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Microbiology Chapter 6: Growth and Culturing of Bacteria

Terms in this set (107)

microbial growth
defined not in terms of cell size but as the increase in the number of cells, which occurs by cell division
cell division
occurs by binary fission or budding in bacteria
budding
a process where small, new cells develop from the surface of an existing cell and subsequently separates from the parent cell
- occurs in cell division in yeast and a few bacteria
medium
a mixture of substances on or in which microorganisms grow
lag phase
the organisms do NOT increase significantly in number, but they are metabolically active
- individual organisms increase in size and they produce large quantities of energy in the form of ATP
- the length is determined in part by characteristics of the bacterial species and conditions in the media
log phase
once organisms have adapted to a medium, population growth occurs at an exponential or logarithmic rate
- growth appears as a straight diagonal line
- logarithmic growth is only for a limited time bc it causes the cells to use a lot of energy
generation time
a regular genetically determined interval
- when organisms divide at their most rapid rate bc the population of organisms double in each generation time
synchronous growth
if cells divided together and the generation time was exactly 20mins, the number of cells in a culture would increase in a stair-step pattern, exactly doubling every 20mins. (hypothetical)
non synchronous growth
each cell divides sometime during the 20min generation time with about 1/20 of the cells dividing each minute (natural)
chemostat
a device that logarithmic growth can be maintained
stationary phase
when cell division decreases to the point that new cells are produced at the same rate as old cells, the number of live cells stays CONSTANT
- limited amount of nutrients
decline (death) phase
as conditions in the medium become less and less supportive of cell division, many cells lose their ability to divide then die
- many undergo involution
- the number of live cells decreases at a logarithmic rate
involution
cells assume a variety of unusual shapes making them difficult to identify
colony
all the descendants of the original cell
- what cells form when they exponentially divide
- non synchronous growth
how is bacterial growth measured?
by estimating the number of cells that have arisen by binary fission during a growth phase
standard plate count
under proper conditions, only a living bacterium will divide and form a visible colony on an agar plate
agar plate
a petri dish containing a nutrient medium solidified with agar
agar
a complex polysaccharide extracted from certain marine algae
serial dilutions
help count the number of colonies on one plate through dilution
serial dilution procedure
1) start with organisms in a liquid medium
2) adding 1ml of this medium to 9ml of sterile water makes a 1:10 dilution
3) pour plate method
4) spread plate method
colony forming unit (CFU)
represented by each bacterium
- if you've made dilutions properly, you should get plates with countable number of colonies
direct microscopic counts
when a known volume of medium is introduced into a specially calibrated, etched glass side called a Petroff-Hauser counting chamber
- does NOT know living vs. non-living
most probable number (MPN)
used when samples contain too few organisms to give reliable measures of population size
- consists of 5 tubes each of 3 volumes
- those that contain an organism will display growth by producing gas bubbles and/or by becoming cloudy when incubated
- best used for testing water purity
filtration
when a known volume of water or air is drawn through a filter with pores too small to allow passage of bacteria
turbidity
a cloudy appearance in a culture tube indicating the presence of organisms
spectrophotometer
obtains accurate estimates of growth by measuring turbidity
- useful in monitoring the rate of growth without disturbing the culture
dye reduction test
measures the direct or indirect uptake of oxygen
- methylene blue is used and remains colorful in presence of oxygen
- not an accurate measure of bacterial mass
dry weight measurements
determines the number of cells in a culture
- cells are separated from the medium by filtration or centrifugation and then dried and weighed
physical factors affecting bacterial growth
pH, temp, oxygen, concentration, moisture, hydrostatic pressure, osmotic pressure, and radiation
nutritional factors affecting bacterial growth
availability of carbon, nitrogen, sulfur, phosphorous, trace elements, and sometimes vitamins
pH
how the acidity or alkalinity of a medium is expressed
- microorganisms have an optimal pH (7)
- bacteria are classified as acidophilus, neutrophiles, or alkaliphiles
acidophiles
acid-loving organisms that grow best at a pH of 0.1-5.4
ex. lactobillus, which produces lactic acid
neutrophiles
exist from pH 5.4-8
- most of these bacteria cause diseases in humans
alkaphiles
base-loving organisms that exist from pH 7.0-11.5
buffers
incorporated into growth media to maintain the proper pH levels
- helps prevent the interference of metabolic by-products
temperature
most species of bacteria can grow over a 30C temp. range
- bacteria are classified as psychophiles, mesophiles, or thermophiles
- bacteria are more apt to survive extremes of cold than extremes of heat
psychophriles
cold-loving organisms grow best at 15-20C
- live mostly in cold water and soil
- NONE can live in the human body
obligate psychophriles
can NOT grow above 20C
facultative psychophriles
grow best BELOW 20C but can grow over
mesophiles
includes most bacteria that grow best at temp. between 25-40C
- human pathogens are included and grow near body temp (37C)
thermophiles
heat-loving organisms grow best at 50-60C
obligate thermophiles
grow ONLY at temp above 37C
facultative thermophiles
grow BOTH above and below 37C
oxygen
bacteria can be divided into aerobes and anaerobes
obligate aerobes
must have free oxygen for aerobic respiration
ex. pseudomonas is a common cause of hospital-acquired infections
- grow near the top in a culture tube
obligate anaerobes
killed by free oxygen
- grows near the bottom in a culture tube
microaerophiles
grow best in the presence of a small amount of free oxygen
- grows below the surface of the medium in a culture tube
capnophiles
carbon dioxide-loving organisms
facultative anaerobes
carries on aerobic metabolism when oxygen is present, but they shift to anaerobic metabolism when oxygen is absent
- can do both making them the most complex enzyme systems
aerotolerant anaerobes
can survive in the presence of oxygen but do NOT use it in their metabolism
superoxide
a highly reactive and toxic form of oxygen that kills obligate anaerobes
moisture
all actively metabolizing cells generally require a water environment
- most vegetative cells can live only a few hrs without moisture
- ONLY spores can exist in a dry environment
hydrostatic pressure
pressure exerted by standing water in proportion to its depth
barophiles
bacteria that live at high pressures
- their membranes and enzymes require pressure to function properly
osmotic pressure
the membranes of all microorganisms are selectively permeable
- environments that contain dissolved substances exert osmotic pressure
- salt is used as a preservative bc the high concentration of dissolved substances exert sufficient osmotic pressure to kill or inhibit microbial growth (same with sugar)
plasmolysis
when cells are in hyper osmotic environments and lose water, the cell shrinks
halophiles
salt-loving organisms require moderate to large quantities of salt
- the cells need Na to maintain a high intracellular K concentration so that their enzymes will function
- typically found in the ocean
radiation
radiant energy, such as gamma rays and UV light, can cause mutations and even kill organisms
- some microorganisms have pigments that screen radiation and help to prevent DNA damage
fastidious
when microbes have special nutritional needs that can be difficult to meet in the lab
- some can be grown in the human body but not easily in lab
ex. gonnorhea
carbon sources
used as an energy source
- many use carbon-containing compounds as building blocks to synthesize cell components
nitrogen sources
all organisms need N2 to synthesize enzymes, other proteins, and nucleic acids
- some obtain N2 from inorganic sources
sulfur
important cell component
- used to make proteins, co-enzymes and other cell components
phosphorous
important cell component
- microorganisms obtain this mainly from inorganic phosphate ions
- they use this to synthesize ATP, phospholipids, and nucleic acids
trace elements
tiny amounts of minerals, such as copper, iron, zinc, and cobalt, usually in the form of ions
- often serve as cofactors in enzymatic reactions
vitamins
organic substances that an organism requires in small amounts and that's typically used as a co-enzyme
- some make their own from simpler substances
- others regulate it in their media bc they lack the enzymes to synthesize them
ex. folic acid vitamin b12, vitamin K
nutritional complexity
the number of nutrients it must obtain to grow
- determined by the kind and number of its enzymes
- fewer enzymes = complex nutritional requirements
- many enzymes = simple nutritional requirements
extracellular enzymes
act in the medium around the organism
- usually produced by gram POS organisms
periplasmic enzymes
act in the periplasmic space
- usually produced by gram NEG organisms
hydrolases
enzymes that add water as they split large molecules of carbs, lipids, or proteins into smaller ones that can't be absorbed
adaptation to limited nutrients
1) some synthesize increased amounts of enzymes for uptake and metabolism of limited nutrients
2) others have the ability to synthesize enzymes needed to use a different nutrient
3) many organisms adjust the rate at which they metabolize nutrients and the rate at which they synthesize molecules required for growth to fit the availability of the least plentiful nutrient
quorum sensing
explains bacterial bioluminescence
- bacteria can produce inducer molecules that will turn on bioluminescent genes, but ONLY when the inducers are present in a high enough concentration
- need a large amount for light
- leads to the production of toxins, digestive enzymes, and strands of adhesive molecules
biofilm
one of the most important results of quorum sensing
- most microbes exist in biofilms made up of many different species
- acts as a kind of "super organism" with different cells responding differently within it
biofouling
bacteria have a "sense of smell", they're able to somehow detect airborne chemicals that indicate rival bacteria are nearby
- they respond to these smells by forming a biofilm with its accompanying slime
sporulation
the formation of endospores that occurs in gram POS genera
- formed during the stationary phase
- protective or survival mechanism
bacterial endospores
form inside the bacterial cell
fungal spores
produced in great numbers and are a form of reproduction
germination
when a spore returns to its vegetative state and occurs in 3 stages
activation
first stage of germination
- usually requires some traumatic agent such as low pH or heat, which damages the coat
germination proper
second stage of germination
- requires water and a germination agent that penetrates the damaged coat
- living cell takes in large amounts of water and loses its resistance to heat and staining and its refractility
outgrowth
third stage of germination
- occurs in a medium with adequate nutrients
- proteins and RNA are synthesized then DNA synthesis begins
vegetative cycle
bacteria cells capable of sporulation are repeated at intervals of 20mins. or more
sporulation cycle
bacteria cells capable of sporulation are initiated periodically
cysts
spherical, thick-walled cells that resemble endospores
- metabolically inactive and resist drying
- germinate into single cells
conidia
chains of aerial spores with thick outer walls
- temporarily dormant but not especially resistant to heat or drying
- DOES contribute to reproduction
endospore septum
consists of a cell membrane but lacking a cell wall
- forms the cortex
spore coat
keratin-like protein that's laid down around the cortex by the mother cell
exosporium
a lipid-protein membrane that's formed outside the coat by the mother cell
- function is unknown
the streak plate method
the accepted way to prepare pure cultures
- uses agar plates
- bacteria are picked up on a sterile wire loop and the wire is moved lightly along the agar surface
- inoculating loop is flamed
- the use of sterile technique ensures that the new medium will contain organisms of only a single species
the pour plate method
makes use of serial dilutions
- made so that the final dilution contains a small number of bacteria, some which will form isolated colonies on the agar
- useful for growing microaerophiles that can NOT tolerate exposure to oxygen in the air at the surface of the medium
natural media
oceans, lakes, soil, and on living or dead organic matter
synthetic media
a medium prepared in the lab from materials of precise or reasonably well-define composition
- generally lab media
defined synthetic media
contains known specific kinds and amounts of chemical substances
complex medium
contains reasonably familiar materials but varies slightly in chemical composition from batch to batch
- aka chemically non defined medium
peptone
a product of enzyme digestion of proteins
- provides small peptides that microorganisms can use
yeast extract
contains a number of vitamins, coenzymes, and nucleoside
- enriches certain media
- commonly used
casein hydrolysate
contains many amino acids
- used to enrich certain media
- made from milk protein
- commonly used
serum
the liquid part of the blood after clotting factors have been removed
- useful in enriching media
- commonly used
blood agar
useful in identifying organisms that can cause hemolysis, the breakdown of rbcs
- commonly used
selective medium
encourages the growth of some organisms but suppresses the growth of others
ex. SPS agar
differential medium
has a constituent that causes an observable change in the medium when a particular biochemical reaction occurs
- this change allows microbiologists to distinguish a certain type of colony from others growing on the same plate
ex. SPS agar or Macconkey agar
enrichment medium
contains special nutrients that allow growth of a particular organism that might not otherwise be present in sufficient numbers to allow it to be isolated and identified
- does NOT suppress others
stock culture
when an isolated organism is maintained indefinitely in a pure culture
- the stock culture itself is NEVER used for lab studies
- organisms in stock go through growth phases, deplete nutrients, and accumulate wastes
aseptic techniques
minimize the chances that cultures will be contaminated by organisms from the environment or that organisms, esp. pathogens, will escape into the environment
- important in making subcultures from stock cultures
preserved culture
a culture in which organisms are maintained in a dormant state
lyophilization
when cells are quickly frozen, dehydrated while frozen, and sealed in vials under vacuum
- most commonly used technique
- can be kept indefinitely at room temp
reference cultures
a preserved culture that maintains the organisms with the characteristics as originally defined
enterotube system
used to identify enteric pathogens
- consists of a tube with compartments containing one or more different media and a sterile inoculating rod
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