Chapter 4: Microbial Growth and Its Control

dont forget to visit equations in notes (p 16 and 17)

binary fusion

cell elongation (2x size) with the formation of a partition that constricts the cell into two daughter cells

septum

partiition betwen two dividing cells formed from the inward growwth of the cytoplasmic membrand and cell wall from oposing directions

generation

1 generation has occured after one division

generation time

time required for binary fission or cell division to occur

budding division

formation of totally new daughter cell with the mother cell retaining its identity. Result of unequal cell growth

polar growth

growth from a single point

intercalary growth

growth throught the whole cell

planktonic growth

suspended lifestyle of bacteria

sessilem growth

attached to a surface lifestyle of bacteria

biofilm development

attachment of planktonic cells, formation of a sticky matrix (made of the glycoprotein layer or the glycocalyx),followed y further growth and development into an almost impenitrable mature biofilm

microbial mats

biofilms composed of different layers, with potentially differeent orgainsims in the individual layers

pros of biofilms

1. prevents penetration of harmful proteins

2. barrior to bacterial grazing by protists

3. stops bacteria from being washed to less favorable habitat

cons of biofilms (for humans)

cna form on implants (i.e. hart valves) and cause difficult to treat infects, causes systic fibrosis by preventing gas exchange in the lung, and can foul water systems

k

instantainous growth rate constant with units h^-1

batch culture

organism growin in an enclosed vessel (i.e. flask or tube)

growth cycel stages

1. lag

2. exponetial

3. stationary

4. death

lag phase

results from the depletion of various essital consittuents in the inoculum culture that has to be replenished before growth can occur. can also happen wen going to a nutrient poor medium

exponetial phase

generally the healthiest state of cells and its rates vary by cell type and growing conditions. Proks tend to grow faster tha euks (small euks faster than larger ones)

stationary phase

when growth stops (no net income. or dec. in number of cells), generally because of nutrient depletion and/or waste accumulation

death phase

cells begin to die faster thna they are being produced at an exponetial rate (slower that growth rate)

continuous culture

mechanism to avoif th limitations of a bach culture. It is an opens system that allows a constant rate sterile medium and soiled medium to flow in and out of the container. allow for a constant volume of medium, nutrient/waste, and number of cells.

steady state

equilibrium state of an open culture system where all the values of cell number, medium volume, and waste/nutrient presence stay constant. (cells are flowwing out with the spoiled medium

chemostat

device where the specific growth rate (cells grown per unit time) and cell density (cells per ml) can be controlled independently

chemostat controles

1. dillution rate (D)

2. concentraiton of limiting nutrient (i.e. carbon or nitrogen)

dillution rate

(D) expressed as F/V

F=flow rate of fresh medium pumped in and spent medium removed

V=culture volume

why use a chemostat

keeps cells in the expontial phase for long periods of time which is the most desirerable phase for physiological experiments

defined media

made by adding precise amounts of pure inorganic or organic chemicals to distilled water (exact composition is known

complex media

made from the digests of microbial, animal, or plant products (i.e milk protien (casein), beef (Extract), soybeans, yeast cells etc)

exact nutrient composition is unknown

enriched media

gerneally used for nutritionally demanding (fastidious) microbes (many are pathogens) is a complex medium with an added highlighy nutrious substance (ie. serum or blood)

selective medium

contains compunds that inhibit the growth of certain microbes but not others

differential medium

indicator (often a dye) is added that changes color whan a particular metabolic reaction has occurred during growth

aseptic technique

series of step sto prevent contmination during manipulations of cultures and sterile culture media (liquid and solid)

microsopic cell count

way of collecting the total counts of microbs in aculture or natural sample on wither adried slide or a liquid sample

microscopic counting cons

1. dead cells genrally can be distiguesed from live ones

2. precision is difficut

3. small cells can be missed

4. debri might be counted

5. cencetration might be too low

DAPI

stain for microscope counts that stains all cells as it interacts with DNA

flourescent stains

can be used to tell live and dead cellls apart because you can see if they cytoplasmic membrane is intact.

these stains cen be speialles to only reacted with certain domains of proteins etc.

If cells are present in low concentration, they cen be concentrated on a filter, then stained, then visualized

viable

cells that are able to divide and form offspring (wgat we are most interested in cell-counting situations generally)

viable count

also known as a plat count as agar plates are required.

spread-plate

volume (< 0.1 ml generally) is spread over an agar plate using a sterile gass spreader

pour-plate

(0.1-1.0 ml) of clutre is popetted into a sterile petri plate then molten agar , just above gelling temp (~50 degreesC) is added and gently mixed before it solidifies

ideal number of colonies

30-300

errors with viable counts

1. plateing inconsistancies

2. nonuniform sample (i.e. clumping)

3. insufficient mixing

4. heat intolerance (for pour plate method)

the great plate count anomaly

plate counts can be very unreliable when used to asses total number of cells in a natural sample because they show less micrones that microscopic counts do

turbidity

measurement that can quickly estimate the number of cells in a lab culture beccause as cell mass increases, cloudiness (turbidity) increases as well (think refractive index)

spectrophotometer

an instrument that passes light of a specific wavelength through a smaple (cell suspension) and measures the unscattered light that emerges. a standard curve msut be generated so that cells that cater light back into the spectrophotometer are controlled for.

common spectrophotometer wavelengths

480 nm (blue), 540 (green), 660 (red)

shorter is more sensitive but longer wavelengths are better for denser suspensions (more accurate)

optical density (OD)

units of turbidity that are wavelength specified

turbudity cons

not effective with clumps/clusters and biofilms, they must be broken up before measurments are taken

Environmental factors that control microbe growth

1. temp

2. pH

3. water availability

4. oxygen

cardinal temps

the three temperatures that determine cell growth rates: minimum, optimal, and maximum temperature

general growth temps

whole range is from -15 to 122 degrees celsius but most organism have a range of 40 degrees celcius

maximum temp

reflects the temperature at which major cell components (i.e. enzymes) are denatured. optiamal temp generallly lies closer to this than to the minimum

minimum temp

don’t know factors that control this as much but thought to do with the fluidity of the cytoplasmic membrane and its ability to do mececular transport.

temperature classes of organism (where there optimal tmp lies)

1. psychophiles (low temps)

2. meseophiles (medium temps)

3. thermophiles (high temps)

4. hyperthermophiles (super high temps)

E. coli cardinal temps

min: 8 degrees C

optimal: 39 degrees C

max: 48 degrees C

psychrophile

min: 0 degrees C or lower

optimal: 15 degrees C or lower

max: below 20 degrees C

psychotolerant

can grow at 0 degrees C but optimua is 20-40 degrees C

enzymatic adapations of psychophiles

1. proteins have more alpha helixes (more flexible) than beta sheets

2. more polar and less hydrophobic regions

3. lower number of weaker h bonds and ionic bonds compareed to corresponding mesophile enzymes

4. higher content of unsaturated and short fatty acid chains (help retain semifluid state at low temps)

5. polyunsaturated (flexibile at very cold temps)

“cold shock” proteins

types of molecular adaptation to cold temperatures. “cold shock” proteins (not limited to psychophiles) act as a molecular chaperonw and can do many things.

ex. maintaining cold-sensitive proteins in an active form or bind to specific mRNA to facilitate translation

cryoprotectants

include dedicated antifreeze proteins/ speciffic solutes like glycerol or some sugars that help prevent the formation of ice crystals (can puncture the cytoplasmic membrane)

exopolysaccharide cell surface slime

produced by high psychrophilic bacteria that confer cryoprotection (protect from puncture by ice crystals)

thermophiles

optimum: 80-45 degrees C

hyperthermophiles

optimum: >80 degrees C

habitats >65 degrees C

only prokaryotes can thrive here

thermophile and hyperthermophile heat resistanceresistance

thought to be from…

• subtle changes in amino acid sequence from comparable mesophile enzymes (resist heat denaturation)

• increased ionic bonding between basic and amino acid

• highly hydrophobic intreiors (resistant to unfolding

Heat denaturing protectant solutes

di-inositol phosphate, diglycoerol phosphate, and mannosylglycerate

characteristics of thermophiles and hyperthermo cyto membrane

higher content of long-chain (higher mp) and saturated fatty acids (give a stronger hydrophobic environment

hyperthermophile membranes

most are archea and dont have fatty acids. Instead, they have C₄₀ hydrocarbons made of repeating isoprene bonded by ether linkage to glycerol phosphate. is a monolayer that covalently links both halves of the membrane and prevents it from melting

microorganisms pH range

2-3 pH units

natural environment pH range

between 3 and 9

neutrophiles

orgs that grow optimally at pH wails 5.5-7.9 (circumneutral)

acidophiles

orgs that grow best below pH 5.5 (different classes)

alkaliphiles

microbes that show pH optima of =/>8

halophiles

organisms that thrive in NaCl environments (it is a requirement for them) and it can’t be replaced by any other salt (i.e. KCl). Different orgs require different amounts of salt.

internal pH

must reamin the same and in the ranfe of about 4 pH units ( 5-9). → the inside of ALL microbes is nearer to neutral so that macromolecules are stable

buffers in media

used to prevent major pH shifts during micobe growth in batch cultures.

neutrophilic buffers

potasoumphosphate (KH₂SO₄) and sodium bicarbonate (NaHCO₃)

water activity (a_w)

ratio of the vapor pressure of air in equilibrium with a substance or solution to the vapor pressure of pure water. varies from 0 (no free water) to 1 (pure water)

diffusion

movement of water from low solute concentration to high solute concentration

positive water balance

cells tend to have a higher solute concentration the their environment so water tends to diffuse into the cell

levels of salt tolerance

haplotolerant (cans servive but beter without), haplophile, extreme haplohiles (capable of growth in very salty environments

osmophiles

grow in environments high in sugar

xerophiles

grow in very dry environments

lower water activity limit for orgs

0.61 (a_w) defined by the constraints on obtaining water in osmotic environments that can’t be overcome through biochemical adaptations by the cell under this level

matric water activity

measuer of water bound to a surface, measure the same as osmatic water activity

compatible solute

a solute that is pumped into the cell or synthesized (compatible solute) to maintain positive water balance by increaseing internal solute concentration

common compatible solutes

sugars, alcohols, amino acid derivatives, and other highly water-soluble organic moleculs

aerobes

microbes that can frow at full osygen (air is 21% O₂) and respire (take in O₂ and breath out CO₂) oxygen in their metabolism

microaerophiles

aerobes hat use O₂ only when present at levels reduced from that in air microoxic conditions

facultative

under the appropriate conditions (nutrient and culture) can grow in the abscence of (O₂) but often grow better with it

anaerobes

grow without O2

aerotolerant anaerobes

microbes that can tolerate oxygen even though they can’t respire

obligated anaerobes

inhibited or even killed by oxygen

culture tech. for aerobes

nee aeration becuase O₂ consumed faster than it is made. Shake or bubble sterilixed air in the medium (thought glass tube or porous glass disk.

culture tech. for anaerobes

use a bottle or tube, filled to the top with a leakproof closure → suitibly anaerobic if not overly sensitive to O₂. can add a reducing agent to reduce oxygen to water

thioglycolate

common reducer when growing anaerobic microbes, indicated by the redox indicator resazurin

resazurin

redox indicator. pink when oxidized and collorless when reduced

O₂ toxic?

byproducts of the reduction of O₂ to H₂O such as: O₂^-, H₂O₂, and OH (radical) can be harmful to the cell unless they are destroyed

enzymes that reduce H₂O₂

catalase and peroxidase and form O₂ and H₂O respectively