gwoth of microbes

8/7/2019 Gwoth of microbes

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DR. RIDDHI DAVE

MICROBIOLOGIST [email protected]

MICROBIAL GROWTH



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



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



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



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



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



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



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



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



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



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



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



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



Estimating Growth Rate



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



Growth Cycle

� Exponential growth cannot continue forever

� Cycle has 4 distinct areas ± lag, exponential,

stationary and death phases



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



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



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



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



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



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



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.



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



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



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



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



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



Chemistat

� Constant growth rate

and population

density

� 2 important factors± dilution rate

± concentration of

limiting nutrient,

usually N or C



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



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



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



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



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



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



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



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



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



Other Types of Organisms

� Osmophiles ± grow in environments with a

high [sugar]

� Xerophiles ± grow in very dryenvironments



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



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)



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



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



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



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



Toxic Forms of O2 and Enzymes



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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