Biology 120 lecture 4 2011 2012

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

Physical

Chemical

Culture Media

REPRODUCTION IN PROKARYOTES

Parungao-Balolong 2011

Binary fission

Budding

Conidiospores

(actinomycetes)

Fragmentation of

filamentsThursday, July 14, 2011

MICROBIAL GROWTH

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

GROWTH

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

MICROBIAL NUTRITION

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

GROWTH FACTORS

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)

MICROBIAL NUTRITION

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

MICROBIAL NUTRITION

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

MICROBIAL NUTRITION

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

THE GROWTH CURVE

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)

THE GROWTH CURVE

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

THE LAG PHASE

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

THE LAG PHASE

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

EXPONENTIAL/LOG PHASE

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

STATIONARY PHASE

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

DEATH PHASE

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

Physical

Chemical

Culture Media

REQUIREMENTS FOR GROWTH:

PHYSICAL

Temperature

Minimum growth temperature

Optimum growth temperature

Maximum growth temperature

INFLUENCE OF LIPID CONTENT

◦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)

TEMPERATURE RANGE

STENOTHERMAL MICROBES◦Narrow range◦Neisseria gonorrhea

EURYTHERMAL MICROBES◦Wide range◦Enterococcus faecalis

Most bacteria grow between pH 6.5 and 7.5

Molds and yeasts grow between pH 5 and 6

Acidophiles grow in acidic environments

REQUIREMENTS FOR GROWTH: PHYSICAL

Osmotic pressure

Hypertonic

environments,

increase salt or sugar,

cause plasmolysis

Extreme or obligate halophiles require high osmotic

pressure

Facultative halophiles tolerate high osmotic pressure

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)

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)

REQUIREMENTS FOR GROWTH: CHEMICAL

Carbon

Structural organic

molecules, energy

source

Chemoheterotrophs use

organic carbon sources

Autotrophs use CO2

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

Oxygen (O2)

Singlet oxygen: O2 boosted to a higher-energy state

Superoxide free radicals: O2–

Peroxide anion: O22–

Hydroxyl radical (OH•)

Physical

Chemical

Culture Media

CULTURE MEDIA

Culture medium:

Nutrients prepared

for microbial growth

Sterile: No living

microbes

Inoculum:

Introduction of

microbes into

medium

Culture: Microbes

growing in/on

culture medium

CULTURE MEDIA

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

RECALL: HISTORY OF

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

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

ANAEROBIC CULTURE METHODS

Reducing media

Contain chemicals (thioglycollate or oxyrase) that combine O2

Heated to drive off O2

ANAEROBIC CULTURE METHODS

SELECTIVE MEDIA & DIFFERENTIAL MEDIA

SELECTIVE: Suppress unwanted microbes and

encourage desired microbes. DIFFERENTIAL

: Make it easy to

distinguish

colonies of

different

SELECTIVE MEDIA & DIFFERENTIAL MEDIA

ENRICHMENT MEDIA

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

Physical

Chemical

Culture Media

MATHEMATICS OF GROWTH

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)

DO THE MATH...

If 100 cells growing for 5 hours produced

1,720,320 cells:

MATHEMATICS OF GROWTH

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

SAMPLE COMPUTATION

Given an initial density of 4 x 104

SAMPLE COMPUTATION

After 2 hours the cell density became 1 x 106

SAMPLE COMPUTATION

Compute for the generation time

SAMPLE COMPUTATION

Solution:

SAMPLE COMPUTATION

Solution:◦t = 2

SAMPLE COMPUTATION

Solution:◦t = 2◦n = log (1 x 106) – log (4 x 104)

SAMPLE COMPUTATION

Solution:◦t = 2◦n = log (1 x 106) – log (4 x 104)' ' ' ' 0.301

SAMPLE COMPUTATION

Solution:◦t = 2◦n = log (1 x 106) – log (4 x 104)' ' ' ' 0.301◦n = 4.65

SAMPLE COMPUTATION

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

MICROORGANISM TEMPERATURE (°C) GENERATION TIME (hours)

Escherichia coli 40 0.35

Bacillus subtilis 40 0.43

Mycobacterium tuberculosis

Euglena gracilis 25 10.9

Giardia lamblia 37 18

Sacharomyces cerevisiae

DIRECT MEASUREMENTS

Plate counts: Perform serial dilutions of a sample

Direct methods

Plate counts

Filtration

Direct microscopic count

Dry weight

DIRECT MEASUREMENTS: Plate Count

Inoculate Petri

plates from serial

dilutions

After incubation, count colonies on plates that have

25-250 or 30-300 colonies

report as (CFUs)

DIRECT MEASUREMENTS: Plate Count

DIRECT MEASUREMENTS: Filtration

DIRECT MEASUREMENTS: Direct Microscopic Count

INDIRECT MEASUREMENTS: Turbidity

Indirect methods

Turbidity

Metabolic

activity

Dry weight

INDIRECT MEASUREMENTS: MPN

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

Physical

Chemical

Culture Media

PURE CULTURE

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

OBTAINING PURE CULTURE: Spread Plating

OBTAINING PURE CULTURE: Pour Plating

COLONY CHARACTERISTICS

Julius Richard Petri (1887)

Easy to use, stackable (saving space), requirement for plating methods

OBTAINING PURE CULTURE: The Essentials

POURING MEDIA ON YOUR DISHES

Physical

Chemical

Culture Media

PRESERVATION METHODS: Long Term

Deep-freezing: –50°to –95°C

Lyophilization (freeze-drying): Frozen (–54° to –72°C) and

dehydrated in a vacuum

REVIVING LYOPHILIZED CULTURES

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