How many arrows do you see in the following shape?
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Transcript of How many arrows do you see in the following shape?
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How many arrows do you see in the following shape?
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Microbial GrowthMicrobial GrowthMicrobiology 2314
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Bacterial GrowthBacterial Growth
• Bacterial Growth is Bacterial Reproduction
• The Numbers of Bacteria are Increasing
• We see:
1. Observable Increases in Colonies
Growing on Solid Media
2. Turbidity, Sediment, Scum or a
Change in Color in Broth Cultures
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Turbidity indicates bacterial numbers are increasing.
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Binary Fission
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Generation Time
The Time Required for a Cell to Divide
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Rapid Growth of Bacterial PopulationRapid Growth of Bacterial Population
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Cockroaches Left Unchecked and Allowed to Multiply At Will
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Generation TimeGeneration Time
N = (log10Nf – log10No ) / .301
N Number of generations
Nf Final Concentration of Cells
No Original concentration of cells
.301 Conversion Factor to Convert Log2 to Log10
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What is a Logarithm?What is a Logarithm?
• Logarithm is a function that gives the exponent in the equation bn = x. It is usually written as logb x = n.
• For example:
34 = 81 Therefore log3 81 = 4
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Example 1Example 1N = (log10Nf – log10No ) / .301
Measure Culture at 9:00 a.m. 10,000 cells / mlMeasure Culture at 3:00 p.m. 100,000 cells / mlCalculate N
N = (log10Nf – log10No ) / .301
N = (5-4)/0.301
N = 1/0.301
N = 3.33 Generations in 6 Hours
We Know: 6 Hours = 360 Minutes
Therefore: Generation Time = 360 Minutes / 3.33 Generations
N = 108 Minutes to Generate
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Example 2Example 2N = (log10Nf – log10No ) / .301
Measure Culture at 9:00 a.m. 10,000 cells / ml
Measure Culture at Noon 1,000,000 cells / ml
Calculate N
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Example 2Example 2N = (log10Nf – log10No ) / .301
Measure Culture at 9:00 a.m. 10,000 cells / mlMeasure Culture at Noon 1,000,000 cells / mlCalculate N
N = (log10Nf – log10No ) / .301
N = (6-4)/0.301
N = 2/0.301
N = 6.64 Generations in 3 Hours
We Know: 3 Hours = 180 Minutes
Therefore: Generation Time = 180 Minutes / 6.64 Generations
N = 27 Minutes to Generate
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Example 3Example 3N = (log10Nf – log10No ) / .301
Measure Culture at 9:00 a.m. 2000 cells / ml
Measure Culture at 1:00 p.m. 18,000 cells / ml
Calculate N
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Example 3Example 3N = (log10Nf – log10No ) / .301
Measure Culture at 9:00 a.m. 2000 cells / mlMeasure Culture at 1:00 p.m. 18,000 cells / mlCalculate N
N = (log10Nf – log10No ) / .301
N = (4.25-3.30)/0.301
N = .95/0.301
N = 3.16 Generations in 4 Hours
We Know: 4 Hours = 240 Minutes
Therefore: Generation Time = 240 Minutes / 3.16 Generations
N = 75.9 Minutes to GenerateN = 1.27 Hours to Generate
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Bacterial Growth Can Be Bacterial Growth Can Be Modeled With Four Different Modeled With Four Different
PhasesPhases
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Phases of Microbial Growth
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Typical bacterial exponential Growth Curve. In a rich culture medium bacteria, grown under aerobic conditions, achieve a final concentration of 2-5 x 109 cells per ml in about 12-18 hours. Although plotted on a different time scale the human growth curve looks the same; the human population at similar points on the growth curve are shown in red.
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Remember the Four Main StagesRemember the Four Main Stages• Lag Phase
Initial Phase / Metabolic Activities• Exponential Phase
2nd Phase / Optimum Growth / Doubling• Stationary Phase
3rd Phase / Exhaustion of Nutrients / Accumulation of Wastes
• Death PhaseFinal Phase / Continued Accumulation90% of Cells Die, then 90% of Remaining Cells Die, etc.
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Quantification of BacteriaQuantification of Bacteria
• Cell Numbers
• Total Mass of the Population
• Population Per Mediacells / ml or cells / gram
• Direct County Methods and Indirect Counting Methods
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Direct Counting MethodsDirect Counting Methods
• Normally Viable Counts• Remember that a Colony Starts Out as 1
Bacteria that Reproduces• Colonies May Not All Be The Same Size
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Types of Direct MeasurementsTypes of Direct Measurements
1. Plate Count
a. Spread (Streak) Plate
b. Pour Plate
2. Direct Observation on Slides
a. Petroff-Hausser Chamber Slide
3. Filtration
4. Most Probable Number
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Direct Count
Spread or Streak Plate
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Advantages to a Streak Plate?Advantages to a Streak Plate?
Disadvantages?Disadvantages?
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Direct Direct CountCount
Pour PlatePour Plate
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Advantages to a Pour Plate?Advantages to a Pour Plate?
Disadvantages?Disadvantages?
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Petroff-Hausser Chamber Slide
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What are You Counting on a Petroff-Hausser Slide?
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Direct Method Direct Method
FiltrationFiltration
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Most Probable NumberMost Probable Number
• Statistical Procedure used to estimate the number of bacteria that will grow in liquid media.
• Gives a 95% probability that the bacterial numbers will fall within a certain range.
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Indirect MeasurementsIndirect Measurements
Turbidity
a. No turbidity = < 107 cells/ml
b. Slight = 107 – 108 cells/ml
c. High = 108 – 109 cells/ml
d. Very High = > 109 cells/ml
Metabolic Activity
Dry Weight
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There Are More Accurate Methods to There Are More Accurate Methods to Determine Turbidity LevelsDetermine Turbidity Levels
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Chemical and Physical Chemical and Physical Requirements for Bacterial Requirements for Bacterial
GrowthGrowth
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Physical Requirements
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Why is Mexican Food Spicy?Why is Mexican Food Spicy?
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Oklahoma is #1Oklahoma is #1
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Cardinal TemperaturesCardinal Temperatures
• Minimum Temperature• Optimum Temperature• Maximum Temperature
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Classification of Bacteria by Classification of Bacteria by Temperature RequirementsTemperature Requirements
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Buffers Are Added to Media to Maintain Proper pH
1. Phosphates
2. Peptones
3. Amino Acids
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Classification of Bacteria by pH Classification of Bacteria by pH RequirementsRequirements
• Acidophiles 1.0 to 5.5
• Neutrophiles 5.5 to 8
• Alkalophiles 8.5 to 11.5
• Extreme
Alkalophiles > = 10.0
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Osmotic Pressure EffectsOsmotic Pressure Effects
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Bacterial response to osmotic effects is the reason we dry food.
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Hams can be sugar cured or salt cured to preserve them.
Are they cooked?
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What About Jerky?What About Jerky?
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Chemical RequirementsChemical Requirementsfor Bacteriafor Bacteria
• Water (80-90%)
• Carbon (Backbone of Hydrocarbons)
• Nitrogen (Amino Acids & Vitamins)
• Sulfur (Amino Acids &Vitamins)
• Phosphorus (Nucleic Acids, ATP)
• Minerals (Fe, Cu, Mg, etc. / as Cofactors)
• Oxygen (aerobes only)
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What About What About Oxygen?Oxygen?
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Special Special Culture Culture TechniquesTechniques
Gas Pack Gas Pack Jar Is Used Jar Is Used for for Anaerobic Anaerobic GrowthGrowth
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Special Culture TechniquesSpecial Culture Techniques
Candle JarCandle Jar
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Peritoneal Fluid Mixed Anaerobic Infection
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Types of MediaTypes of Media
• Media can be classified on three primary levels
1. Physical State
2. Chemical Composition
3. Functional Type
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Physical States of MediaPhysical States of Media
• Liquid Media
• Semisolid
• Solid (Can be converted into a liquid)
• Solid (Cannot be converted into a liquid)
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Liquid MediaLiquid Media
• Water-based solutions• Do not solidify at
temperatures above freezing / tend to be free flowing
• Includes broths, milks, and infusions
• Measure turbidity• Example: Nutrient
Broth, Methylene Blue Milk, Thioglycollate
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Semi-Solid Semi-Solid MediaMedia
• Exhibits a clot-like consistency at ordinary room temperature
• Determines motility• Used to localize a
reaction at a specific site.
• Example: SIM for hydrogen sulfide production and indole reaction
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Solid MediaSolid Media
• Firm surface for discrete colony growth• Advantageous for isolating and culturing• Two Types
1. Liquefiable (Reversible)
2. Non-liquefiable• Examples: Gelatin and Agar (Liquefiable)
Rice Grains, Cooked Meat Media,
Potato Slices (Non-liquefiable)
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Chemical Composition of Culture MediaChemical Composition of Culture Media
1. Synthetic Media • Chemically defined
• Contain pure organic and inorganic compounds
• Exact formula (little variation)
2. Complex or Non-synthetic Media • Contains at least one ingredient that is not
chemically definable (extracts from plants and animals)
• No exact formula / tend to be general and grow a wide variety of organisms
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Selective MediaSelective Media
• Contains one or more agents that inhibit the growth of a certain microbe and thereby encourages, or selects, a specific microbe.
• Example: Mannitol Salt Agar encourages the growth of S. aureus.
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Differential MediaDifferential Media
• Differential shows up as visible changes or variations in colony size or color, in media color changes, or in the formation of gas bubbles and precipitates.
• Example: Spirit Blue Agar to detect the digestion of fats by lipase enzyme. Positive digestion (hydrolysis) is indicated by the dark blue color that develops in the colonies.
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Growth of Staphylococcus aureus on Manitol Salt Agar results in a color change in the media from pink to yellow.
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Enrichment MediaEnrichment Media
• Is used to encourage the growth of a particular microorganism in a mixed culture.
• Ex. Manitol Salt Agar for S. aureus
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Microbes are Managed and Characterized Microbes are Managed and Characterized by Implementing the Five I’sby Implementing the Five I’s
1. Inoculation
2. Incubation
3. Isolation
4. Inspection
5. Identification
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InoculationInoculation
• Sample is placed on sterile medium providing microbes with the appropriate nutrients to sustain growth.
• Selection of the proper medium and sterility of all tools and media is important.
• Some microbes may require a live organism or living tissue as the inoculation medium.
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IncubationIncubation
• An incubator can be used to adjust the proper growth conditions of a sample.
• Need to adjust for optimum temperature and gas content.
• Incubation produces a culture – the visible growth of the microbe on or in the media
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IsolationIsolation
• The end result of inoculation and incubation is isolation.
• On solid media we may see separate colonies, and in broth growth may be indicated by turbidity.
• Sub-culturing for further isolation may be required.
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InspectionInspection• Macroscopically observe cultures to note color,
texture, size of colonies, etc.
• Microscopically observe stained slides of the culture to assess cell shape, size, and motility.
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FYI
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IdentificationIdentification
• Utilize biochemical tests to differentiate the microbe from similar species and to determine metabolic activities specific to the microbe.
• Utilize immunologic tests and genetic analysis.
The Major Purpose of the 5 I’s is to Encourage Growth of a Microorganism so that the Lab Worker Can Determine the Type of Microbe, Usually to the Level of Species.