Biol 319 exam 2

Culture media

nutrient media that enhances the growth of microorganisms

solid medium

has agar and other solidifying agents

liquid medium

contains specific nutrients but has no gelling agents

minimal medium

chemically defined medium

- you know how much of each is present in each individual component

-Components are limited to only those nutrients that the organisms needs in order to grow

complex medium

chemically non-defined medium

- don't really know what the complex medium is made up of

heterotrophs

rely on other organisms to make the organic compounds that they use as carbon sources

autotrophs

use the CO2 discarded by heterotrophs to make complex cel constituents made up of C, H, and O, such as carbohydrates

phototrophs

extract energy from absorption of light

chemotrophs

extract energy from oxidation-reduction reactions that remove electrons from high energy compounds to produce lower energy compounds

- lithotrophy

- organotrophy

obtaining nitrogen

-Nitrogen is a critical component of proteins, nucleic acids, and other cellular constituents and is required in large amounts by living organisms

-N2 makes up nearly 79% of Earth's atmosphere, but the nitrogen in N2 is unavailable for use by most organisms

-Nitrogen from N2 must be "fixed" or converted to ammonium ions (NH4+) through the nitrogen cycle

nitrogen cycle

1. nitrogen is removed from air and converted to ammonia

2. Ammonia is converted to nitrate

3. Nitrogen is removed from nitrate and converted to nitrogen gas

nitrogen fixation

Bacteria can't use N2 alone they have to go through nitrogen fixation to form NH4+ (ammonium ions)

Binary fission

one parent cell splits into 2 equal daughter cells

-most bacteria grow by binary fission

budding in a planctomyces species

-form of asexual reproduction

-growing a smaller new cell (called a bud) off the side of the parent

-doesn't split in two equal halves like most bacteria

budding process

1. Bud formation

2. DNA transfer: parent DNA --> Bud

3. growth of the bud

4. separation: free independent daughter cell

Epulopiscium

a giant bacterium that has a viviparous life cycle

viviparous lifecycle

it gives birth to live offsprings

the Epulopiscium life cycle

1. epulopiscium forms one or two daughter cells inside its own cytoplasm

-- they grow while inside the other cell

-- mother cell nourishes the offspring

2. once the offsprings are fully developed, the mother cell dies and lyses (breaks open)

-- this releases the daughter cell into the environment (gut)

3. the process repeats

batch culture system

the growth curve of a bacterial culture is represented by the logarithm of the number of live cells plotted as a function of time the graph can be divided into four phases according to the slope, each of which matches events in the cell

-Lag

-log

-stationary

-death

lag phase

no increase in number of living bacterial cells

log

exponential increase in number of living bacterial cells

stationary

plateau in number of living bacterial cells; rate of cell division and death roughly equal

death

exponential decrease in number of living bacterial cells

exponential growth

continuous culture

in open systems where fresh medium is continually added to a culture and an equal amount of culture is constantly siphoned off, bacterial populations can be kept in exponential phase at a constant cell mass for long periods of time

-culture media is constantly added (feed) and constantly removed (effluent)

chemostat

a continuous culture system in which the diluting medium contains a growth-limiting amount of an essential nutrient

chemostat biofilms

Complex and dynamic ecosystems that common form on surfaces

biofilm growth occurs in 5 stages

1. Cellular flagella attach to the monolayer

2. Microcolonies form

3. Cells produce exopolysaccharides (EPSs)

4. The biofilm matures

5. The biofilm dissolves and cells disperse

biofilm

grow in continuous culture system while attached to a surface and their growth consists of 5 developmental stages

biofilm structure

-extracellular polymeric substances (EPS)

-channels

-micro-colonies

-streamers

physical requirements on microbial growth

-pH

-temperature

-moisture

-hydrostatic pressure

-osmotic pressure and radiation

chemical requirements on microbial growth

-availability of carbon

-nitrogen

-sulfur

-phosphorus

-trace elements

-organic compounds

-oxygen

classification of growth temperatures

-psychrophiles

-mesophiles

-thermophiles

-hyperthermophiles

psychrophiles

microorganisms that can grow at cold temperatures from -5℃-20℃

mesophiles

microorganisms that can grow in temperature from 15℃-45℃

theermophiles

microorganisms that can grow in temperature from 45℃-80℃

hyperthermophiles

microorganisms that can grow in temperatures from 65℃-105℃

psychrotrophs

microorganisms that can grow at cold temperatures 0-30° (best at 20-30°) which is not too different from psychrophiles which can grow at 0-20° (best at 10°)

temperature and growth rate

whether or not a cell can survive in a temperature depends on the enzymatic activity

-enzymatic activity is low at lower temps and high at optimal temps

barophiles (weight loving) or piezophiles (pressure loving)

organisms that have adapted to grow at high pressures

water activity

water availability is measured as water activity which is a quantity approximated by concentration

-Interactions with solutes (such as NaCl) lower water activity. The more solutes in a solution, the less water is available for microbes to use for growth

osmolarity

a measure of the number of solute molecules in a solution

-The more particles in a solution, the greater the osmolarity and the lower the water activity

-Most microorganisms are relatively tolerant of the external environment relative to the osmolarity or tonicity. Halophiles grow only in high salt concentrations

plasmolysis

hypertonic environments, or an increase in a salt or sugar, cause plasmolysis

halophiles

grow in high salt concentration

extreme or obligate halophiles

require high osmotic pressure

facultative halophiles

tolerate high osmotic pressure

hypersaline habitats

Hypersaline habitats for halophilic Archaea. Aerial view near San Francisco Bay, California, of a series of seawater evaporation ponds where solar salt is prepared. The red-purple color is predominantly due to bacterioruberin and bacteriorhodopsin in cells of Halobacterium

microbial responses to changes in pH

-pH influences growth by altering protein shape, which in turn changes protein activity

-Extreme concentrations of either hydrogen or hydroxide ions in solution will limit growth

-Concentration of hydrogen ions [H+] also have a direct effect on the cell's macromolecular structures

-Microbes have adapted to inhabit diverse pH environments from 0-11.5

strategies for pH homeostatis in bacterial

-Increased metabolic acid production through amino acid deaminases and sugar fermentation

-Increased ATP synthase that couples H+ entry to ATP generation

-Changes in cell surface properties

-Increased expression and acidity of monovalent cation/proton antiporters.

-Among these strategies, monovalent cation/proton antiporters play an essential and dominant role in alkaline pH homeostasis in many bacteria in addition to roles in Na+ and volume homeostasis

most bacteria grow..

between pH 6.5 and 7.5

molds and yeast grow ...

between pH 5 and 6

acidophiles

grow in acidic environments. they are often chemoheterotrophs that oxidize reduced metals and generate strong acids such as sulfuric acid

-grow below pH 3

alkaliphiles

occupy the opposite end of the pH spectrum, growing best at values ranging from pH 9 to pH 11

neutrophiles

bacteria that generally grow between pH 6 and pH 8 and include most humans pathogens

pH homeostasis mechanisms

-E.coli can reverse proton influx by importing a variety of cations such as K+ and Na+

-Under extremely alkaline conditions the cells can use Na+/H+ antiporters to recruit protons into the cell in exchange for expelling Na+

-Some organisms can change the pH of the medium by using various amino acid decarboxylases and deaminases, producing alkaline and acidic products, respectively

hot, acidic, and rich in sulfur

Habitats of hyperthermophilic archaea.

-Sulfur-rich hot spring, a habitat containing dense populations of Sulfolobus. The acidity in solfataras and sulfur springs comes from the oxidation of H2S and SO to H2SO4 (sulfuric acid) by Sulfolobus and related prokaryotes

facultative anaerobes

posses the enzymes to detoxify oxygen radicals and also the machinery for both fermentation and aerobic respiration

-growth with or without oxygen

strict aerobe

microorganism that grow only in oxygen

strict anaerobe

microorganisms that grow only without oxygen

aerotolerant anaerobes

use only fermentation to provide energy but contain superoxide dismutase and catalase or peroxidase to protein them from ROS

Microaerophiles

will grow only at low oxygen concentrations. they possess a decreased level of superoxide dismutase and/or catalase

- superxode dismutase and catalase are 2 enzymes required for a microorganism to be able to grow and survive under oxygenated conditions. so microaerophiles has low levels of these since they only grow in low oxygen

capnophiles

microorganisms that thrive in the presence of high concentrations of carbon dioxide. species of Campylobacter are bacterial capnophiles that are more easily identified because they are also microaerophiles, organisms that can grow in high carbon dioxide as long as a small amount of free oxygen is present but at a dramatically reduced concentration

problem with oxygen

-Oxygen is a powerful oxidant and can oxidize numerous chemicals in the cell, leading to cell injury or death

enzymes that protect the cell from toxic forms of oxygen

1. Catalase

2. Peroxide

3. Superoxide Dismutase

Dipicolinic acid (DPA)

-helps the endospore by:

1. removing water from core: makes the spore super dry, which protects proteins & DNA

2. stabilizes DNA which prevents damage from heat or radiation

3. increases resistance to chemicals, heat, & other environmental factors

agar

-complex polysaccharide

-used a solidifying agent for culture media

-in Petri plates, slants, and deeps

-generally not metabolized by microbes

-liquefies at 100°C

-solidifies at ~40°C

-agar itself has no nutritional function or value

selective media

Encourage the growth of certain organisms and discourage others (e.g. Media containing antibiotics)

-contains growth-inhibiting addictive, such as bite salts, crystal violet or an antibiotic, which will limit growth on the medium to only those organisms that are desired

MSA (mannitol-salt agar)

both selective and differential; selects for salt-tolerant organisms and differentiates mannitol fermenters from non-mannitol fermenters

selective media examples

1. Mannitol Salt agar: high concentration of salt (Is selective against Gram-negative and most Gram-positive bacteria)

2. MacConkey Agar: crystal violet, neutral red and bile salts and lactose (Is selective against Gram-positive organisms)

differential media

enable different species to be distinguished from each other (Mannitol Salt Medium)

Enrichment media

encourage the growth of a specific organism (brain Heart Infusion Medium)

Selective vs. Differential

-Selective medium: will have a growth-inhibiting addictive, such as bite salts, crystal violet or an antibiotic, which will limit growth on the medium to only those organisms that are desired

-Differential medium: allows for differentiation of particular chemical reaction yield an observable characteristic associated with the growth a particular organism or group of organisms that are able to grow on the medium

differential medium example

-blood agar

-beta-hemolysis

-alpha-hemolysis

-Y-hemolysis

blood agar

-Blood agar distinguishes bacterial species that can break down the red blood cells included in the blood agar medium. Typically used to differentiate between Streptococcus species

-allows for differentiation of particular chemical reaction yield an observable characteristic associated with the growth a particular organism or group of organisms that are able to grow on the medium

beta-hemolysis

complete lysis, resulting in a clear zone around the colony

-ex. Streptococcus pyogenes

Alpha-hemolysis

partial lysis, resulting in greenish coloration around the colony

-ex. Streptococcus pneumonia

Y-hemolysis

no lysis, no color change

-ex. Enterococcus faecalis

Winogradsky Column

In the latter part of the 19th century, Sergei Winogradsky (b1856-d1953), a Russian microbiologist developed an early form of enrichment culture to study interactions between soil microorganisms

Top layer of Winogradsky Column

-Aerobic & light

-common microbes: algae, cyanobacteria

-- aerobic heterotrophs

Upper middle layer of Winogradsky Column

-microaerophilic

-common microbes: purple non-sulfur bacteria

-- photoheterotrophs

Lower middle layer of Winogradsky Column

-anaerobic & light

-common microbes: green sulfur bacteria, purple sulfur bacteria

bottom layer of Winogradsky Column

-anaerobic & dark

-common microbes: sulfate reducers

-- fermentative heterotrophs

pure cultures

one bacterium gives rises to one colony

enumeration of bacterial cells

spread-plate method and pour-plate method

spread-plate method

pour-plate method

Serial dilutions

CFU/mL= (number of colonies per volume plated in μL) (1000μL/volume plated) (1/dilution factor)

example of serial dilutions

-CFU/mL= (389)(1000μL/100μL)(1/(10^-3)= 3891010^3= 3.89*10^6

-CFU/mL= (2)(1000/100)(1/10^-5)= 21010^5=2000000= 2*10^6

-CFU/mL= (50)(1000/100)(10^4)= 5*10^5

-the average CFU/mL is the average of the 3 plates

-between 30 to 300 colonies is good

colony

1 colony is a population of bacterial cells from the same mother cell, thus they are genetically identical

total cell count

-accounts for all cells, regardless of viability and culturability

-may not accurately represent potential for infection or food spoilage

total cell count importance

-useful for monitoring overall microbial load in a sample

viable cell count

-accounts only for cells capable of reproducing and forming colonies

viable cell count importance

-crucial for assessing food safety, water safety and pathogenicity

-used to evaluate the effectiveness of sterilization and antimicrobial treatments

counting chambers

fluorescence labelling

-live/dead stain

-propidium iodide

-syto 9 green

propidium iodide

is unable to cross the cell membrane of intact/live cells. But can stain the nucleic acid of lysed cells red

Syto 9 green

is membrane permeable and will bind both live and dead cells

-Live cells are stained green, dead cells are stained red

Enumeration of bacterial cells

direct counting without a microscope

Fluorescence-activated cell sorter (FACS)

enumeration can be accomplished with an electronic instrument called FACS, which can count and separate bacterial cells with different properties

-used for viable cell count

turbidity

-Population can be calculated by measuring the turbidity of a cell culture. The degree to which the liquid medium has become cloudy because of microbial growth

-can be measured in real time using a spectrophotometer, which passes a beam of light through a sample of the culture