Microbial Growth Curve Stages

REPRODUCTION &
GROWTH
Lecture 4

Reference: Chapter 6 (Tortora)

Parungao-Balolong 2011
Thursday, July 14, 2011

LECTURE OUTLINE
Reproduction & Growth

Requirements for Growth

Physical

Chemical

Measurement of Microbial Growth

Culture Media

Obtaining Pure Cultures

Preservation Methods

REPRODUCTION IN
PROKARYOTES
 Binary ﬁssion
 Budding
 Conidiospores
(actinomycetes)
 Fragmentation of
ﬁlaments

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  PYRIDOXINE or VIT B6
◦Carboxylation (Leuconostoc) ◦Transamination (Lactobaci!us)
 CYANOCOBALAMIN or VIT  NIACIN
B12 ◦Precursor of NAD and NADP
◦Molecular rearrangements (Bruce!a)
(Euglena)
 RIBOFLAVIN or VIT B2
 FOLIC ACID ◦Precursor of FAD and FMN
◦One-carbon metabolism (Caulobacter)
(Enterococcus)
 THIAMINE or VIT B1
 PANTOTHENIC ACID
◦Aldehyde group transfer (Baci!us
◦Fatty acid metabolism (Proteus) anthracis)


MICROBIAL NUTRITION
CARBON SOURCES
Autotrophs CO2 sole or principal biosynthetic carbon source

Heterotrophs Reduced, preformed, organic molecules from
other organisms

ENERGY SOURCES
Phototrophs Light

Chemotrophs Oxidation of organic or inorganic compounds

HYDROGEN AND ELECTRON SOURCES
Lithotrophs Reduced inorganic molecules

Organotrophs Organic molecules


MICROBIAL NUTRITION
MAJOR NUTRITIONAL TYPES SOURCES OF ENERGY, REPRESENTATIVE
HYDROGEN/ELECTRONS AND MICROORGANISMS
CARBON

PHOTOLITHOTROPHIC Light energy Algae
AUTOTROPHY Inorganic hydrogen/electron Purple and green sulfur
donor bacteria
CO2 carbon source Blue-green algae
(cyanobacteria)

PHOTOORGANOTROPHIC Light energy Purple non-sulfur bacteria
HETEROTROPHY Organic hydrogen/electron Green non-sulfur bacteria
donor
Organic carbon source (CO2
may also be used)


MICROBIAL NUTRITION
MAJOR NUTRITIONAL TYPES SOURCES OF ENERGY, REPRESENTATIVE
HYDROGEN/ELECTRONS AND MICROORGANISMS
CARBON

CHEMOLITHOTROPHIC Chemical energy source Sulfur-oxidizing bacteria
AUTOTROPHY (inorganic) Hydrogen bacteria
Inorganic hydrogen/electron Nitrifying bacteria
donor Iron bacteria
CO2 carbon source

CHEMOORGANOTROPHIC Chemical energy source Protozoa
HETEROTROPHY (organic) Fungi
Organic hydrogen/electron Most non-photosynthetic
donor bacteria
Organic carbon source


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 depleted
 must be synthesized first before growth can begin
◦Medium maybe different from the one the microorganism was growing
previously
 new 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

 Temperature
 Minimum growth temperature REQUIREMENTS
 Optimum growth temperature FOR GROWTH:
 Maximum growth temperature PHYSICAL


INFLUENCE OF LIPID CONTENT
◦PSYCHROPHILY
HIGH CONTENT OF
UNSATURATED FATTY ACIDS
HELP MAINTAIN A SEMI-FLUID
MEMBRANE STATE AT LOW
TEMPERATURE

◦THERMOPHILY
PROTEINS 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


pH
 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

REQUIREMENTS FOR
GROWTH: PHYSICAL
WATER SOURCE BACTERIA FUNGI ALGAE
ACTIVITY

1.00 blood Most Gram none none
(pure water) negative and
non-halophiles

0.90 ham Most cocci and Fusarium, Mucor,
Bacillus Rhizopus

0.60 Chocolate none Saccharomyces rouxii none

0.55
(DNA
disordered)


REQUIREMENTS FOR
GROWTH: PHYSICAL
 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)


REQUIREMENTS FOR
GROWTH: CHEMICAL
 Carbon
 Structural organic
molecules, energy
source
 Chemoheterotrophs use
organic carbon sources
 Autotrophs use CO2

REQUIREMENTS FOR
GROWTH: CHEMICAL
 Nitrogen
 Trace elements
 In amino acids and proteins
 Inorganic elements required
 Most bacteria decompose proteins
in small amounts
 Some bacteria use NH4+ or NO3–
 Usually as enzyme cofactors
 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

REQUIREMENTS FOR
GROWTH: CHEMICAL
 Oxygen (O2)


REQUIREMENTS FOR
GROWTH: CHEMICAL
 Singlet oxygen: O2 boosted to a higher-energy state

 Superoxide free radicals: O2–

 Peroxide anion: O22–

 Hydroxyl radical (OH•)


LECTURE OUTLINE


Physical

Chemical

Culture Media

Measurement of Microbial Growth



 Culture medium:

CULTURE Nutrients prepared

MEDIA
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  GELATIN
◦Liquid medium ◦Frederick Loeﬄer
◦Meat extract
 POTATO SLICES medium + gelatin
◦Robert Koch (1881) ◦But gelatin liquid at
◦Used boiled potato, room temperature
sliced
 AGAR
◦Not all bacteria grew
well ◦Fannie Eilshemius
Hesse (1882)
◦Agar used for jams
and jelly


AGAR
 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

LECTURE OUTLINE


Physical

Chemical

Culture Media

Measurement of Microbial
Growth



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 number
 Nt = the population at time t
 n = 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


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 hoursParungao-Balolong 2011
(t/n)

GENERATION TIME
MICROORGANISM TEMPERATURE (°C) GENERATION TIME
(hours)
Escherichia coli 40 0.35

Bacillus subtilis 40 0.43

Mycobacterium 37 12
tuberculosis
Euglena gracilis 25 10.9

Giardia lamblia 37 18

Sacharomyces 30 2
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


Plate Count
 After incubation, count colonies on plates that have
25-250 or 30-300 colonies
 report as (CFUs)


Filtration


Direct Microscopic Count


INDIRECT
MEASUREMENTS: Turbidity
Indirect methods
 Turbidity
 MPN
 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. Parungao-Balolong 2011

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 Mixed

OBTAINING PURE
CULTURE: Streak Plating


OBTAINING PURE
CULTURE: Spread Plating


OBTAINING PURE
CULTURE: Pour Plating


COLONY
CHARACTERISTICS


OBTAINING PURE
CULTURE: The Essentials

 Julius Richard Petri (1887)

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

POURING MEDIA ON
YOUR DISHES


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

http://www.jcm.riken.jp

Microbial Growth Curve Stages

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