacteria decompose proteins – Some bacteria use SO42 or H2S Phosphorus – In DNA, RNA, ATP, and membranes – PO43 is a source of phosphorus • Inorganic elements required in small amounts- potassium, magnesium and calcium • Trace Elements - iron, copper, molybdenum and zinc - Usually as enzyme cofactors Chemical Requirements • Organic Growth Factors – Essential organic compounds an organism is unable to synthesize – Organic compounds obtained from the environment – Vitamins, amino acids, purines, pyrimidines Chemical Requirements - Oxygen (O2) Toxic Forms of Oxygen • Singlet oxygen: O2 boosted to a higher-energy state • Superoxide free radicals: O2 • Peroxide anion: O22 • Hydroxyl radical (OH) Figure 6.6 A jar for cultivating anaerobic bacteria on Petri plates. Lid with O-ring gasket Clamp with clamp screw Envelope containing sodium bicarbonate and sodium borohydride Anaerobic indicator (methylene blue) Petri plates Palladium catalyst pellets Biofilms • Microbial communities • Form slime or hydrogels – Bacteria attracted by chemicals via quorum sensing Clumps of bacteria adhering to surface Migrating clump of bacteria Surface Water currents Obtaining Pure Cultures • A pure culture contains only one species or strain • A colony is a population of cells arising from a single cell or spore or from a group of attached cells – A colony is often called a colony-forming unit (CFU) • The streak plate method is used to isolate pure cultures • Exercise 9: Aseptic Technique • Each person make 3 inoculations: 1. Broth to broth - Escherichia coli - 37°C 2. Slant to slant - E. coli - 30°C 3. Plate to slant - Serratia marcescens - 30°C (changed) • Exercise 10: Pure Culture Technique – Each person make 2 streak plates: Quadrant Streak Method B, page 82. – Incubate at 25°C • Turn in cultures from home for incubation • Finish microscope worksheet if necessary Streak Plate Flame the loop, cool it Flame the loop, cool it Flame the loop, cool it Figure 6.10a, b • Exercise 9: Aseptic Technique • Each person make 3 inoculations: 1. Broth to broth - E. coli - 37°C 2. Slant to slant - E. coli - 37°C 3. Plate to slant - S. marcescens 30°C (changed) • Exercise 10: Pure Culture Technique – Each person make 2 streak plates – Quadrant Streak Method B, page 85. – Incubate plates at 25°C Each pair will need: 1 broth culture Escherichia coli, 1 slant culture Escherichia coli 1 plate culture Serratia marcescens Each person will need: 1 Nutrient Broth/BHI tubes, 2 Nutrient Agar/BHIA slants Each pair will need: 1 mixed culture (which contains: Escherichia coli, Serratia marcescens and Micrococcus luteus) Each person will need: 2 Nutrient Agar/BHIA plates Get help now, today, if you are having any difficulty with oil immersion lens use or specimen measurements using the ocular micrometer!!!