gwoth of microbes
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DR. RIDDHI DAVE
MICROBIOLOGIST riddhisnewworld@yahoo.co.in
MICROBIAL GROWTH
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Bacterial Cell Division
� Cell growth is defined as an increase in
the number of cells, requires continued
growth to maintain species� ~2000 chemical reactions with a wide
variety of types
± main rxn is polymerization reaction
� monomer to polymer
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Binary Fission� Cell growth continues until
divides into 2 new cells
� Cells create a septumbetween new cells± starts as cytoplasmic
membrane and eventually
becomes cell wall� Each batch of new cells is a
generation
� Cellular components increaseproportionally so each cell getsenough of everything to the
new cell� Time to generate new cells isdependent on nutritional andgenetic factors± division is tied to chromosomal
replication
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Fts Proteins
� Filamentous temperature sensitive
proteins ± mutation in genes that encode
the Fts proteins± bacteria without FtsZ have difficulty dividing
± FtsZ is universally distributed in all
prokaryotes
± see FtsZ-like proteins in mitochondria andchloroplasts, also similar to tubulin in
eukarotes
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Fts Complexes =
Division Apparatus
� Fts interact to form thedivision apparatus calledthe divisome
� FtsZ attach in a ring tothe cell at the membraneand then attracts FtsAand Zip A± FtsA ± ATP hydrolyzing
enzymes for proteins indivisome
± ZipA ± anchor attachmentof FtsZ to membrane
� Also contain Fts proteinsinvolved in peptidoglycansynthesis ± FtsI is a
penicillin-binding protein(activity site for penicillin)
� Divisome makes newcytoplasmic membraneand cell wall in bothdirections until largeenough to divide
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DNA Replication
� Occurs prior to FtsZ ring formation and
when done get the ring formation between
the 2 nucleoid regions using min proteins� min C inhibits cell division until exact
center of the cell is found
� min E inhibits min C activity and attached
at center of cell, recruits the FtsZ and ring
formation
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Cell Shape
� Morphology = cell shape� Peptidoglycans thought to dictate shape but now
know only a minor role
� Protein for shape is homologous to actin
� Major protein ± MreB ± forms an actin-likecytoskeleton± filamentous, spiral-shaped bands in cell under
cytoplasm membrane
± cocci lack MreB and its gene, default shape ± sphere
� Bacteria make FtsZ and MreB ± tubulin- andactin-like proteins± evolutionary similarities between eukaryotes and
prokaryotes
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Cell Wall Formation
� Precursors to the cell wall are spliced into
existing peptidoglycan
� If the precursors aren¶t coordinated withthe old, the cell goes through spontaneous
autolysis ± cell ruptures
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Biosynthesis of Peptidoglycan
� Cut pre-existing peptidoglycans by autolysins with
simultaneous insertion of precursors ± bactoprenol ±
lipid carrier molecule, hydrophobic C55 alcohol� Bactoprenol makes the peptidoglycan precursors
hydrophobic so they can cross membrane to be
inserted, spend time in the periplasm to build cell wall
and make glycosidic bonds
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Transpeptidation and Penicillin
� Final step ± need to insert the peptide
components of the cell wall between the
muramic acid (refer to cell wall structure from
before)� This reaction is inhibited by penicillins ± prevent
cell wall formation by binding to FtsI, autolysins
continue to weaken the cell wall and leads to
lysis
± used in humans� since we do not have cell walls, can use drug at high levels
� virtually all bacterial pathogens have peptidoglycan so works on
most bugs
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Final Interactions
� Interaction with severalamino acids based on the
organism
± E coli ± between
diaminopimelic acids and D-
Ala on adjacent peptides
� Removal of the 2nd D-Ala
drives the rxn as there is not
ATP (outside the cell)
� In gram +, glycineinterbridge is usaully
present, cross-link accur
across the interbridge on L-
Lys and D-Ala
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Growth of Bacterial Populations
� Increase in the number of organisms in a
population
� Terminology
± 1 cell to 2 cells is a generation
± time for the new cell to form is the generation time,
mass also doubles so also called doubling time
� These vary between organisms and are based
on growth medium, growth conditions
± usually differ out in nature vs. the test tube
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Exponential Growth
� Where number of cellsdouble during a regular
tine interval
� Graph on linear scale,
see a dramatic increasein the numbers over time
� Graph on semi-log
paper, you get a straight
line, meaning
exponentially growing
± use to estimate growing
time
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Estimating Growth Rate
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Growth
� In exponential growth ± rate increase is
slow initially but increases in cell number
± in non-sterile, nutrient rich environments, suchas milk ± slow growth is good, leave milk out
an hour, not to many bacteria, but if leave out
several hours, the level of bacteria will be
much higher
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Growth Cycle
� Exponential growth cannot continue forever
� Cycle has 4 distinct areas ± lag, exponential,
stationary and death phases
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Lag Phase
� Delay in growth of bacteria
� Interval may be different
± based on organism and growth conditions
� See when using old or stationary phase
cultures to start your growth curve
� Lag is caused by cells being depleted in
essential constituents, must also repair if damaged by heat or radiation, etc
± also see if moving from a rich medium to a
poorer medium
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Exponential Phase
� Cells divide for a brief time ± based on
resources and other factors
� Rate of growth vary greatly± influenced by environmental conditions and
genetic characteristics of the organism
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Stationary Phase
� Limitation on growth caused by 2 factors± essential nutrients of culture medium is used up
± some waste products of the organism build up in
medium and inhibit growth± can be a combination of both
� Exponential growth stops and there is no netincrease or decrease in the cell number ± maybe slow growth
� Cellular functions continue ± energy metabolismand biosynthetic processes± some divide and some die ± cryptic growth
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Death Phase
� Cells will die eventually
� Death accompanied by cell lysis
� ³Exponential death´ ± but slower thangrowth
� Figure 6.8 is of a POPULATION and not asingle cell, this process does NOT apply to
them
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Total Counts
� Total count using a microscope andhemocytometer ± a special counting chamber with a square on surface of glass with a
known volume under a cover slip± count the number of cells on the grid and then
calculate the number of cells based on thevolume on the chamber
± also count dead cells, miss small cells, precisionis hard to achieve, requires phase contrastmicroscopy when not stained, not good a lowdensity and motile cells must be immobilized
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Viable Counts
� Viable cells can divide and make offspring
� Determine whether capable of forming colonies onsuitable agar ± plate count or colony count, assume each cell can
yield a colony� 2 methods ± spread plate and pour plate
± spread plate ± use small volume of diluted cells andspread over surface of agar, count colonies andcalculate number using dilution
± pour plate ± add volume of culture into Petri dish,add melted agar, mix by swirling ± colonies formthroughout the agar, not just on top like abovemethod, examine carefully
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Dilutions
� Use dilutions to determine the number of colonies in a countable range ± count/plateshould be between 30 and 300 colonies
� Must determine optimum conditions to growthe bacteria ± temp, medium, time, etc
� Perform serial dilutions to get into the³countable´ range
� Sources of error ± not using correct growingconditions, errors also in technique ±pipeting, mixing, etc.
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Serial Dilutions
� Usually do serial 10-folddilutions by mixing 0.5 ml of sample with 4.5 ml of freshmedium (1 part in 9 parts =10)
� Do consecutive dilutions inthe same manner and platea volume on the agar
� After growth, count thecolony forming units (cfu)and calculate the number of
bacteria using dilution andvolume placed on plates
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Colony Forming Units
� Use colony forming units because occasionallyyou may have 2 bacteria in the same area thatmake a single colony you can¶t tell apart
� Can use selective media to count only a particular organism
� Great plate anomaly ± may be unreliable toassess total number of cells in natural samples ±soil, water plate count is usually lower than directcount± organisms may have really different nutrient needs
± may need selective media to get a better count
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Indirect Measurements
� Use turbidity as an indirect measurement ± more cellsmean it is more cloudy
� Use a photometer or spectrophotometer ± similar in use ± light scattered by cells and all light that passes
thru will be collected
± differences is with the light source ± photometer is a broad passfilter and spec is prism or diffraction grating
± both measure only unscattered light but report in Klett units or optical density
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Generation of a
Standard Curve� Can substitute turbidity for
direct counting methods butneed to make a standard curve
� Relate direct count to indirect
method± may use cell number or cell mass
� Must use within limits ± really³dense´ samples may deflectlight and then another cell re-
deflects them to the detector ± makes things non-linear
± can check sample repeatedlywithout altering test outcome
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Continuous Culture - Chemistat
� Previous growth was based on batch cultures ±
fixed volume of medium ± altered by metabolism
in culture ± closed system
� Constant environment needed over long periodsof time to generate a continuous supply of
exponential phase bacteria ± continuous
culture ± open system
± add fresh medium and remove the ³old´ medium in a
chemistat
± volume, cell number and nutrient state are constant ±
steady state
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Chemistat
� Constant growth rate
and population
density
� 2 important factors± dilution rate
± concentration of
limiting nutrient,
usually N or C
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Affect of Nutrient on Growth and
Yield
� In batch cultures [nutrient]
can affect growth rate
and growth yield
� At low concentrationsonly the rate is reduced
± cannot meet the needs of
organisms
� [Moderate] to [high] may
not change the rate butthe yield will increase
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Chemistat ± Control of Rate and
Yield� Both rate and yield can
be controlledindependently by alteringthe dilution rate whicheffects the [] of nutrientspresent
� Dilution rate ± at high andlow rates the steady statebreaks down, at [high] ±bacteria aren¶t growing
fast enough and at [low] ±not feeding fast enoughso cells are dying
� Cell density (cells/ml) ±controlled by level of limiting nutrient
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Environmental Effects -
Temperature
� Most important as alter the temperature to
drastically the bacteria will die
� Raising the temperature may speed up growth
rates but over a limited range ± may bedetrimental if too high ± maximum temperature
or too low ± minimum temperature
� Optimum temperature ± temperature that growth
occurs most rapidly ± usually nearer to the
maximum than the minimum
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Cardinal Temperatures
� Maximum, optimum andminimum are the cardinaltemperatures
� Cardinal temps are not fixedand may fluctuate depending
on growth medium� Maximal temperatures reflectsdenaturation of 1 or moreproteins
� Not sure what causes minimaltemperature but may be thecomposition of the cytoplasmic
membrane± alter composition resulted in a
change in the maximum andminimum temperatures
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Temperature Classes of Organisms
� Psychrophile ± very low temperatures
� Mesophiles ± moderate temperatures
� Thermophiles ± high temperatures
� Hyperthermophiles ± very high temperatures
� All but mesophiles can also be classified as
extremophiles
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pH
� Each organism has a range that it can grow in(external pH) ± usually 2-3 pH units andbetween pH 5-9
� Acidophiles usually live at < pH 2, fungi are
more tolerant of low pH, some obligateacidophiles as they need a large amount of H+ tomaintain membrane structure
� Alkaliphiles usually > pH 10, some are alsohalophilic (love salt) ± use the Na+ to± proteases and lipases from alkaliphile bacteria seen
in household cleaners
� Neutrophiles live between pH 6-8
� Internal pH must remain close to neutral
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Buffers
� We add buffering chemicals to the media
to insure to proper pH for the organisms
� Metabolic reactions will increase or decrease the pH depending on what is
happening in the cell
� Potassium phosphate is used quite
frequently, use others depending on the
pH range needed for the bacteria
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Osmotic Effects
� Water availability is expressed as water activity
� Water diffuses from [high] to [low] thru a
membrane ± osmosis� [Solute] usually higher outside the
organism so water moves into the cell
± cell in a positive water balance, in an area of low water activity, then water leaves the cells� causes many problems
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Halophile
s
� Osmotic effects seen in habitats with high [salt]
� Mild halophile ± 1-6%, moderate halophile ± 7-15% NaCl
� Halotolerant ± can adjust to increase in solute by
decreasing water in the cell
� Extreme halophiles ± 15-30% NaCl
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Other Types of Organisms
� Osmophiles ± grow in environments with a
high [sugar]
� Xerophiles ± grow in very dryenvironments
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Compatible Solutes
� Organisms grown in an area of low water activity
need to adjust to this
� Gain water by increasing the concentration of
internal solutes
� Accomplish this by
± pumping inorganic ions into cell from environment
± synthesizing or concentrating and organic solute
� Solute must not inhibit the biochemical
processes in the organism
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Where do the Solutes Come
From?� Synthesize or take up solute ± genetically
determined by the organism
� Staphylococcus species are salt tolerant ±
use to select over salt intolerant organismand use proline as a compatible solute
� See glycine betaine in halophilic bacteria
and cyanobacteria� Extreme halophiles produce ectoine (cyclic
derivative of aspartate)
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Oxygen and Microbial Growth
� Anoxic organisms ± grow without oxygen
� Classes of microorganisms ± vary in use of oxygen andtolerance
� Aerobe ± grow in 21% O2 ± and respire O2 in metabolism
� Microphiles require less than 21% O2 ± may contain anO2 labile protein, limited capacity to respire
� Facultative aerobe ± under appropriate nutrient andculture conditions either grow anoxic or oxic condition
� Anaerobes cannot respire in O2± 2 kinds ± aerotolerant anaerobes ±can tolerate O2 and grow in
the presence of O2 but do not use it and obligate or strictanaerobes ± inhibited or killed by O2
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3 Types of Obligate Anaerobes
� Prokayotes ± important one is clostridium
family that is gram positive spore forming
rob that causes food poisoning
� Some fungi
� Few protozoans
� Sensitivity to O2
varies in all these groups
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Culture
Techniques� Anaerobes need the O2
removed form the culture
± use a reducing agentsuch as thioglycolate inbroth to determineoxygen requirements
� Growth at the top is
obligate aerobes,facultative organismsgrow throughout themedium and anaerobesgrow only at the bottom of
the tubes� Also use resazurin in the
medium to indicate if O2
is present ± should seeonly near the top
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Anoxic Jar or Anaerobic Hood
� Use a tightly sealed
jar or bag that you
use a chemical
reaction to remove allthe O2 from it to grow
anaerobes
� Hood uses a series of
vacuum pumps toremove O2 and
replace usually with
N2
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Toxic Forms of O2 and Enzymes
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Enzymes� Catalase is the most
common enzyme toremove H2O2
� Used in conjunction withsuperoxide dismutasewhich generates H2O2
when combining 2superoxide ions, alsomakes O2
� Peroxidase removesH2O2 but requires NADHto make water
� Superoxide reductase inArchaea ± reducesuperoxide to H2O2
without the production of O2, remove H2O2 with
id lik
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