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REPRODUCTION & GROWTH
Lecture 4
Reference: Chapter 6 (Tortora)
Parungao-Balolong 2011Thursday, July 14, 2011
LECTURE OUTLINEReproduction & Growth
Requirements for Growth
Physical
Chemical
Measurement of Microbial Growth
Culture Media
Obtaining Pure Cultures
Preservation MethodsParungao-Balolong 2011
Thursday, July 14, 2011
LECTURE OUTLINEReproduction & Growth
Requirements for Growth
Physical
Chemical
Measurement of Microbial Growth
Culture Media
Obtaining Pure Cultures
Preservation MethodsParungao-Balolong 2011
Thursday, July 14, 2011
REPRODUCTION IN PROKARYOTES
Parungao-Balolong 2011
Binary fission
Budding
Conidiospores
(actinomycetes)
Fragmentation of
filamentsThursday, July 14, 2011
MICROBIAL GROWTH
Parungao-Balolong 2011
Microbial growth = increase in number of cells, not cell size
Nutrients = substances used in biosynthesis and energy production (required for microbial growth)
Environmental Factors = temperature, oxygen levels, osmotic concentration
Thursday, July 14, 2011
GROWTH
Parungao-Balolong 2011
GROWTH◦Increase in cellular constituents◦Leads to a rise in cell number
Budding, Binary Fission
For coenocytic organisms (multinucleate)◦Growth results in increased cell size not number
Thursday, July 14, 2011
MICROBIAL NUTRITION
Parungao-Balolong 2011
Macroelements or Macronutrients◦Carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium, calcium, magnesium and iron
Trace elements or Micronutrients◦Manganese, zinc, cobalt, molybdenum, nickel and copper
Thursday, July 14, 2011
GROWTH FACTORS
Parungao-Balolong 2011
BIOTIN◦Carboxylation (Leuconostoc)
CYANOCOBALAMIN or VIT B12◦Molecular rearrangements (Euglena)
FOLIC ACID◦One-carbon metabolism (Enterococcus)
PANTOTHENIC ACID◦Fatty acid metabolism (Proteus)
PYRIDOXINE or VIT B6◦Transamination (Lactobaci!us)
NIACIN◦Precursor of NAD and NADP (Bruce!a)
RIBOFLAVIN or VIT B2◦Precursor of FAD and FMN (Caulobacter)
THIAMINE or VIT B1◦Aldehyde group transfer (Baci!us
anthracis)
Thursday, July 14, 2011
MICROBIAL NUTRITION
Parungao-Balolong 2011
CARBON SOURCESCARBON SOURCES
Autotrophs CO2 sole or principal biosynthetic carbon source
Heterotrophs Reduced, preformed, organic molecules from other organisms
ENERGY SOURCESENERGY SOURCES
Phototrophs Light
Chemotrophs Oxidation of organic or inorganic compounds
HYDROGEN AND ELECTRON SOURCESHYDROGEN AND ELECTRON SOURCES
Lithotrophs Reduced inorganic molecules
Organotrophs Organic molecules
Thursday, July 14, 2011
MICROBIAL NUTRITION
Parungao-Balolong 2011
MAJOR NUTRITIONAL TYPES SOURCES OF ENERGY, HYDROGEN/ELECTRONS AND CARBON
REPRESENTATIVE MICROORGANISMS
PHOTOLITHOTROPHIC AUTOTROPHY
Light energyInorganic hydrogen/electron donorCO2 carbon source
AlgaePurple and green sulfur bacteriaBlue-green algae (cyanobacteria)
PHOTOORGANOTROPHIC HETEROTROPHY
Light energyOrganic hydrogen/electron donorOrganic carbon source (CO2 may also be used)
Purple non-sulfur bacteriaGreen non-sulfur bacteria
Thursday, July 14, 2011
MICROBIAL NUTRITION
Parungao-Balolong 2011
MAJOR NUTRITIONAL TYPES SOURCES OF ENERGY, HYDROGEN/ELECTRONS AND CARBON
REPRESENTATIVE MICROORGANISMS
CHEMOLITHOTROPHIC AUTOTROPHY
Chemical energy source (inorganic)Inorganic hydrogen/electron donorCO2 carbon source
Sulfur-oxidizing bacteriaHydrogen bacteriaNitrifying bacteriaIron bacteria
CHEMOORGANOTROPHIC HETEROTROPHY
Chemical energy source (organic)Organic hydrogen/electron donorOrganic carbon source
ProtozoaFungiMost non-photosynthetic bacteria
Thursday, July 14, 2011
THE GROWTH CURVE
Parungao-Balolong 2011
Population growth is studied by analyzing the growth curve of microorganisms
Growth of microorganisms reproducing by binary fission can be plotted as the logarithm of cell number versus the incubation time (Growth curve)
Thursday, July 14, 2011
THE GROWTH CURVE
Parungao-Balolong 2011
The Growth Curve can be obtained via a Batch Culture
◦Microorganisms are cultivated in a liquid medium◦Grown as a closed system◦Incubated in a closed culture vessel with a single batch of medium◦No fresh medium provided during incubation◦Nutrient concentration decline and concentrations of waste increase during the incubation period
Thursday, July 14, 2011
THE LAG PHASE
Parungao-Balolong 2011
No immediate increase in cell mass or cell number (Cell is synthesizing new components)
The necessity of a lag phase:◦Cells may be old and ATP, essential cofactors and ribosomes depletedmust be synthesized first before growth can begin◦Medium maybe different from the one the microorganism was growing
previouslynew enzymes would be needed to use different nutrients◦Microorganisms have been injured and require time to recover
Cells retool, replicate their DNA, begin to increase in mass and finally divide
Thursday, July 14, 2011
THE LAG PHASE
Parungao-Balolong 2011
LONG LAG PHASE◦Inoculum is from an old culture◦Inoculum is from a refrigerated source◦Inoculation into a chemically-different medium
SHORT LAG PHASE (or even absent)◦Young, vigorously growing exponential phase culture is transferred to fresh medium of same composition
Thursday, July 14, 2011
EXPONENTIAL/LOG PHASE
Parungao-Balolong 2011
Microorganisms are growing and dividing at the maximal rate possible given their genetic potential, nature of medium and conditions under which they are growing
Rate of growth is constant
Microorganism doubling at regular intervals
The population is most uniform in terms of chemical and physiological properties
Why the curve is smooth:◦Because each individual divides at a slightly different moment
Thursday, July 14, 2011
STATIONARY PHASE
Parungao-Balolong 2011
Population growth ceases and the growth curve becomes horizontal (around 109 cells on the average)
Why enter the stationary phase:◦Nutrient limitation (slow growth)◦Oxygen limitation◦Accumulation of toxic waste products
Thursday, July 14, 2011
DEATH PHASE
Parungao-Balolong 2011
Detrimental environmental changes like nutrient depletion and build up of toxic wastes lead to the decline in the number of viable cells
Usually logarithmic (constant every hour)
DEATH: no growth and reproduction upon transfer to new medium
Death rate may decrease after the population has been drastically reduced due to resistant cells
Thursday, July 14, 2011
LECTURE OUTLINEReproduction & Growth
Requirements for Growth
Physical
Chemical
Measurement of Microbial Growth
Culture Media
Obtaining Pure Cultures
Preservation MethodsParungao-Balolong 2011
Thursday, July 14, 2011
REQUIREMENTS FOR GROWTH:
PHYSICAL
Parungao-Balolong 2011
Temperature
Minimum growth temperature
Optimum growth temperature
Maximum growth temperature
Thursday, July 14, 2011
INFLUENCE OF LIPID CONTENT
Parungao-Balolong 2011
◦PSYCHROPHILYHIGH CONTENT OF
UNSATURATED FATTY ACIDSHELP MAINTAIN A SEMI-FLUID MEMBRANE STATE AT LOW TEMPERATURE
◦THERMOPHILYPROTEINS OR ENZYMES = INCREASED NUMBER OF SALT BRIDGES (RESIST UNFOLDING IN THE AQUEOUS MILIEU)
MEMBRANES = RICH IN SATURATED FATTY ACIDS (STABLE AT HIGH TEMPERATURES)
Thursday, July 14, 2011
TEMPERATURE RANGE
Parungao-Balolong 2011
STENOTHERMAL MICROBES◦Narrow range◦Neisseria gonorrhea
EURYTHERMAL MICROBES◦Wide range◦Enterococcus faecalis
Thursday, July 14, 2011
pH
Parungao-Balolong 2011
Most bacteria grow between pH 6.5 and 7.5
Molds and yeasts grow between pH 5 and 6
Acidophiles grow in acidic environments
Thursday, July 14, 2011
REQUIREMENTS FOR GROWTH: PHYSICAL
Parungao-Balolong 2011
Osmotic pressure
Hypertonic
environments,
increase salt or sugar,
cause plasmolysis
Extreme or obligate halophiles require high osmotic
pressure
Facultative halophiles tolerate high osmotic pressure
Thursday, July 14, 2011
REQUIREMENTS FOR GROWTH: PHYSICAL
Parungao-Balolong 2011
WATER ACTIVITY
SOURCE BACTERIA FUNGI ALGAE
1.00 (pure water)
blood Most Gram negative and non-halophiles
none none
0.90 ham Most cocci and Bacillus
Fusarium, Mucor, Rhizopus
0.60 Chocolate none Saccharomyces rouxii none
0.55(DNA disordered)
Thursday, July 14, 2011
REQUIREMENTS FOR GROWTH: PHYSICAL
Parungao-Balolong 2011
1atm
BAROTOLERANT◦Increased pressure does adversely affect them but not as much as it does non-tolerant bacteria
BAROPHILIC◦Grow more rapidly at high pressures
TRIVIA: one barophile has been recovered from the Mariana trench near the Philippines (10, 500m depth) ◦Can only grow at pressure greater than 400-500 atm (at 2°C)
Thursday, July 14, 2011
REQUIREMENTS FOR GROWTH: CHEMICAL
Parungao-Balolong 2011
Carbon
Structural organic
molecules, energy
source
Chemoheterotrophs use
organic carbon sources
Autotrophs use CO2
Thursday, July 14, 2011
REQUIREMENTS FOR GROWTH: CHEMICAL
Parungao-Balolong 2011
Nitrogen
In amino acids and proteins
Most bacteria decompose proteins
Some bacteria use NH4+ or NO3
–
A few bacteria use N2 in nitrogen fixation
Sulfur
In amino acids, thiamine and biotin Most bacteria decompose proteins
Some bacteria use SO42– or H2S
Phosphorus
In DNA, RNA, ATP, and membranes
PO43– is a source of phosphorus
Trace elements
Inorganic elements required
in small amounts
Usually as enzyme cofactors
Thursday, July 14, 2011
REQUIREMENTS FOR GROWTH: CHEMICAL
Parungao-Balolong 2011
Oxygen (O2)
Thursday, July 14, 2011
REQUIREMENTS FOR GROWTH: CHEMICAL
Parungao-Balolong 2011
Singlet oxygen: O2 boosted to a higher-energy state
Superoxide free radicals: O2–
Peroxide anion: O22–
Hydroxyl radical (OH•)
Thursday, July 14, 2011
LECTURE OUTLINEReproduction & Growth
Requirements for Growth
Physical
Chemical
Culture Media
Measurement of Microbial Growth
Obtaining Pure Cultures
Preservation MethodsParungao-Balolong 2011
Thursday, July 14, 2011
CULTURE MEDIA
Parungao-Balolong 2011
Culture medium:
Nutrients prepared
for microbial growth
Sterile: No living
microbes
Inoculum:
Introduction of
microbes into
medium
Culture: Microbes
growing in/on
culture medium
Thursday, July 14, 2011
CULTURE MEDIA
Parungao-Balolong 2011
TYPES: Chemically-Defined and Complex
Chemically defined media: Exact chemical composition is known
Complex media: Extracts and digests of yeasts, meat, or plants
Nutrient broth
Nutrient agar
Thursday, July 14, 2011
RECALL: HISTORY OF
Parungao-Balolong 2011
BEFORE AGAR◦Liquid medium
POTATO SLICES◦Robert Koch (1881)◦Used boiled potato, sliced◦Not all bacteria grew well
GELATIN◦Frederick Loeffler◦Meat extract medium + gelatin◦But gelatin liquid at room temperature
AGAR◦Fannie Eilshemius Hesse (1882)◦Agar used for jams and jelly
Thursday, July 14, 2011
Parungao-Balolong 2011
Fannie, wife of Walther Hesse, was
working in Koch's laboratory as her
husband's technician and had
previously used agar to
Complex polysaccharide
Used as solidifying agent for culture
media in Petri plates, slants, and deeps
Generally not metabolized by
microbes
Liquefies at 100°C
AGAR
Thursday, July 14, 2011
ANAEROBIC CULTURE METHODS
Parungao-Balolong 2011
Reducing media
Contain chemicals (thioglycollate or oxyrase) that combine O2
Heated to drive off O2
Thursday, July 14, 2011
ANAEROBIC CULTURE METHODS
Parungao-Balolong 2011Thursday, July 14, 2011
SELECTIVE MEDIA & DIFFERENTIAL MEDIA
Parungao-Balolong 2011
SELECTIVE: Suppress unwanted microbes and
encourage desired microbes. DIFFERENTIAL
: Make it easy to
distinguish
colonies of
different
Thursday, July 14, 2011
Parungao-Balolong 2011
SELECTIVE MEDIA & DIFFERENTIAL MEDIA
Thursday, July 14, 2011
ENRICHMENT MEDIA
Parungao-Balolong 2011
Encourages growth of desired microbe used when the population of your target microbe is low used when your target microbe is damaged
MRS = lactic acid bacteria Lactose Broth = enterics
Thursday, July 14, 2011
LECTURE OUTLINEReproduction & Growth
Requirements for Growth
Physical
Chemical
Culture Media
Measurement of Microbial Growth
Obtaining Pure Cultures
Preservation MethodsParungao-Balolong 2011
Thursday, July 14, 2011
MATHEMATICS OF GROWTH
Parungao-Balolong 2011
GENERATION TIME◦The time required for a
microbial population to double in number
MEAN GROWTH RATE CONSTANT(k)◦The rate of microbial
population growth expressed in terms of the number of generations per unit time
MEAN GENERATION TIME (g)
Thursday, July 14, 2011
DO THE MATH...
Parungao-Balolong 2011
If 100 cells growing for 5 hours produced
1,720,320 cells:
Thursday, July 14, 2011
MATHEMATICS OF GROWTH
Parungao-Balolong 2011
N0 = initial population numberNt = the population at time tn = the number of generations in time t
Nt = N0 x 2n
To solve for n:◦log Nt = log N0 + n ⋅ log 2◦n = log Nt – log N0 = log Nt – log N0' ' ' log 2'' ' 0.301
Thursday, July 14, 2011
SAMPLE COMPUTATION
Parungao-Balolong 2011Thursday, July 14, 2011
SAMPLE COMPUTATION
Parungao-Balolong 2011
Given an initial density of 4 x 104
Thursday, July 14, 2011
SAMPLE COMPUTATION
Parungao-Balolong 2011
Given an initial density of 4 x 104
After 2 hours the cell density became 1 x 106
Thursday, July 14, 2011
SAMPLE COMPUTATION
Parungao-Balolong 2011
Given an initial density of 4 x 104
After 2 hours the cell density became 1 x 106
Compute for the generation time
Thursday, July 14, 2011
SAMPLE COMPUTATION
Parungao-Balolong 2011
Given an initial density of 4 x 104
After 2 hours the cell density became 1 x 106
Compute for the generation time
Solution:
Thursday, July 14, 2011
SAMPLE COMPUTATION
Parungao-Balolong 2011
Given an initial density of 4 x 104
After 2 hours the cell density became 1 x 106
Compute for the generation time
Solution:◦t = 2
Thursday, July 14, 2011
SAMPLE COMPUTATION
Parungao-Balolong 2011
Given an initial density of 4 x 104
After 2 hours the cell density became 1 x 106
Compute for the generation time
Solution:◦t = 2◦n = log (1 x 106) – log (4 x 104)
Thursday, July 14, 2011
SAMPLE COMPUTATION
Parungao-Balolong 2011
Given an initial density of 4 x 104
After 2 hours the cell density became 1 x 106
Compute for the generation time
Solution:◦t = 2◦n = log (1 x 106) – log (4 x 104)' ' ' ' 0.301
Thursday, July 14, 2011
SAMPLE COMPUTATION
Parungao-Balolong 2011
Given an initial density of 4 x 104
After 2 hours the cell density became 1 x 106
Compute for the generation time
Solution:◦t = 2◦n = log (1 x 106) – log (4 x 104)' ' ' ' 0.301◦n = 4.65
Thursday, July 14, 2011
SAMPLE COMPUTATION
Parungao-Balolong 2011
Given an initial density of 4 x 104
After 2 hours the cell density became 1 x 106
Compute for the generation time
Solution:◦t = 2◦n = log (1 x 106) – log (4 x 104)' ' ' ' 0.301◦n = 4.65
◦Generation time = 2/4.65 or 0.43 hours (t/n)Thursday, July 14, 2011
GENERATION TIME
Parungao-Balolong 2011
MICROORGANISM TEMPERATURE (°C) GENERATION TIME (hours)
Escherichia coli 40 0.35
Bacillus subtilis 40 0.43
Mycobacterium tuberculosis
37 12
Euglena gracilis 25 10.9
Giardia lamblia 37 18
Sacharomyces cerevisiae
30 2
Thursday, July 14, 2011
DIRECT MEASUREMENTS
Parungao-Balolong 2011
Plate counts: Perform serial dilutions of a sample
Direct methods
Plate counts
Filtration
Direct microscopic count
Dry weight
Thursday, July 14, 2011
DIRECT MEASUREMENTS: Plate Count
Parungao-Balolong 2011
Inoculate Petri
plates from serial
dilutions
Thursday, July 14, 2011
Parungao-Balolong 2011
After incubation, count colonies on plates that have
25-250 or 30-300 colonies
report as (CFUs)
DIRECT MEASUREMENTS: Plate Count
Thursday, July 14, 2011
DIRECT MEASUREMENTS: Filtration
Parungao-Balolong 2011Thursday, July 14, 2011
DIRECT MEASUREMENTS: Direct Microscopic Count
Parungao-Balolong 2011Thursday, July 14, 2011
INDIRECT MEASUREMENTS: Turbidity
Parungao-Balolong 2011
Indirect methods
Turbidity
MPN
Metabolic
activity
Dry weight
Thursday, July 14, 2011
INDIRECT MEASUREMENTS: MPN
Parungao-Balolong 2011
Multiple Tube
Fermentation Test as
measured in MPN or
Most probable Number
Count positive tubes and
compare to statistical
MPN table.Thursday, July 14, 2011
LECTURE OUTLINEReproduction & Growth
Requirements for Growth
Physical
Chemical
Measurement of Microbial Growth
Culture Media
Obtaining Pure Cultures
Preservation MethodsParungao-Balolong 2011
Thursday, July 14, 2011
PURE CULTURE
Parungao-Balolong 2011
A pure culture contains only one species or strain.
A colony is a population of cells arising from a single cell
or spore or from a group of attached cells.
A colony is often called a colony-forming unit (CFU).
PURE MixedThursday, July 14, 2011
OBTAINING PURE CULTURE: Streak Plating
Parungao-Balolong 2011Thursday, July 14, 2011
Parungao-Balolong 2011
OBTAINING PURE CULTURE: Spread Plating
Thursday, July 14, 2011
Parungao-Balolong 2011
OBTAINING PURE CULTURE: Pour Plating
Thursday, July 14, 2011
Parungao-Balolong 2011
OBTAINING PURE CULTURE: Pour Plating
Thursday, July 14, 2011
COLONY CHARACTERISTICS
Parungao-Balolong 2011Thursday, July 14, 2011
Parungao-Balolong 2011
Julius Richard Petri (1887)
Easy to use, stackable (saving space), requirement for plating methods
OBTAINING PURE CULTURE: The Essentials
Thursday, July 14, 2011
POURING MEDIA ON YOUR DISHES
Parungao-Balolong 2011Thursday, July 14, 2011
LECTURE OUTLINEReproduction & Growth
Requirements for Growth
Physical
Chemical
Measurement of Microbial Growth
Culture Media
Obtaining Pure Cultures
Preservation MethodsParungao-Balolong 2011
Thursday, July 14, 2011
PRESERVATION METHODS: Long Term
Parungao-Balolong 2011
Deep-freezing: –50°to –95°C
Lyophilization (freeze-drying): Frozen (–54° to –72°C) and
dehydrated in a vacuum
Thursday, July 14, 2011
REVIVING LYOPHILIZED CULTURES
Parungao-Balolong 2011http://www.jcm.riken.jp
Thursday, July 14, 2011