Biology 120 lectures for 2nd exam 2012 2012 (part 1 microbial growth)

MICROBIAL
GROWTH

AY
2012-2013
Friday, July 27, 2012

DEFINITION OF
MICROBIAL GROWTH

• NUMBER OF CELLS
• NOT CELL SIZE
• e.g. Growing microbes
= increase in
numbers, accumulating
colonies


DEFINITION OF
MICROBIAL GROWTH
• Note: for coenocytic
organisms
(multinucleate):
growth = increased
cell size


FOR YOU TO GROW....


HOW ABOUT
THEM?


RECALL 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

RECALL 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)


RECALL 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


REQUIREMENTS FOR
MICROBIAL GROWTH
•PHYSICAL •CHEMICAL
REQUIREMENTS REQUIREMENTS

• TEMPERATURE • CARBON

•
• pH NITROGEN, SULFUR &
PHOSPHORUS

• OSMOTIC • TRACE ELEMENTS
PRESSURE
• OXYGEN

• ORGANIC GROWTH
FACTORS

REQUIREMENTS FOR MICROBIAL
GROWTH: TEMPERATURE

• “Most microorganisms grow well at temperatures
favored by humans”
• 3 primary groups (on the basis of temperature
preference)
• psychrophiles (cold-loving)
• mesophiles (moderate-temperature-loving)
• thermophiles (heat-loving)

GROWTH: TEMPERATURE

MINIMUM, OPTIMUM, MAXIMUM

GROWTH: TEMPERATURE

• Psychrotrophs:
grow between 0°C
and 20-30°C; cause
food spoilage
• Hyperthermophiles
: extreme
temperatures
(members of the
archaea)


GROWTH: TEMPERATURE


GROWTH: pH
• RECALL: pH acidity or
alkalinity of a solution

• acidophiles

• neutrophiles

• alkaliphiles


GROWTH: OSMOTIC PRESSURE
• Reactions of microorganism in solution based on solute
concentration: hypertonic, isotonic, hypotonic

• e.g. based on osmotic pressure requirement: Halophiles
(obligate/extreme or facultative)

• Water activity (aw): water that is available for metabolic
processes; i.e. water in food which is not bound to food
molecules can support the growth of bacteria, yeasts and molds
(fungi) or unbound and available water


GROWTH: OSMOTIC PRESSURE


REQUIREMENTS FOR
MICROBIAL GROWTH:
CARBON
• one of the most important
requirements for microbial
groth
• structural backbone of living
matter
• e.g. Chemoautotrophs (carbon
dioxide) and
Chemoheterotrophs (organic
materials)

GROWTH: NITROGEN

• ACCESS: amino acids
and proteins
• Most bacteria
decompose proteins

• Some bacteria use NH4+
or NO3–

• A few bacteria use N 2 in
nitrogen ﬁxation


GROWTH: SULFUR

• ACCESS: amino
acids, thiamine
and biotin
• Most bacteria
decompose
proteins
• Some bacteria
use SO42– or H2S


GROWTH: NITROGEN, SULFUR
AND PHOSPHORUS

• ACCESS: In
DNA, RNA,
ATP and
membranes
• PO is a
4
3–

source of
phosphorus


GROWTH: TRACE ELEMENTS

• iron, copper,
molybdenum,
zinc
• essential for the
function of co-
factors


GROWTH: TRACE ELEMENTS
• BIOTIN • PYRIDOXINE or VIT B6

• Carboxylation (Leuconostoc) • Transamination (Lactobacillus)

• CYANOCOBALAMIN or VIT B12 • NIACIN

• Molecular rearrangements (Euglena) • Precursor of NAD and NADP
(Brucella)
• FOLIC ACID
• RIBOFLAVIN or VIT B2
• One-carbon metabolism
(Enterococcus) • Precursor of FAD and FMN
(Caulobacter)
• PANTOTHENIC ACID
• THIAMINE or VIT B1
• Fatty acid metabolism (Proteus)
• Aldehyde group transfer (Bacillus


GROWTH: OXYGEN

• “microbes that
use molecular
oxygen produce
more energy
from nutrients
than microbes
that do not use
oxygen”


GROWTH: OXYGEN

• aerobic bacteria
• anaerobic
bacteria

• microaerophilic
bacteria

GROWTH: OXYGEN

• Microbes can be harmed by toxic forms
of oxygen
• singlet oxygen ( 2
1O -): normal molecular
oxygen that has been boosted into a higher-
energy state; extremely reactive
• hydroxyl radical (OH•): most reactive
intermediate form of oxygen formed in
cellular cytoplasm by ionizing radiation


GROWTH: OXYGEN
• Microbes can be harmed by toxic forms of
oxygen

• peroxide anion (O22-): toxic; active ingredient in
hydrogen peroxide and benzoyl peroxide

• SOLUTION: catalase and peroxidase


GROWTH: OXYGEN
• Microbes can be harmed by toxic forms of
oxygen

• superoxide free radicals (O2-): toxicity is caused by
their great instability; they steal an electron from a
neighboring molecule, which in turn becomes a free
radical, and the cycle continues

• SOLUTION: production of superoxide dismutase
(SOD): aerobic, FA and aerotolerant anaerobes

• convert superoxide free radicals to molecular
oxygen and hydrogen peroxide


GROWTH: ORGANIC GROWTH
FACTORS
• VITAMINS: Unlike
humans, most bacteria
can synthesize all their
own vitamins and are
not dependent on
outside sources
• Some bacteria lack the
enzymes needed for the
synthesis of certain vitamins,
amino acids, purines and
pyrimidines

CULTURE MEDIA

CULTURE MEDIA
• nutrient material prepared for the growth of
microorganisms in a laboratory
• IMPORTANT TERMS:
• inoculum: microbes introduced into a
culture medium
• culture: microbes that grow and multiply
in a culture medium
• sterile medium: a pre-requisite = no
living microorganisms

AGAR
• solidifying agent
• only a few microbes can degrade
it

• liquiﬁes at 1000C and solidiﬁes
below 400C

• pouring temperature: 500C
(prevents injury to microbes)

• used for the preparation of slants,
stabs/deeps, plates

TYPES OF CULTURE MEDIA:
Chemically-deﬁned Media

• exact chemical
composition is known

• mostly for
autotrophic bacteria,
fastidious bacteria

• Contents: organic
growth factors
(carbon and energy)

Complex Media

• made up of nutrients
including extracts from
yeasts, meat or plants, or
digests of proteins

• exact chemical
composition varies from
batch to batch

• mostly for heterotrophic
bacteria and fungi

Anaerobic Growth Media

• “reducing media”
• sodium thioglycollate:
chemically combine with
dissolved oxygen and
deplete the oxygen in the
culture medium

• heated ﬁrst before use to
drive off absorbed oxygen


ANAEROBIC CULTURE
TECHNIQUES


Selective & Differential Media
• Goal: to detect the
presence of speciﬁc
microorganisms
associated with disease
or poor sanitation

• SELECTIVE: suppress
growth of unwanted
bacteria and encourage
the growth of desired
microbes


•Why it can select:
• BSA: Bismuth Sulﬁte Indicator
and Brilliant Green are
complementary, inhibiting Gram-
positive bacteria and coliforms,
allowing Salmonella spp. to grow

• SDA: pH 5.6 where fungi can
outgrow bacteria

• Goal: to detect the
presence of speciﬁc
microorganisms associated
with disease or poor
sanitation

• DIFFERENTIAL:
distinguish colonies of
desired organisms when
grown together with
others

Differential Media


Enrichment Media
• mostly for soil and fecal
samples or when desired
microbe is injured

• may also be selective
• e.g. MRS agar (deMann,
Rogosa and Sharpe agar or
Lactobacillus agar)

• e.g. lactose broth

PURE
CULTURE

PREPARING PURE CULTURE

• Julius Richard Petri
(1887)
• Easy to use, stackable
(saving space),
requirement for
plating methods

OBTAINING PURE CULTURES:
Streak Plating


PURE VS MIXED
CULTURE

CHARACTERIZING
COLONIES


CULTURE
PRESERVATION

WAYS TO PRESERVE YOUR
CULTURE

•subculturing
• mineral oil overlay
• freezing as glycerol
stocks

• liquid nitrogen storage
• lyophilization

CULTURE

• subculturing
•mineral oil overlay
• freezing as glycerol stocks
• lyophilization

CULTURE

• subculturing
•freezing as glycerol stocks
• lyophilization

CULTURE

• subculturing
•liquid nitrogen storage
• lyophilization

CULTURE

• subculturing
•lyophilization

REVIVAL OF PRESERVED L-
DRIED CULTURES

http://www.jcm.riken.jp

GROWTH OF BACTERIAL
CULTURES

BACTERIAL DIVISION


OTHER FORMS OF DIVISION BY OTHER
MICROBES

Budding = Chains of conidiospores
Rhodopseudomonas carried externally at the
tips of the ﬁlaments =
Actinomycetes Fragmentation of
ﬁlaments = Actinomycetes


THE MATHEMATICS
OF GROWTH

CELL DIVISION
• Generation
time: time
required for a
microbial
population to
double

• g = mean
generation time

• g = t/n

GENERATION
TIME

• g = t/n


SAMPLE...
• Given an initial • Solution: t = 2
density of 4 x 104
• n = [ log (1 x 10 ) –
6

• After 2 hours the log (4 x10 4)]/

cell density became 0.301; n = 4.65
1 x 10 6

• Generation time =
• Compute for the (t/n); 2/4.65 or 0.43
generation time hours OR 25.8
minutes

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

THE GROWTH CURVE


OBTAINING A
GROWTH CURVE
• The Growth Curve can be obtained via a Batch
Culture

• Microorganisms are cultivated in a liquid medium and
grown as a closed system

• Incubated in a closed culture vessel with a single batch
of medium and NO fresh medium provided during
incubation

• SCENARIO: Nutrient concentration decline and
concentrations of waste increase during the incubation
period

1. THE LAG
PHASE
• No immediate
increase in cell mass
or cell number

• Cell is synthesizing
new components

• Cells retool,
replicate their
DNA, begin to
increase in mass and
ﬁnally divide

1. THE LAG PHASE

• 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

SHORT LAG PHASE
• SHORT LAG PHASE (or
even absent)
• Young, vigorously
growing exponential
phase culture is
transferred to fresh
medium of same
composition


LONG LAG PHASE
• LONG LAG PHASE
• Inoculum is from an old
culture
• Inoculum is from a
refrigerated source
• Inoculation into a
chemically-different
medium


2. THE LOG/
EXPONENTIAL 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: 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

3. 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

4. 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
• NOTE: Death rate may decrease after the
population has been drastically reduced due
to resistant cells

DIRECT MEASUREMENT

• Plate counts
• Filtration
• Most Probable Number (MPN)
• Direct Microscopic Count

PLATE COUNTS


RECALL: HOW TO
COMPUTE CFU

FILTRATION


INDIRECT MEASUREMENTS:
ESTIMATING BACTERIAL NUMBERS
• Turbidity: spectrophotometry estimates
• Metabolic Activity
• e.g. MBRT for Milk = Class 1. Excellent, not
decolorized in 8 hours; Class 2. Good, decolorized in
less than 8 hours but not less than 6 hours; Class 3.
Fair, decolorized in less than 6 hours but not less than
2 hours; Class 4. Poor, decolorized in less than 2
hours

• Dry Weight: for ﬁlamentous molds


NEXT MEETING:
MICROBIAL METABOLISM
& PHYSIOLOGY

Biology 120 lectures for 2nd exam 2012 2012 (part 1 microbial growth)

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