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How many arrows do you see in the following shape?. Microbial Growth. Microbiology 2314. Bacterial Growth. Bacterial Growth is Bacterial Reproduction The Numbers of Bacteria are Increasing We see: 1. Observable Increases in Colonies Growing on Solid Media

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How many arrows do you see

in the following shape?

### Microbial Growth

Microbiology 2314

Bacterial 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

Generation Time

The Time Required for a Cell to Divide

Generation 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?
• Logarithm is a functionthat gives the exponent in the equation bn = x. It is usually written as logbx = n.
• For example:

34 = 81 Therefore log3 81 = 4

Example 1N = (log10Nf – log10No ) / .301

Measure Culture at 9:00 a.m. 10,000 cells / ml

Measure Culture at 3:00 p.m. 100,000 cells / ml

Calculate 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 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 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

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 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 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

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 Generate

N = 1.27 Hours to Generate

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 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 Phase

Final Phase / Continued Accumulation

90% of Cells Die, then 90% of Remaining Cells Die, etc.

Quantification of Bacteria
• Cell Numbers
• Total Mass of the Population
• Population Per Media

cells / ml or cells / gram

• Direct County Methods and Indirect Counting Methods
Direct 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 Measurements
• Plate Count

b. Pour Plate

2. Direct Observation on Slides

a. Petroff-Hausser Chamber Slide

3. Filtration

4. Most Probable Number

Direct Count

Direct Count

Pour Plate

Direct Method

Filtration

Most 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 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

Cardinal Temperatures
• Minimum Temperature
• Optimum Temperature
• Maximum Temperature
Classification of Bacteria by pH Requirements
• Acidophiles 1.0 to 5.5
• Neutrophiles 5.5 to 8
• Alkalophiles 8.5 to 11.5
• Extreme

Alkalophiles > = 10.0

Chemical Requirementsfor 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)
Special Culture Techniques

Gas Pack Jar Is Used for Anaerobic Growth

Types of Media
• Media can be classified on three primary levels

1. Physical State

2. Chemical Composition

3. Functional Type

Physical States of Media
• Liquid Media
• Semisolid
• Solid (Can be converted into a liquid)
• Solid (Cannot be converted into a liquid)
Liquid 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 Media
• 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 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 Media
• Synthetic Media
• Chemically defined
• Contain pure organic and inorganic compounds
• Exact formula (little variation)
• 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 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 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 Media
• Is used to encourage the growth of a particular microorganism in a mixed culture.
• Ex. Manitol Salt Agar for S. aureus
• Inoculation
• Incubation
• Isolation
• Inspection
• Identification
Inoculation
• 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.
Incubation
• 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
Isolation
• 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.
Inspection
• 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.
Identification
• 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.