Chapter 4: Microbial growth and its control Flashcards
Nutrients
Supply of elements required by cells for growth
Macronutrients
Nutrients required in large amounts
Micronutrients
Nutrients required in small amounts
Hetrotrophs
Obtain carbon from breakdown of organic polymers or uptake of monomers
Autotrophs
Synthesise organics from CO2
Nitrogen
-mostlu proteins, ammonia, nitrate or nitrogen gas
-Nealry all microbs us NH3
-many use nitrate
-Some use organics like amino acids or N2 (nitrogen fixing)
Other macronutrients
Oxygen and hydrogen from water
-Phosphorous: nucleic acids and phospholipids (usually inorganic phosphate)
-sulfur: sulfur containing amino acids, vitamins like biotin and microbes assimilate sulfate, sulfide or organics
-Potassium
-Magnessium: stabilises ribisomes, membranes, nucliec acids and required by many enzymes
-Calcium and sodium
Trace metals
Many enzymes require metal ion or small organic as a cofactor for catalysis
-Iron is used in cellular respiration, related oxidation-reduction reactions
-required in small amounts
Growth factors
vitamins: most function as coenzymes
others: aa, purines, pyramidines
Cobalt
Vitamin B12, transcarboxylase
Culture
Nutrient solution used to grow microbes
sterilised in an autoclave
Aseptic
Something that is contamination free
Sterile
It is the use of physical/chemical procedure to destroy all micro-organisms including spores
types of media
defined
complex
selective
differential
Defined media
Exact chemical composition is known
Complex media
Composed of digests of microbial, animal, or plant products (you know whats in but not how much)
Selective media
Contains compounds that selectively inhibit growth of some microbes but not others
Differential medium
Contains an indicator, usually a dye that detects particular metabolic reactions during growth
MacConkey is a medium that discrimanates between lactose fermenting and non fermenting bacteria
solid media
prepared by addition of gelling agent agar to liquid media
cells form colonies on this media
Morphology can be used to identify micro organisms and to check for contamination
Aseptic technique
Microscopic cell count
Direct count
Observing and enumerating cells present
dried on slides or on liquid samples
counting chambers with squares etched on a slide used for liquid samples
limitation is that you dont know if cells are alive or dead
Trpan blue
Azo dye
it selectively colours dead cells
it allows for a viable direct cell count as bacteria arent fixed
Colony counting limits
25-250 colonies
reported in colony-forming units (cfu/ml)
spread-plate method
Pour-plate method
Applications of the Plate count
quick and easy
used in food, dairy, medical and aquatic microbiology
high sensitivity
can target particular species in mixed samples
common in wastewater and other water analyses
Great plate count anomaly
Can only culture 1% of microbes and so culture is not an actual representative
Microscope vs plate count
Plate counts are always better as can indicate all microbes present
Indirect counts
Give an estimate
1. turbidity
2. Metabolic activity
3. dry weight
Turbidity
Cell suspensions are turbid because cells scatter light
More cells=more scattered light and increased turbidity
Turbidity measurements are rapid and used for estimates
Optical density
used to relate turbidity to cell numbers
measuered with a spectrophotometer
Unit is OD at a specific wavelenght
for unicellular organisms, OD is proportional to cell number up to 2 units
To relate a direct cell count to a turbidity value, a standard curve must first be established
Ad of turbity measurements
Quick and easy
do not require destruction or significant disturbance of sample
sample can be checked repeatedely
sometimes problematic when clumps or biofilms are formed
Metabolic activity
Method assumes that the CO2 meausered is directly proprtional to cell numbers
higher CO2 = higher cell pop
Dry weight
Used to track growth of filamentous fungi
fungus is filtered out of medium, dried in an oven or dessicator and then weighed.
dried ensures moisture content does not affect results
Binary fission
Cell division following enlargement of a cell to twice original size
-Quicker than mitosis because it does not have to generate mitotic spindle or dissolve nuclear membrane
septum
partiton dividng cells, pinches off between daughter cells
generation time
Time required for microbial cells to double in number
Batch culture
A closed-system microbial culture of fixed volume
Phases of growth curve
Lag phase
exponential phase
stationary phase
death phase
lag phase
Interval between inoculation of a culture and beginning of growth
-new conditions require alternating metabolic state
-Time needed for biosynthesis of new enzymes and to produuce required metabolites before growth can begin
Exponential phase
Doubling at regular intervals
close to metabolically identical
rates vary greatly, influenced by media, conditions, organism itself
continues until conditions can no longer sustain growth
Stationary phase
growth limited by nutrient depletion or waste accumulation
growth rate is zero
metabolism continues at greatly reduced rate
Decline phase
Total number decreases due to cell death
Cryptic growth
subpopulations adapt
Diauxic growth
Growth characterised by cellular growth in 2 phases
caused by presence of 2 sugars in a culture growth media, one of which is easier to metabolise
Doubling time formula
Td= 0.693/B
B is the number from equation on graph
Planktonic growth
Growth in suspension of free-floating/free-swimming cells
Sessile growth
Attached to surface
can develop into biofilms
NB in medical and industrial applications
Biofilm
cells emmeshed polysaccharide matrix attached to surface
stages of biofilm growth
Planktonic cells attach (fimbriae, flagella, pili)
-Colonisation: growth and extracellular polysaccharide (EPS) production
-Development: metabolic changes
-Dispersal: colonise new sites
Can study in flow chamber
Microbial mats
Multilayered sheets with differnt organisms in each layer
Humans and biofilms
Implicted in join infections, implanted medical devices
-Responsible for cavities and cause gum disease
-Foul, plug, corrode pipes
-Form in fuel tanks and on ship hulls
Biofilm lethality
Resistant to antibiotics and infections
Optimum temperature for most microbes
Less than 40 degrees
psychrophile
low, found in cold environments
optimal groth temp is below 15 and max 20 and min 0
constantly cold environments
found in polar regions
Mesophile
midrange, commonly studied
thermophile
high, hot environments
hyperthermophiles
Very high, found in extremely hot habitats such as hot springs and deep-sea hydrothermal vents
Extremophiles
Organisms that grow under extreme conditions (hot and cold)
psychotolerant
can gro at 0 but optimum is 20-40
more widely distributed in nature
isolated from soils and water in temperate climates and food at 4 degrees
Molecular adaptions for cold
More alpha helixes than B sheets: greater flexibility for catalysis at cold temp
More polar and fewer hydrophobic aa
fewer weak bonds
Cytoplasmic membrane has higher unsaturated and shorter fatty acid content as wll as polyunsaturated fatty acids which remain flexible at very low temp
-cold shock proteins
-Cryoprotectants prevent formation of ice crystals
-Expolysaccharide cell surface slime
Hyperthermophile diet
Chemoorganotrophic and chemolithotrophic
Thermophile
Growth temperature beteen 45 and 80
Hyperthermophile
Growth greater than 80
inhabit hot springs and hydrothermal vents with temps greater than 100
above 65 only pro survives
Adaptions to survive heat
-amino acid substitiutions that resist denaturation
-increased ionic bonding and hydrophobic interiors
-production of solutes like dyglycerol phosphate to help stabilise proteins
-saturated fatty acids increased
useful thermophile
Taq polymerase for PCR
adaptions of hyperthermophile
-They have C40 hydrocarbons made of repeating isoprene units bonded by ethers to glycerol phosphate and they have a monolayer memebrane
Neutrophile
pH> 5.5 and <8
Acidophile
grow best at low pH <5.5
governed by stability of cytoplasmic membrane
at neutral pH, acidophiles lyse as the require protons for stability
Akaliphiles
pH>8
found in soda lakes and high carbonante soil
used commercially in detergents
have sodium motive force
water activity
ratio of water vapor pressure of air equilibrium with a substance or solution to vapor pressure of pure water
positive water balance
cytoplasm has higher solute concentration than outside otherwise water flows out
Halophiles
grow best at aw=0.98 which is sea water
have requirement for NaCl
Halotolerant
Tolerate some dissolved solutes but grow best in absence
Extreme halophiles
Need 15-30% NaCl and cant grow at lower concentrations
Compatible solutes
Microbes pump these solutes into cell to maintain positive water balance
they do not inhibit biochemical processes
Osmophile
Live in high sugar
Xerophile
live in dry environment
Why add reducing agents to culture?
To reduce oxygen to water
resazurin
dye that indicates Oxygen concentration
singlet oxygen
molecular O2 that has been boosted into higher energy state- extremely reactive
Superoxide radical
formed in normal amounts due to respiration
toxic
Peroxide anion
Byproduct of conversion of superoxide radicals
extremely toxic
Hyrdoxyl radical
Formed by IONIZING RADIATION in cytoplasm
most reactive
Superoxide dismutase (SOD)
Converts superoxide to H peroxide and O2
Catalase and peroxidase
Convert H peroxide to O2 and H2O
Facultative anaerobes
Both aerobic and anaerobic growth, greater with O2
-Growth is best where most O2 is but occurs everywhere
-Presence of SOD and catalase allows toxic forms of O2 to be neutralised
Obligate aerobes
-Oxygen required
-growth occurs in high O2 concentrations
-Presence of SOD and catalase allows toxic forms of O2 to be neutralised
Superoxide reductase
Converts superoxide to H peroxide without producing O2
Obligate anaerobes
-Only grows without O2
-growth occurs where little O2
-lacks enzymes to neutralise O2
Micro-aerophiles
-Only aerobic growth with low O2 concentration
-Growth occurs in low concentration
-produces lethal amounts of byproducts if exposed to atmospheric O2
Aerotolerant anaerobes
-only anaerobic growth
-growth occurs evenly, O2 has no effect
-Presence of SOD partially neutralises O2
Decontamination
Treatment of object to make safe to handle
Disinfection
-directly targets pathogens
-kills or inhibts growth
Heat sterilisation
most used
higher heat kills faster
moist heat penetrates better than dry heat
Decimal reduction time
Amount of time reuired at a given temp to reduce viability 10-fold
Thermal death time
time to kill all cells at a given temp and is affected by pop size
Autoclave
Steam under pressure gets to temp of 121
kill endospores
Ionising radiation
EM radiatipn that produces ions and reactive molecules
used for surgical supplies, labwear, etc
Pasterurisation
uses heat to only kill pathogenic bacteria but not all organisms
UV
-affects DNA leading to death
-Used for decontaminating surfaces
-poor penetration
Filter sterilisation
used for heat sensitive liquid and gas
pores too small for organisms but does not trap viruses
Depth filters
fibrous sheet made of overlapping paper or glass
-HEPA filters
Bacteriostatic agent
Inhibit biochemical properties and bind weakly
Bactericidal agents
bind tightly and kill without lysis
Bacteriolytic agent
kills by lysis
sterilants
destroys all micro including endospores
Minimum inhibitory concentration
smallest amount of agent needed to inhibt growth
-can be determined via Kirby-Bauer assay
Kirby-Bauer assay
-Antimicrobe agent added to filter paper
-diffuses into agar
-MIC is reached at some distance
sanitizers
reduce M number but dont sterilise
antiseptics
kill or inhibt growth but are non toxic enough to apply to living tissues
