3. Microbial Growth and Nutrition Flashcards
Macronutrients
- elements required in large amounts to build macromolecules
- CHONPS
- make up more than 90% of the dry weight cell
Types of macromolecules
lipids, carbs, proteins and nucleic acid
Proteins
- CHON and sometimes S
- polymer made of building blocks
- more than 50% of dry weight
Lipids
- CHO and sometimes P
- building blocks of fatty acids and glycerol
Carbohydrates
- CHO and sometimes N
- building blocks of sogars
Nucleic acids
- CHONP
- building blocks of nucleotides
If the macromolecular composition of a cell has a lipopolysaccaride what kind of gram is it
gram negative
Other macronutrients - inorganic ions
K, Mg, Ca, Fe
What are inorganic ions used for
metabolic cofactors
- used in addition to macronutrients
- its the non-protein component required for enzyme function
K+, Fe2+, Mg2+, Ca2+
K+ - enzymes for protein synthesis
Fe2+ - cytochromes to carry
Mg2+ - stabilize membranes and nucleic acids
Ca2+ - stabilize cell walls and heat stability for endospores
How can you remember all macronutrients?
C HOPKNS CaFe Mg - the c hopkins cafe is mighty good
Micronutrients
- trace elements required in very small amounts
- cofactors for enzyme
Growth factors
- small organic molecules required for growthh
What happens if an organism cannot synthesize a growth factor?
it must be added to medium to grow that microbe in the lab
Three classes of growth factors
- Amino acids
- Purines and pyrimidines
- Vitamins
Amino acids
needed for protein synthesis
Purines and pyrimidines
AG - 2 rings
TCU - 1 ring
needed for nucleotides, building blocks of RNA and DNA
Vitamins
- small molecules used to make organic cofactors
- non-protein components required by some enzymes
Growth factor requirements
- many have none (E.coli) some require alot (Leuconostoc mesenteroides)
-some may require a medium to promote growth
Nutrient sources of O and H
- no specific nutrient
- Found in H2O and organic media components
Nutrient sources of P
- usually a phosphate salt
Limiting nutrient
In relatively low concentration compared to other nutrients
- when it runs out, growth stops despite other nutrients present
Nutrient sources of N
- inorganic - salt - reduced to NH3 to make amino acids
- organic - rich organic molecules - doesn’t need to be reduced
- atmospheric N2 - N2 is reduced to NH3 - nitrogen fixation - energy expensive - only bacteria and archaea
Nutrient sources of S
- inorganic - salt - reduced to S2- to make amino acids - assimilative sulfate reduction
- organic - pre-made amino acids - less energy to assimilate
Nutrient sources of C
- Depends on the type of organism
- If heterotrophs - organic carbon (C and H) - 1 or more C is reduced
- If autotrophs - inorganic carbon (CO2) as their sole source of carbon - needed energy to assimilate - photosynthesis
Metabolism
the sum total of all the chemical rxns in a cell
Catabolic reactions
energy releasing metabolism - break down
- fermentation, respiration
anabolic reactions
energy-requiring metabolism - build up
- biosynthesis
Microorganisms can be categorized by
- energy source
- electron source
- carbon source
energy sources
chemo - chemical
photo - light
electron sources
organo - organic
litho - inorganic
carbon sources
hetero - pre-existing
auto - inorganic
Chemoorganotrophs
energy from chemical reactions involving organic material
chemolithotrophs
energy from inorganic chemical reactions
phototrophs
energy from light
heterotrophs vs autotrophs
h - use organic carbon for building cell carbon and biomass
a - use co2 to synthesize cell carbons
Basic medium
nitrogen fixing photolithoautotroph
medium 1
allows Non-nitrogen fixing photolithoautotrophs to grow
medium 2
reduced organic carbon source - energy and e- for chemoorganotrophs
- some chemoorganoheterotrophs
medium 3
allows growth of chemoorganoheterotrophs with a growth factor requirement
medium 4
vitamins! - N source, alternate source of carbon energy and e-
defined medium
exact chemical composition is known
- used to study metabolism
minimal medium
a type of defined medium that provides the minimum nutritional requirements for growth
complex medium
exact chemical composition is not known
- usually made from meat or yeast
- supply a variety of growth factors
differential medium
allows different bacteria to be distinguished
Example of differential medium
Blood agar - tsoy plate - 5% sheeps blood
Hemolytic bacteria (destroying red blood cell)
alpha - incomplete destruction of blood cells
beta - complete destruction
gamma - no destruction (not hemolytic)
Selective medium and example
contain ingredients that inhibit the growth of unwanted microbes - allow only specific microbes to grow
- Ex) mannitol salt agar - contains very high salt so only halotolerant bacteria can grow - isolate staphylococci from skin
Enriched medium
- supplemented with special nutrients to encourage the growth of fastidious bacteria
- complex nutrient requirements
- Ex) blood agar, chocolate agar
how is growth measured for microbio?
increase in the number of cells of apopulation not individual growth
- increase through binary fission - cell division following enlargement of a cell to twice its mini size
generation time (G)
time required for microbial cells to double in number
cell division - euk vs pro
pro: growth in cell size, chromosome replication and septum formation occur at the same time - no mitosis
euk: growth, replication, seperation are at differnt times
bacteria have shorter generation times than eukaryotes
what is generation time dependent on
growth medium and incubation conditions
exponential growth
growth of a microbial population in which cell numbers double at a constant and specific time interval
relationship between initial number vs final number
Nf = N0x2^n - final cell number is equal to inital cell number multiplied by 2 to the power of number of generations
exponential growth for this one
0.5 hour
What is this graph showing?
because cells increase exponentially the increase in cell number is initially slow but increases at an ever faster rate following an exponential curve - cells are doubling at a constant rate
Why is unlimited growth called exponential growth
it generates a curve whose slope increases conitnuously
Growth rate (k)
- rate of increase in population number or biomass
Why is pro growth rate expressed as the number of doublings per hour
they grow by binary fission - always doubling
N0 = 0.5 x 107 cells
NF = 1.00 x 107 cells
Dt = 4.2 - 3.5 = 0.7 hour
K? and G?
k = (log1x107 - log0.5x107)/(.301)(0.7)
k = 1.43 gen/hr
g=1/k
g= 1/1.43 = 0.7hr/gen
specific growth rate
for each organism, they have a growth rate that is the fastest in the best growth medium at optimal temperature
batch culture
a closed-system microbial culture of fixed volume - once inoculated - thats all you get - because theres a fixed number of resources
Growth curve for population of cells phases
- lag phase
- exponential phase
- stationary phase
- death phase
lag phase
interval between inoculation of a culture and beginning of growth
- adapting to new environment
- ex) when you add bacteria to a tsoy plate
- small increase in cells is due to transition - elongating cells but not division
exponential phase
- LOG phase - doubling happens here
- cells in this phase are typically in the healthiest state
which is why we use it for calculating growth rate
stationary phase
- cells metabollically active but growth rate of population is zero
- either an essential nutrient is used up or wast product of organism accumulates in medium
death phase
- if incubation continues after cells reach stationary phase, cells will eventually die
- not all bacteria die, some bacteria form spores of cysts or dormant stages that allow a significant proportion of cells to survive a long time
continuous culture
an open system microbial culture of fixed volume
- new nutrients and wastes are consistent
chemostat
most common type of continuous culture device
- growth rate and population density of culture can be controlled independently and at the same time
- dilution rate - rate at which fresh medium is pumped in and spent medium is pumped out
- concentration of a limiting nutrient controls the population size and the growth rate
microbial counts
direct microscopic observations using a counting chamber
- each square corresponds to a calibrated volume
-results can be unreliable
limitations of microscopic counts
- cannot distinguish between live and dead cells without special stain
- small cells can be overlooked
- precision is difficult
- needs a specific microscope
- low density cells are hard to count
- hard to count motile cells
- debris can be mistaken for cells
- cells may move and form clumps
flow cytometry
automaticlaly counts the total number of cells based on laser beams, dyes and electronics
viable cell counts and how?
- measure only living cells - cells that are capable of growingto form a population
- 2 main ways: spread plate or pour plate
issues with viable cell counts
- needs alot of preparation and incubation time for a culture
- plate counts are unreliable when used to assess total cell numbers of natural samples
- selective culture media and growth conditions target only particular species - a single medium cant grow every microbe - can only count the types of bacteria that can grow in the medium selected
great plate anomaly
- microscopic counts reveal more organisms than plate counts
- genome techniques suggest that only 1-10% of microbial diversity is culturable from most environment samples because
1. microscopic methods count dead cells and viable methods dont
2. differnt organisms may have vastly different requirements for growth
3. we dont know the specific requirements for all organisms
spectrophotometric counts
used for turbidity measurements
- optical density measurement
- bacteria are like small particle and absorb and scatter light
- only a portion of the incident light makes it to the photocell
- the larger the number of particles, the greater the absorbance, the lower the light transmission to the photocell
- can’t distinguish dead cells from living cells
what does this graph say
standard curve: viable cell counts and weight of biomss produced
- optical density has a fixed linear range of measurement
- it only works if the cells are evenly distributed throughout the medium
dry weight measurement (total mass of cells)
a specific aliquot cells are concentrated, washed to remove media components, concentrated and dried
other spectrometric techniques are used for
to measure compoenets of the cell, protein, DNA
cardinal temps
the minimum, optimum and maximum temps at which an organism grows
psychrophile
low temp is optimum
mesophile
midrange temp is optimum
- warm blooded animals, trrestrial and aquatic, temperate and tropical latitudes
thermophile
high temp optimum
hyperthermophile
very high temp optimum
cold loving microorgnaism
extremophiles - organisms that grow under very hot or very cold conditions
psychrphiles - organisms with cold temperature optima - inhabit permanently cold environments
psychotolerant - organisms that can grow at 0 but have optimum of higher
adaptations that support cold loving microorganisms
- enzyme; function optimally in the cold
- modified cytoplasmic membranes - high unsaturated fatty acid content
heat loving microorganisms
- only prokaryotic life forms exists
including chemoorganotrophic and chemolithotrophic species - high diversity - thermophiles: organisms with growth temperature optima between 45 and 80 - hot springs, compost
-hypothermophiles: organisms with optima greater than 80 - hot environments like boiling hot springs, seafloor hydrothermal vents
adaptations that support heat loving microorganisms
- cytoplasm membrane:
bacteria - lipids rich in saturated fatty acids
archaea - lipid monolayer rather than bilayer - hyperthermophiles produce enzymes widely used in industrial microbiology - hydrolytic enzymes like proteases, cellulases and lipases
- enzymes of thermophiles are more stable and have higher activity than mesophilic counterparts
what is the dying temp for life?
140-150
_________ closest descendants of ancient microbes
hyperthermophiles - deepest and shortest branches of the phylogenetic tree
- oxidation of H2 is common
- maybe first energy-yielding metabolism
pH and microbial growth
some organisms grow best at high or low pH
- most organisms grow between 6-8 - neutrophiles
- less than 6 (low ph) - acidophiles
- higher than 9 (high ph) - alkaliphiles
bottom line of pH and adaptations
- cytoplasmic membrane maintains its integrity at the growth pH
- the internal ph must stay relatively close to neutral even though the external ph is highly acidic or basic
how do microbial culture media maintain ph
buffers!
- some bactera produce acids to decrease ph
some bacteria grow on amino acids to increase ph
water activity
water avaialbility is expressed in physical terms
- ratio of vp of air in equil w a substance/solution to vp of pure water
- reflects the amount of water thats interacting with ions and polar compounds in solution
cytoplasm has a ____ solute concentration than the surroundings
higher; water wants to move into the cell creating turgor pressure
halophiles
grow best at reduced water potential and have a specific requirement for NaCl
- many marine microbes
- high solute concentration
extreme halophils:
require high levels of nacl for growth
15-20%
- microbes in the dead sea
halotolerant
can tolerate some reduction in water activity of environment but generally grow best at lower solute concentrations
- staphy aureus
osmphiles
organisms that grow with high sugar as solute
xerophils
organisms able to grow in very dry environments
specialized and rare organisms
honey, jams and jellies, beef jerky and salted cod
high osmolarity created with nacl is used to _________
select for acid producing microorganisms
- fermentation
- high salt and low ph prevents growth of MOST pathogens in the finished product
mechanisms for combatting low water activity in surroundings:
- pumping inorgnic ions from environment into cell
- synthesizing or concentrating organic solutes
- compatible solutes: compounds used by cell to counteract low water activity in surrounding environment
obligate aerobes
require oxygen to live
strict anaerobes
don’t require oxygen to live and may die from exposure to oxygen
facultative aerobes
can live with or without oxygen - they use it if its available
aerotolerant anaerobes:
can tolerate oxygen and grow in its presence even though they cannot use it
microaerophiles
can use oxygen only when it is present in a small amount
how can oxygen tolerance be distinguished?
thioglycolate broth
name the type in each
a) obligate
b) strict
c) facultative
d) microaerophile
e) aerotolerant
how to grow anaerobic techniques
reducing agents to reduce oxygen
- removal of air and replacement with an inert gas
toxic forms of oxygen formed in cell
- superoxide anion
- hydrogen peroxide
- hydroxyl radical
enzymes that neutralize toxic oxygen and who lacks some/all of these
catalase, peroxidase, superoxide dismutase, superoxide reductase
obligate anaerobes
sterilization
killing of all viable organisms within a growth medium
inhbiition
effectively limitng microbial growth
- no killing taking place
decontamination
the treatmeent of an object to make it safe to handle
disinfection
directly targets the removal of all pathogens
not necessarily all microorganisms
heat sterilization
- controlling microbial growth
- high temperatures denature macromolecules
What cells are resistant to sterilization and what would D be for this
- endospores because they have lipids, sugars, nucleic acids that decrease penetration
- D would be linear
What happens to time when temp decreases for sterilization
increase in temp decreases D or time
pasteurization
using precisely controlled heat to reduce the microbial load in heat-sensitive liquids
- this does not kill all organisms - like endospores
-
milk and pasteurization and disease
can use diff temps
- LTLT - low temp but for a long time - 63 degrees for 30 mins
- HTST - high temp for a short time - 72 degrees for 15 seconds
- both processes kill Coxiella bernetii - the causative agent of Q fever
autoclave
a sealed device that uses steam under pressure
- allows temp of water to get 100 degrees
- 121 degrees for 15 minutes at 15 pounds per square inch of pressure is typically used
- the point it takes the longest to heat must stay at 121 for 15 minutes
what can kill better and faster? dry or moist
moist
ionizing radiation
- electromagnetic radiation that produces ions and other reactive molecules generates electrons, hydroxyl radicals, hydride radicals
sources of radiation and what its used for in the medical and food industry
- cathode ray tubes, xrays, radioactive nuclides
- approved by who and is used in the usa to decontaminate foods
filtration
- avoids the use of heat on sensitive liquids and gases
- pores of filters are too small for organisms to pass through - only liquids and gases
3 membrane filter types
- syringe - small volume
- pump - large - pushing out
- vacuum - large - sucking in
nucleapore filtration
pore size 5 micrometer - bacteria can pass through
pore size 0.2 micrometer - prevents bacteria from passing through
antibacterial agents
- bacteriostatic
- bacteriocidal
- bacteriolytic
bacteriostatic
- prevents cell growth as long as the antimicrobial agent is present
bacteriocidal
kills the cell but doesn’t lyse them
bacteriolytic
- kills and lyses them
why is the cell count still high in bacteriocidal
because the defining cell count is the cytoplasmic membrane - so if its not lysed its still there
MIC
minimum inhibitory concentration
- the smallest amount of an agent needed to inhibit growth of a mircoorganism
- because its expensive to use alot and it could also be toxic
- varies with the organism, inoculum, temp and ph
- lowest concentration with no growth
- it may still have living organisms that arent growing (bacteriostatic)
MLC
minimal lethal concentration
- when plating the broth from MIC and tubes with higher concentrations, do colonies form?
- the one where colonies stop forming is the MLC
whats higher, MLC or MIC
MLC
disc diffusion assay
- solid media
- antimicrobial agent is added to filter
- MIC is reached
- forms a zone of inhibition
- area of no growth around disc
antimicrobial agents 2 categories
- products used to control microorganisms in commercial and industrial applications
- products desgined to prevent grwoth of human pathogens in animate environments and on external body surfaces
ex of products used to control microorganisms in commercial and industrial applications
chemicals in foods, air conditioning cooling towers, textile and peper products, fuel tanks
ex of products designed to prevent growth of human pathogens in inaminate environments and on external body surfaces
- sterilants - destroys all microorganisms including endospores
- disinfectant; kills microorganisms but not all endospores
- sanitizer - reduced the numbers of microorganisms on surfaces
- antiseptic - kills or inhibits the growth of microorganisms but non toxic enough to be applied on living tissue like mouthwash
