how many arrows do you see in the following shape?
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How many arrows do you see in the following shape?
Microbial GrowthMicrobial GrowthMicrobiology 2314
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
Turbidity indicates bacterial numbers are increasing.
Binary Fission
Generation Time
The Time Required for a Cell to Divide
Rapid Growth of Bacterial PopulationRapid Growth of Bacterial Population
Cockroaches Left Unchecked and Allowed to Multiply At Will
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
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
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
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
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
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
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
Bacterial Growth Can Be Bacterial Growth Can Be Modeled With Four Different Modeled With Four Different
PhasesPhases
Phases of Microbial Growth
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.
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.
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
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
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
Direct Count
Spread or Streak Plate
Advantages to a Streak Plate?Advantages to a Streak Plate?
Disadvantages?Disadvantages?
Direct Direct CountCount
Pour PlatePour Plate
Advantages to a Pour Plate?Advantages to a Pour Plate?
Disadvantages?Disadvantages?
Petroff-Hausser Chamber Slide
What are You Counting on a Petroff-Hausser Slide?
Direct Method Direct Method
FiltrationFiltration
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.
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
There Are More Accurate Methods to There Are More Accurate Methods to Determine Turbidity LevelsDetermine Turbidity Levels
Chemical and Physical Chemical and Physical Requirements for Bacterial Requirements for Bacterial
GrowthGrowth
Physical Requirements
Why is Mexican Food Spicy?Why is Mexican Food Spicy?
Oklahoma is #1Oklahoma is #1
Cardinal TemperaturesCardinal Temperatures
• Minimum Temperature• Optimum Temperature• Maximum Temperature
Classification of Bacteria by Classification of Bacteria by Temperature RequirementsTemperature Requirements
Buffers Are Added to Media to Maintain Proper pH
1. Phosphates
2. Peptones
3. Amino Acids
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
Osmotic Pressure EffectsOsmotic Pressure Effects
Bacterial response to osmotic effects is the reason we dry food.
Hams can be sugar cured or salt cured to preserve them.
Are they cooked?
What About Jerky?What About Jerky?
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)
What About What About Oxygen?Oxygen?
Special Special Culture Culture TechniquesTechniques
Gas Pack Gas Pack Jar Is Used Jar Is Used for for Anaerobic Anaerobic GrowthGrowth
Special Culture TechniquesSpecial Culture Techniques
Candle JarCandle Jar
Peritoneal Fluid Mixed Anaerobic Infection
Types of MediaTypes of Media
• Media can be classified on three primary levels
1. Physical State
2. Chemical Composition
3. Functional Type
Physical States of MediaPhysical States of Media
• Liquid Media
• Semisolid
• Solid (Can be converted into a liquid)
• Solid (Cannot be converted into a liquid)
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
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
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)
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
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.
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.
Growth of Staphylococcus aureus on Manitol Salt Agar results in a color change in the media from pink to yellow.
Enrichment MediaEnrichment Media
• Is used to encourage the growth of a particular microorganism in a mixed culture.
• Ex. Manitol Salt Agar for S. aureus
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
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.
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
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.
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.
FYI
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.