2 Lab 2 Goals and Objectives : Lecture: Chapter 6 (Microbial Growth)Learn aseptic technique and pure culture isolationExercise 9: Aseptic TechniqueExercise 10: Pure Culture TechniqueTurn in cultures from home for incubationFinish microscope worksheet if necessary
3 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.
4 Growing microorganisms 1. Culture Medium: Nutrients prepared for microbial growthSterile: No living microbes2. Inoculation: Introduction of microbes into mediumInoculum: The material used in an inoculation3. Culture: Microbes growing in/on culture medium
5 Culture Media Nutrient broth- liquid form Nutrient agar- solid form Agar - complex polysaccharideUsed as solidifying agent for culture media in Petri plates, slants, and deepsGenerally not metabolized by microbesLiquefies at 100°CSolidifies ~40°C
7 Bacterial growth in culture Bacteria multiply by binary fissionThe population grows in geometric progression122 43 84 165 326 647 1288 2569 51810 103611 206412 412813 8256………-Lag phase: initial period of little to no cell division as bacteriaacclimate to new media-Log phase: period of exponential growth with a constant generationtime-Stationary phase: cell growth is equal to cell death-Death phase: cell death exceeds cell growth
9 Generation timeGeneration time - the time required for a cell to divide, to undergo one round of binary fissionIf 100 cells growing for 5 hours produced 1,720,320 cells:E. coli has a generation time of 20 minCommon bacterial generation times range 1-3 hrs
10 Quantifying Microbial Growth Direct MeasurementsPlate CountsFiltrationMost Probable Number (MPN)Direct Microscopic CountIndirect EstimationsTurbidityMetabolic ActivityDry Weight
11 Microbial Growth The requirements for microbial growth Physical TemperaturepHOsmotic pressureChemicalOxygenCarbonNitrogenSulfurPhosphorusTrace elementsOrganic factors
12 Physical Requirements -Temperature Minimum growth temperaturethe lowest temperature at which the species will growOptimum growth temperaturespecies grow bestMaximum growth temperaturethe highest temperature at which growth is possible.
13 Physical Requirements - pH Most bacteria grow between pH 6.5 and 7.5Molds and yeasts grow between pH 5 and 6Acidophiles grow in acidic environments
14 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 osmoticpressureExtreme or obligate halophiles requirehigh osmotic pressure
15 Chemical Requirements CarbonStructural organic molecules, energy sourceAutotrophs use CO2Chemoheterotrophs use organic carbon sourcesNitrogenIn amino acids, proteinsMost bacteria decompose proteinsSome bacteria use NH4+ or NO3A few bacteria use N2 in nitrogen fixationSulfurIn amino acids, thiamine, biotinSome bacteria use SO42 or H2SPhosphorusIn DNA, RNA, ATP, and membranesPO43 is a source of phosphorusInorganic elements required in small amounts- potassium, magnesium and calciumTrace Elements - iron, copper, molybdenum and zinc - Usually as enzyme cofactors
16 Chemical Requirements Organic Growth FactorsEssential organic compounds an organism is unable to synthesizeOrganic compounds obtained from the environmentVitamins, amino acids, purines, pyrimidines
18 Toxic Forms of OxygenSinglet oxygen: O2 boosted to a higher-energy stateSuperoxide free radicals: O2Peroxide anion: O22Hydroxyl radical (OH)
19 Biofilms Microbial communities Form slime or hydrogels Bacteria attracted by chemicals via quorum sensingClumps of bacteria adhering to surfaceMigrating clump of bacteriaSurfaceWater currents
20 Culture Media Complex Media: Extracts and digests of yeasts, meat, or plantsChemically Defined Media:Exact chemical composition is knownDifferential Media:Make it easy to distinguish colonies of different microbes.Enrichment MediaEncourages growth of desired microbeReducing media for anaerobic culture,Contain chemicals (thioglycollate or oxyrase) that combine O2Heated to drive off O2
21 Envelope containing sodium bicarbonate and sodium borohydride Figure 6.6 A jar for cultivating anaerobic bacteria on Petri plates.Clamp with clamp screwLid withO-ring gasketEnvelope containing sodium bicarbonate and sodium borohydridePalladium catalyst pelletsAnaerobic indicator (methylene blue)Petri plates
22 Obtaining Pure Cultures A pure culture contains only one species or strainA colony is a population of cells arising from a single cell or spore or from a group of attached cellsA colony is often called a colony-forming unit (CFU)The streak plate method is used to isolate pure cultures
23 Exercise 9: Aseptic Technique Each person make 3 inoculations:Broth to broth - Escherichia coli - 37°CSlant to slant - E. coli - 30°C3. Plate to slant - Serratia marcescens - 30°C (changed)Exercise 10: Pure Culture TechniqueEach person make 2 streak plates: Quadrant Streak Method B, page 82.Incubate at 25°CTurn in cultures from home for incubationFinish microscope worksheet if necessary
24 Streak Plate Flame the loop, cool it Flame the loop, cool it Figure 6.10a, b
25 Exercise 9: Aseptic Technique Each person make 3 inoculations: Broth to broth - E. coli - 37°CSlant to slant - E. coli - 37°C3. Plate to slant - S. marcescens - 30°C (changed)Exercise 10: Pure Culture TechniqueEach person make 2 streak platesQuadrant Streak Method B, page 85.Incubate plates at 25°CEach pair will need:1 broth culture Escherichia coli,1 slant culture Escherichia coli1 plate culture Serratia marcescensEach person will need:1 Nutrient Broth/BHI tubes,2 Nutrient Agar/BHIA slantsEach pair will need:1 mixed culture (which contains: Escherichia coli, Serratiamarcescens and Micrococcus luteus)Each person will need:2 Nutrient Agar/BHIA platesGet help now, today, if you arehaving any difficulty with oilimmersion lens use orspecimen measurements usingthe ocular micrometer!!!