Microbiology Lecture 2 - Microbial Growth and Nutrition 2023 PDF

MICROBIAL GROWTH AND NUTRITION BMS 100 Week 5 MICROBIAL GROWTH CURVE IN A CLOSED SYSTEM Stationary phase Log10 number viable cells Death phase Exponential phase Lag phase Time Long-term stationary phase MICROBIAL GROWTH CURVE – LAG PHASE Stationary phase Death phase Log10 number viable cells...

MICROBIAL GROWTH AND NUTRITION BMS 100 Week 5 MICROBIAL GROWTH CURVE IN A CLOSED SYSTEM Stationary phase Log10 number viable cells Death phase Exponential phase Lag phase Time Long-term stationary phase MICROBIAL GROWTH CURVE – LAG PHASE Stationary phase Death phase Log10 number viable cells Most cells do not reproduce immediately even though it’s a nutrient rich environment. WHY? Exponential phase Lag phase Time Long-term stationary phase MICROBIAL GROWTH CURVE – EXPONENTIAL PHASE Stationary phase Death phase Log10 number viable cells Cells grow at their maximum rate and there are no limitations to nutrients. Exponential phase Lag phase Time Long-term stationary phase Yield (cells or mg/ml) Growth rate (hr–1) MICROBIAL GROWTH CURVE – EXPONENTIAL PHASE Nutrient concentration Nutrient concentration MICROBIAL GROWTH CURVE – STATIONARY PHASE Stationary phase Death phase Log10 number viable cells As nutrients are depleted and wastes accumulate and we run out of space, the rate of reproduction decreases. Eventually, # of dying cells equals # of cells being produced, and the size of the population remains constant Exponential phase Lag phase Time Long-term stationary phase MICROBIAL GROWTH CURVE – DEATH PHASE Stationary phase Death phase Log10 number viable cells If nutrients are not added and wastes are not removed, a population reaches a point at which cells die at a faster rate than they are produced. Exponential phase Lag phase Time Long-term stationary phase MICROBIAL GROWTH CURVE – LONG-TERM STATIONARY PHASE Stationary phase Death phase Log10 number viable cells Dying cells release nutrients and the remaining cells feed off these nutrients Exponential phase Lag phase Time Long-term stationary phase MATHEMATICS OF GROWTH 50 log10Nt 1.000 40 ( 2n Number of cell3 (N0×2n) ) Time2 Generation Number ) 60 An Example of Exponential Growth1 20=1 1 0.000 20 1 21=2 2 0.301 40 2 22=4 4 0.602 60 3 23=8 8 0.903 10 80 4 24=16 16 1.204 0 30 0.500 20 0.000 0 20 40 60 Minutes of incubation 80 Log10 number of cells ( 0 Number of cells 0 DETERMINING THE GENERATION TIME 3.00 Exponential phase 2.00 Generation time is the time required for a population of cells to double in number. Number of cells (X107) 1.00 0.50 Lag phase 0.10 0 1 2 3 4 g Time (hours) 5 EXAMPLES OF GENERATION TIMES Examples of Generation Times1 Microorganism Incubation Temperature (0C) Generation Time (Hours) Bacteria Escherchiɑ Coli 40 0.35 Stɑphylococcus ɑureus 37 0.47 Clostridium botulinum 37 0.58 Mycobɑcterium tuberculosis 37 ~12 Treponemɑ pɑllidum 37 33 Ignicoccus hospitɑlis 90 1 Pyrococcus ɑbyssi 90 0.67 Sulfolobus tododɑii 75 6 Methɑnosphɑerɑ sedulɑ 75 8 Nitrososphɑerɑ viennensis 37 45 Archaea ENVIRONMENTAL FACTORS AFFECTING GROWTH: 1. SOLUTE CONCENTRATION/OSMOSIS Halophiles Compatible Solutes Growth rate Osmophiles 0 1 2 3 NaCl concentration (M) Nonhalophile Halotolerant Moderate halophile Extreme halophile 4 ENVIRONMENTAL FACTORS AFFECTING GROWTH: 2. PH pH [H+] Acidophiles (pH 0.0-5.5) Neutrophiles (5.5-8.0) Alkalophiles (8-11.5) 1M ACIDIC 10-1M 0 Ferroplɑsmɑ spp. (A) 1 Human stomach fluid 10-2M 2 10-3M 3 pH optima of some microbes Lemon juice Acid mine drainage Dunɑliellɑ ɑcidophilɑ (E) Cyɑnidium cɑldɑrium (E) Thiobɑcillus thiooxidɑns (B) Sulfolobus ɑcidocɑldɑrius (A) Grapefruit juice Oranges 10-4M 4 Beer 10-5M 5 Cheese Physɑrum polycephɑlum (E) 10-6M 6 Beef Lɑctobɑcillus ɑcidophilus (B) E. coli, Pseudomonɑs ɑeruginosɑ (B) Tomato juice Milk 10–7M NEUTRAL 7 10-8M 8 10-9M 9 10-10M 10 10-11M 11 10-12M 12 10-13M 13 Pure water Human blood Stɑphylococcus ɑureus (B) Seawater Nitrosomonɑs spp. (B) Baking soda Soap Bɑcillus ɑlcɑlophilus (B) Household ammonia Bleach 10-14M ALKALINE 14 Microcystis ɑeruginosɑ (B) ENVIRONMENTAL FACTORS AFFECTING GROWTH: 3. TEMPERATURE Temperature Ranges for Microbial Growth CARDINAL TEMPERATURES (0C) Minimum Optimum Maximum Piɑnococcus hɑlocryophilus -15 25 37 Bɑcillus psychrophilus 0-3 25 30 4 25-30 40 Escherichiɑ coli 10 37 45 Neisseriɑ gonorrhoeɑe 30 35-36 38 Thermus ɑquɑticus 40 70-72 79 Pyrolobus fumɑrii 90 106 113 Chlɑmydomonɑs nivɑlis -36 0 4 Amoebɑ proteus 4-6 22 35 Trichomonɑs vɑginɑlis 25 32-39 42 Cyclidium citrullus 18 43 47 0 4-15 15 1-3 28 40 21-23 45-50 50-58 Microorganism Bɑcteriɑ ɑnd Archɑeɑ Optimum Growth rate Pseudomonɑs fluorescens Protists Minimum Maximum Temperature Fungi Cɑndidɑ scotti Sɑcchɑromyces cerevisiɑe Mucor pusillus ENVIRONMENTAL FACTORS AFFECTING GROWTH: 3. TEMPERATURE Thermophiles Growth rate Psychrotolerants -10 Psychrophiles 0 10 Mesophiles 20 30 40 Hyperthermophiles 50 60 Temperature (oC) 70 80 90 100 110 120 ENVIRONMENTAL FACTORS AFFECTING GROWTH: 4. OXYGEN CONCENTRATION Can only survive with oxygen present Oxygen is the final electron acceptor at the end of their catabolism PROBLEM: produces peroxides as a result Solution: either the enzyme catalase or peroxidase or superoxide dismutase DISTINGUISHING AEROBIC BACTERIA: THE CATALASE TEST Catalase 2 H2O2 H2O2 + NADH + H+ 2 H2O + O2 Peroxidase H2O + NAD+ Superoxide dismutase Drop H2O2 on slides with bacteria E. Faecalis Catalase negative S. epidermidis Catalase positive GROUPINGS BY OXYGEN REQUIREMENTS: Obligate aerobes (peroxides)—have catalase and peroxidase Obligate anaerobes (e.g., clostridia) —have catalase and peroxidase Facultative anaerobes (e.g. E. coli)—aerobes that maintain life via fermentation or anaerobic respiration Aerotolerant anaerobes (e.g. Lactobacilli)--do not use aerobic metabolism, but they tolerate oxygen Microaerophiles (e.g. H. pylori)—require oxygen levels of 2% to 10% LIQUID THIOGLYCOLLATE GROWTH MEDIA CAN IDENTIFY OXYGEN REQUIREMENTS Oxic zone Anoxic zone Obligate aerobe Microaerophile Facultative anaerobe Aerotolerant anaerobe Strict anaerobe + SOD +/– Catalase (low levels) + SOD + Catalase + SOD – Catalase – SOD – Catalase Enzyme content + SOD + Catalase BIOFILMS microbial communities—have synergistic relationships among numerous microorganisms, attached to surfaces such as teeth, rocks in streams, shower scurtains, implanted medical devices 70% of bacterial diseases in industrialized nations are caused by biofilms Plaque Stapylococcus aureus biofilm on catheter CULTURING MICROORGANISMS DEFINITIONS Innoculum – the sample you are trying to grow Medium - Collection of nutrients innoculum can grown on Broth – liquid media Colonies – cultures visible on solid media AGAR – THE BASIS OF ALL SOLID MEDIA a complex polysaccharide derived from the cell walls of certain red algae Dissolves in water at 100°C, which does not kill most nutrients Solidifies at temperatures below 40°C (so can add more temperature-sensitive nutrients before solidifying) DEFINED (SYNTHETIC) MEDIA Exact chemical composition is known Difficult to prepare COMPLEX MEDIA Often contain nutrients released from partial digestion of beef, yeast, soy, or proteins like casein from milk. Often supplemented with blood Good for growing fastidious microorganisms e.g., Blood Agar, TSA SELECTIVE MEDIA contain substances that either favor the growth of particular microorganisms or inhibit the growth of unwanted ones. Bacterial colonies pH 7.3 Fungal colonies pH 5.6 Sabouraud Dextrose Agar OTHER EXAMPLES OF SELECTIVE MEDIA Eosin, Methylene Blue, Bile Salts – all kill gram positives but are harmless to gram negatives Increase NaCl to ensure only halophiles grow Or take a defined or complex medium and remove something (e.g., glucose) DIFFERENTIAL MEDIA either the presence of visible changes in the medium or differences in the appearance of colonies help us differentiate among the kinds of bacteria growing on the medium Beta-hemolysis Alpha-hemolysis e.g. utilization of RBC’s in blood agar No hemolysis (gamma-hemolysis) DIFFERENTIAL MEDIA e.g. carbohydrate utilization tubes: each tube contains a single kind of simple carbohydrate as a carbon source and the dye phenol red as a pH indicator. Durham tube (inverted tube to trap gas) No fermentation Acid fermentation with gas ANAEROBIC MEDIA MEDIA Clamp Airtight lid Palladium pellets to catalyze reaction removing O2 Chamber Envelope containing chemicals to release CO2 and H2 Petri plates Stab culture Methylene blue (anaerobic indicator) Anaerobic culture system MACCONKEY AGAR – SELECTIVE AND DIFFERENTIAL they enhance the growth of certain species that can then be distinguished from other species by variations in appearance Shape USING SOLID CULTURE TO IDENTIFY MICROORGANISMS Circular Rhizoid Irregular Filamentous Spindle Margin Entire Undulate Lobate Elevation Size Flat Raised Punctiform Small Curled Convex Moderate Filiform Pulvinate Umbonate Large Texture Smooth or rough Appearance Glistening (shiny) or dull Pigmentation Nonpigmented (e.g., cream, tan, white) Pigmented (e.g., purple, red, yellow) Optical property Opaque, translucent, transparent EXAMPLE Shape Circular Rhizoid Irregular Filamentous Spindle Margin Entire Elevation Flat Undulate Lobate Raised Curled Convex Filiform Pulvinate Size Punctiform Small Moderate Texture Smooth or rough Appearance Glistening (shiny) or dull Pigmentation Nonpigmented (e.g., cream, tan, white) Pigmented (e.g., purple, red, yellow) Optical property Opaque, translucent, transparent Large Umbonate CLINICAL SAMPLING: 1. SKIN, ACCESSIBLE MEMBRANE, WOUND CLINICAL SAMPLING: 2. BLOOD Fine Needle Aspiration from Vein CLINICAL SAMPLING: 3. CEREBROSPINAL FLUID Lumbar Puncture CLINICAL SAMPLING: 4. LUNGS CLINICAL SAMPLING: 5. OTHER EXAMINATIONS For stomach microbes, intubation is necessary A catheter is needed for true aseptic collection of urine – or the “clean catch” method Significantly diseased tissues will require biopsy. OBTAINING PURE CULTURES Pure culture – a culture in which all microbes come from a single progenitor cell or isolated colony The key – all cells in pure culture are genetically identical Requires high degree of aseptic technique and sterilized equipment THE STREAK PLATE used to isolate the organisms from a mixed population into a pure culture. inoculum is diluted by streaking it across the surface of the agar plate. while streaking in successive areas of the plate, the inoculum is diluted to the point where only one bacterial cell is deposited every few millimeters on the surface of the agar plate. MEASURING POPULATION SIZE: SERIAL DILUTIONS ( L A R G E P O P U L AT I O N ) MEASURING POPULATION SIZE: MEMBRANE FILTRATION ( S M A L L P O P U L AT I O N ) Sample to be filtered Membrane transferred to culture medium Membrane filter retains cells To vacuum Incubation Colonies MEASURING POPULATION SIZE: TURBIDITY ( I N D I R EC T & F O R L A RG E P O P U L AT I O N S ) THANK YOU! A N D H AV E A L O V E LY D AY !