Micro 4

Robert Koch

• process by which prokaryotic cells multiply
• After cell increase in size, DNA replicates
• DNA moved into each future daughter cell, cross wall forms
• Cell divides into two cells
• cells separate

• the time it takes for a population of cells to double in number
• Varies greatly from species to species, influenced by conditions in which cells are grown

• N_t = N₀ x 2ⁿ
• N_t = # of cells in population at a given time
• N₀ = original number of cells in population
• n  = number of division those cells have undergone during that time

• polymer-encased communities of microorganisms in aqueous environments
• cause the slipperiness of rocks in stream beds, slimy gunk on kitchen drains, and dental plaque on gums

• Begins when planktonic (free-floating) bacteria move to a surface and adhere
• Bacteria multiply and produce extracellular polymeric substances (is mesh like accumulation of polymers, gives biofilm it's slimy look)
• Other bacteria may attach to the EPS and grow
• Cells communicate and create channels in the EPS that allow nutrients and waste products to pass
• Some cells detach and then move to other surfaces to create additional biofilms

• Dental plaque leads to tooth decay and gum disease
• persistent ear infections
• complications with cystic fibrosis
• majority of bacterial infections
• Tx is difficult as microbes with protective EPS often resistant to antibiotics as well as body's defenses
• Also resistant to disinfectants

• Prokaryotes in environment regularly grow in close association with many diff species
• Some interactions are cooperative, even fostering growth of species that otherwise couldn't survive
• Ex: organisms that cant multiply in presence of O2 will grow in mouth if neighboring microbial cells consume that gas; one species creates a microenvironment in which the other can thrive
• Metabolic wastes of one can be nutrients for another

• a population descended from a single cell and therefore separated from all other species
• Working with pure cultures makes it easier to ID and study activities of a particular species
• However, organisms in lab do behave different than in nature

• procedures that minimize the chance of other organisms being accidentally introduced
• Medium cells are grown in is a culture medium
• Consists of nutrients dissolved in water, can be liquid broth or solid gel

• distinct mass of cells
• about 1 million cells are required for a colony to be easily visible to the naked eye

• a polysaccharide extracted from marine algae
• Used to solidify culture media
• Unlike gelatin and other gelling agents, very few microbes can degrade agar
• not destroyed at high temps so can be sterilized by heating

another name for petri dish

• simplest and most commonly used technique for isolating prokaryotes
• Sterile loop is dipped in sample, then lightly drawn several times across surface of agar, creating set of parallel streaks covering appr 1/3 of agar 
• Loop sterilized, new series of parallel streaks made across at angle from previous ones, covering another surface. This drags some cells from first portion over fresh portion.
• Loop sterilized, another set of parallel streaks are made, etc
• By third set, cells should be separated enough so that distinct, well-isolated colonies will form

Stock cultures can be stored on agar slants in the fridge, frozen or freeze-dried

• also called closed systems because nutrients are not renewed, nor are wastes removed
• As cells grow in this type of system, population increases in distinct pattern of stages and then declines
• Characteristic pattern is called Growth Curve

• Also called open system
• nutrients are added continuously and waste products removed to maintain cells in a state of continuous growth

• characterized by 5 distinct stages:
• Lag Phase
• Exponential Phase (Log phase)
• Stationary Phase
• Death Phase

• First phase of growth curve
• When a dilute culture is transferred into a different medium, cell # don't immediately increase. 
• This is the "lag phase," where cells begin to synthesize enzymes required for growing
• Length of lag phase depends on conditions in original culture and new medium
• If cells are transferred into a medium with fewer nutrients, lag phase will be longer

• Also called Log phase
• cells divide at a constant rate
• this is when generation time is measured
• Medically important as bacteria are most sensitive to antimicrobial meds at this stage
• Also important phase to commercial standpoint, as some molecules made by growing cells are valuable. Ex: amino acids, ethanol, etc

• the small molecules made by cells as they multiply during log phase
• *Occurs during active multiplication

• Compounds that are formed in the later stages of exponential growth
• As nutrients gradually become depleted and waste products accumulate, cells activities shift
• Compounds that begin to accumulate are made for purposes other than growth
• Commercially, the most valuable of these are antibiotics

• Cells enter this stage when nutrient levels are too low to sustain growth
• Total number remains relatively constant; some cells are dying while others are multiplying
• Dead cells often burst, releasing nutrients that then fuel the growth of other cells

• the period when the total number of viable cells in the population decreases as cells die off at a constant rate
• Like cell growth, death is exponential, but usually much slower

• In many cases, a fraction of cell population survives the death phase by using nutrients released from dead cells
• Most survivors die

• a device which continually drips fresh medium into a broth culture contained in a chamber
• Keeps microbial cells in a state of continuous growth
• Makes it possible to study a uniform populations response to different nutrient concentration or environmental conditions

• phile means loving,
• are in the domain Archaea

Microbes that live in harsh environments that would kill most other organisms

• temp - thermalstability appears to be due to protein structure
• atmosphere
• pH
• water availability

• Psychrophiles
• Psychrotrophs
• Mesophiles
• Thermophile
• Hyperthermophile

• "Phycho" likes cold
• Optimum btwn -5° and 15° C
• Grow in cold Arctic and Antarctic regions and in lakes fed by glaciers

• The next "phycho" that likes cold
• Optimum btwn 20° and 30° C, but will grow at lower temps
• Important cause of spoilage in refrigerated foods

• Optimum btwn 25° and 45° C
• E. Coli and most common bacteria are in this group
• Pathogens, adapted to growth in human body, typically btwn 35 & 40°C
• Mesophiles that inhabit soil, a colder environment, generally have a lower optimum, close to 30°

• Optimum btwn 45° and 70° C
• Commonly live in hot springs and compost heaps

• Optimum of 70° C or greater
• Usually members of Archaea

Fridge timps (approx 4° C) slow spoilage as they limit multiplication of otherwise fast-growing mesophiles

• Obligate aerobe
• Facultative anaerobe
• Obligate anaerobe
• Microaerophile
• Aerotolerant anaerobe

• Grows only when O2 is available
• Requires O2 for respiration (an energy-harvesting process)
• Protects against ROS: Produces superoxide dismutase and catalase
• *Will grow at top of tube
• Ex: Micrococcus luteus, common in environment

• "Facultative" means the organism is flexible
• Grows best when O2 is available, but also grows w/o it
• Uses O2 for respiration, if available; but resort to alternative types of metabolism if it is not
• Produces superoxide dismutase and catalase (against ROS)
• E. coli is most common facultative anaerobes in large intestine
• *Will grow throughout tube, but likes top

• Cant grow when O2 is present; in fact, they are killed by even brief exposure to air
• Does not produce superoxide dismutase or catalase
• Will grow at bottom of tube
• Ex: most inhabitant of large intestine are obligate anaerobes, as is the bacteria that causes botulism - Clostridium botulinum

• Requires SMALL amounts of O2 (2% to 10%) for aerobic respiration
• Requires O2 for respiration
• Produces some superoxide dismutase and catalase
• Grows NEAR top, but not on top of tube
• Ex: H. pylori which causes gastric and duodenal ulcers

• Grows equally well with or without O2
• Do not use O2 to harvest energy. 
• Also called obligate fermenters, as fermentation is their only metabolic option
• Ex: Streptococcus pyogenes, which causes strep throat

• Reactive oxygen species
• harmful derivatives the form as by-products when organisms use O2 in aerobic respiration
• Examples are superoxide and hydrogen peroxide
• Can damage cell components, so cells must have mechanisms to protect against them
• *Obligate anaerobes typically do not have these mechanisms, but there are exceptions

• An enzyme which inactivates superoxide by converting it to O2 and hydrogen peroxide
• Virtually all organisms that grow in presence of O2 produce it

• nearly all organisms that grow in presence of O2 and produce superoxide dismutase also produce catalase as well
• Converts hydrogen peroxide into O2 and water
• *important exception is aerotolerant anaerobes- don't produce catalase

• Includes most microbes 
• They live and multiply within the range of pH5 (acidic) to pH8 (basic), and have a pH optimum near neutral pH7
• Food preservation methods (ex: pickling) inhibit growth by increasing acidity of food

• H. pylori
• grows in stomach, sometimes causing ulcers
• Decreases the acidity of it's surroundings by producing urease, which is an enzyme that splits urea into carbon dioxide and ammonia
• The ammonia neutralizes any stomach acid surrounding the cell

• Grow optimally at pH below 5.5
• Ex: P. oshimae (member of Archaea) has optimum pH of less than 1. found in Japan in acid soils of gas-emitting volcanic fissure

• Grow optimally at a pH above 8.5
• Often live in alkaline lakes and soils

• Phenomenon where solute concentration (whether sugar or salt) is higher in medium outside of cell than in the cell
• This causes water to diffuse out of cell due to osmosis
• Causes cytoplams to dehydrate and shrink from the cell wall

• microbes that tolerate high concentrations of salt, up to approx 10% NaCl
• Halo means salt
• Ex: Staphylococcus species, which live on dry salty environment of skin

• Microbes that require high levels of sodium chloride
• Ex: marine bacteria

• Includes certain member of the Archaea that require 9% sodium chloride or more
• found in environments such as salt flats of utah and dead sea

• Carbon, oxygen, and hydrogen: component of amino acids, lipids, nucleic acids, and sugars
• Nitrogen: component of amino acids and nucleic acids
• Sulfur: Component of some amino acids
• Phosphorus: Component of nucleic acids, membrane lipids, and ATP
• Potassium, magnesium, calcium: Required for functioning of certain enzymes; additional functions as well
• Iron: part of certain enzymes

• Heterotrophs
• Autotrophs

• use organic carbon
• hetero means different
• troph means nourishment

• use inorganic carbon in the form of carbon dioxide
• auto means self

• converting inorganic carbon to an organic form
• Done by autotrophs
• Critical to life, as earth would quickly run out of organic carbon w/o it

• unique to prokaryotes
• Some prokaryotes use nitrogen gas (N2) as nitrogen source, converting it to ammonia and then incorporating that into cellular material
• Also essential to life, as once converted other organisms can use it as nitrogen source too

• they are available at the lowest concentration relative to need
• *think of cookbook example. One ingredient would dictate number of batches of cookies you could make

• required in such small amounts that most natural enviroments, including water, have sufficent levels to support microbial growth
• Includes cobalt, zinc, copper, molybdenum, and manganese

*Tracy mostly comes making Zoey crazy.

• Refers to certain molecules, such as amino acids, vitamins, purines, or pyrimidines, that a microbe CAN NOT synthesize
• Therefore, the microbe only grows if the molecule is available in the surrounding environment

• refers to microbes that have complicated nutritional requirements
• Used to measure quantity of vitamins in food products

Phototrophs obtain energy from sunlight. Include plants, algae, and photosynthetic bacteria

Chemotrophs extract energy from chemical compounds. Include mammalian cells, fungi, and other types of prokaryotes

• Photoautotroph
• Photoheterotroph
• Chemolithoautotroph
• Chemoorganoheterotroph

• Use sunlight for energy along with CO2 in atmosphere to make organic compounds
• Are primary producers, meaning they support other forms of life by fixing carbon

• Use sunlight for energy
• Derive carbon from organic compounds
• Some are facultative in nutritional capabilities

• lith means rock, auto means self
• use inorganic compounds for energy and derive their carbon from CO2
• live in seemingly inhospitable places like sulfur hot springs

• Use organic compounds for energy source (sugars, amino acids, etc)
• Also uses organic compounds for carbon

• contains a variety of ingredients such as meat juices and digested proteins, forming a tasty soup for microbes
• Easy to make and used for routine purposes
• Common ingredient is peptone, a mix of amino acids and short peptides produced by digesting proteins

• Complex medium used routinely in clinical labs
• Differential because colonies of hemolytic organisms are surrounded by a zone of red blood cell clearing 
• not selective

• complex medium used to culture fastidious bacteria, particularly those found in clinical specimens
• Not selective or differential

• chemically defined medium
• used in lab experiments to study nutritional requirements of bacteria
• Not selective of differential

• Complex medium used to isolate Gram-neg rods that typically reside in the intestine
• Selective cause bile salts and dyes inhibit gram pos organisms and gram neg cocci
• Differential cause the pH indicator turns pink-red when sugar in medium, lactose, is fermented

• complex medium used for routine lab work
• Supports growth of variety of nonfastidious bacteria 
• not selective or differential

• Complex medium used to isolate Neisseria species, which are fastidious
• Selective because it contains antibiotics that inhibit most organisms except Neisseria species
• Not differential

• composed of exact amts of pure chemicals
• type of medium generally used only for specific research experiments when type and quantity of nutrients must be precisely controlled

• Inhibits the growth of certain species, making it easier to isolate the one being sought
• Ex: Thayer-Martin, MacConkey agar

• Contains substances that certain microbes change in a recognizable way
• Ex: Blood agar, MacConkey agar,

• Refers to using a blood agar plate
• In beta hemolysis, produces a clear zone of hemolysis
• In alpha hemolysis, colonies are surrounded by a zone of greenish partial clearing

• Aerobic: Most can be incubated in open air. If need additional CO2, can use candle jar.
• Microaerophilic: use gastight container
• Anaerobic: use anaerobe container anaerobic chamber (an enclosed compartment maintained as anaerobic envir)

used to isolate an organism present as only

• Used to determine total # of cells, included living and dead
• Includes: 
• Direct microscope count
• Cell-counting instruments

• One of most rapid methods of determining cell concentration
• Liquid specimen added to glass slide with special grid, use microscope
• at least 10 million (10⁷) must be present

• Coulter counters (counts cells through narrow tube) and flow cytometers count total cells in dilute solutions
• Flow cytometers used to count fluorescent dyes/tags

• used to determine # of viable microbes in sample, but # includes only those that can grow in given conditions
• Requires incubation period of approx 24 hr or longer
• Selective & differential media can be used
• Includes:
• Plate count
• Membrane filtration
• Most probable number

• Time consuming but technically simple not requiring sophisticated equipment
• Ideal # on plate btn 30 & 300
• #'s outside of range inaccurate
• 2 techniques used: spread-plate method & pour plate method
• Can then figure CFU (colony-forming units)

• colony-forming units
• used to measure viable cells in plate count
• When calculating, 3 things must be considered:
• # of colonies
• amount the sample was diluted before plated
• volume plated

• used for liquid samples that contain relatively few cells, as might occur in dilute environments as natural waters
• concentrated microbes by filtration before plated

• not precise measurement
• method for estimating concentration of cells in a specimen
• uses a series of dilutions to determine the point at which subsequent dilutions receive no cells
• 3 sets of 3 or 5 tubes prepared, each set received measured amt of sample
• *second set received 10 fold less than first, third 100 fold less than 2nd...

instead of measuring the number of cells, the cell mass is determined

• the cloudiness of a microbial suspension is proportional to the concentration of cells
• measured with spectrophotometer
• Instrument shines light through specimen and measure % that reaches light detector

can be used to measure growth of a culture, but method is tedious and time consuming

• Products including acid, gas, and ATP can indicate growth
• Acid can be detects by pH indicators in culture medium
• Gas production can be detected by gas bubbles in broth