Micro Exam 2

macronutrients & micronutrients

carbon, nitrogen, phosphorus, sulfur, oxygen and hydrogen
--> potassium, sodium, magnesium, calcium, iron

micronutrients (trace elements)
-> Mn, Zn, Co, Mo, Ni, and Cu

C, H, O, N, S

C, H, O, P

C, H, O

C, H, O, N, P

Carbon, hydrogen, oxygen, and electrons

carbon

electrons

assimilate carbon from inorganic sources

assimilate carbon in preexisitng organic form

autotrophs & heterotrophs

phototrophs and chemotrophs

capture light energy to produce ATP

capture energy from oxidation of reduced organic or inorgranic compounds

organotrophs & lithotrophs

acquire their electrons from organic molecules

"rock-eaters" acquire their electrons from inorganic sources

some organisms have great metabolic flexibility and alter their metabolism in response to environmental changes
*can be advantageous

amino acids, purines, pyrimidines, some carbs, lipids, enzyme cofactors etc.

metabolism of amino acids, nitrates, ammonia, from atmosphere
*nitrogen fixation

nucleic acids, phospholipids, coenzymes, some proteins

inorganic phosphate
organic molecules containing a phosphoryl group

amino acids, coenzymes

aminio acids: cysteine and methonine

sulfate to sulfite to hydrogen sulfite

organic compounds that cannot be synthesized by an organism but are essential for growth

amino acids: protein synthesis
purines/pyrmidines: nucleic acids synthesis
vitamins: enzyme cofactors, needed in small amounts

growth rate depends on the amounts of nutrients in the environment
*one key nutrient, available in the lowest amount, will dictate how much growth can occur over time

aerobes

obligate aerobes REQUIRE oxygen
*microaerophiles grow best when there is less oxygen than normal

aren't harmed by oxygen but don't use it, either

cannot grow when oxygen is present

CAN use oxygen but can also grow in the absence of oxygen

often determined by what defenses are available against oxygen's negative effects in the cell

oxygen easily reduced to toxic reactive oxygen species (ROS)

superoxide dismutase, catalase, peroxidase

superoxide dismutase, catalase

work station with incubator
gaspak anaerobic system

incubator, work gloves, airlock for transferring materials into and out of the chamber

*catalyst chamber (contains palladium pellets)
*gas generator envelop (water is added to chemicals in envelope to generate H2 and CO2, carbon dioxide promotes more rapid growth of microorganisms
*anaerobic indicator strip (methylene blue becomes colorless in absence of O2)

microbes have an optimal pH range for growth
* acidophiles = pH < 5.5
* neutrophiles = pH 5.5 to 8.5
* alkalophiles = pH > 8.5

higher osmotic concentration outside
* water enters the cell
* cell swells may burst

only open when pressure is on it, "relief pressure" relieve pressure

lower osmotic concentration outside
* water leaves the cell
* membrane shrinks from the cell wall (plasmolysis) may occur

grow optimally in the presence of NaCl or other salts at a concentration above about 0.2M

require salt concentrations of 2M and 6.2M

molecules that can be kept at high internal concentration

require > 2M, found in ocean, great lake, dead see

amount of water available to organisms

1.0

0.97

0.5

can grow over wide ranges of water activity
ex -> Staphylococsus aeureus 3M NaCl

archaea

0° to 20° C

0° to 35° C

they can grow in cold temperatures, like arctic, food in fridge may not be killed

20° to 45°C

55° to 85°C

85° to 113°C

volcanic vents in ocean, compost piles, hot waterlines

-protein structure stabilized by a variety of means
- histone-like proteins stabilize DNA
- membrane stabilized by variety of means
---e.g., ether linkages (archaeal membranes)

help bind things that can't sustain heat

DNA doesn't contain histones, proteins bind to paired strands

manipulate saturation of carbon chains
-- fully saturated: no double bonds
--all chains fully saturated
--at low temps: increase in saturation, more double bond

solid or liquid preparation
**grow micro-, store, and transport

depend on the organism being cultured
* growth factors *

chemical constituents(what theyre made of), physical nature(solid or liquid), function

1: powdered agar is added to a flask containing liquid media to a final concentration of 1.5g/100 mL (1.5%)
2: the medium is heated to above 85° to melt the agar
3: the medium is allowed to cool around to 45°C, then poured into a plate and allowed to solidify

supportive (general purpose), enriched, selective, differential

dont know whats in it

can identify

protein hydrolysates prepared by partial digestion of various protein sources

aqueous extracts, usually of beef or yeast

sulfated polysaccharide used to solidify liquid media; most microorganisms cannot degrade it - usually extracted from red algae

nutrient broth, tryptic soy broth, MacConkey Agar

supportive, enriched, selective, differential

grow majority bacteria

to grow vistidious m/o, picky organisms
* supply & nutrients

blood agar and chocolate agar

enriched and differential
RBCs

RBCs, but heated
*enriched

favor the growth of some microorganisms and inhibit the growth of others

distinguish between different groups of microorganisms based on their biological characteristics: biochemical / physiological pathway

ferments lactose
** show pink on MacConkey Agar

Doesn't ferment lactose
** no color change on MacConkey Agar

selective and differential

* solid medium-cells are held in place on the surface and can be isolated

streak plate method, spread plate method, pour plate method

spreading mixture of cells on agar surface so that individual cells are well separated from each other
* involves use of bacteriological loop
(T STREAK)

take diluted bacterial suspension and spread on solid agar medium, incubate, bacterial colonies on surface

diluted bacterial suspension into molten agar medium, vortex tube, pour liquid agar into plate, incubate, bacterial colonies grow on agar & in agar

not the right environment, can't replicate conditions

DNA from unculturable bacteria can be amplified and sequenced by PCR
* sequences can be used to produce fluorescent probes that will bind to complementary DNA (fluorescent in situ hybridization or FISH)

1: Fix cells in place and permeabilize
2: Add fluorescently labeled probes, then wash
3: View using on epifluorescence microscope

-DNA is isolated from an environmental sample and sequenced
-Metagenomic information must still be confirmed in cultured organisms

direct counts, viable (living) cell counting, turbidity (cloudiness) measurements

counting cells

count living cells colonies, only cells that can replicate are alive

the density of suspended particles in the water

1: add a drop or two of bacterial suspension to the center of the chamber
2: lower a cover slip over the grid. The suspension spreads out evenly and excess flows into side channels.
3: use microscope to count the cells in the grid

-microbial suspension forced through small orifice with a laser light beam
-movement of microbe through orifice impacts electric current that flows through orifice
-instances of disruption of current are counted
-specific antibodies can be used to determine size and internal complexity

colony forming units (CFU)

(# of colonies)/(dilution factor)(volume plated)

filter apparatus can concentrate the cells

a spectrophotometer sends light through a culture
-- it can give a rough measure of cell density in the tube

- dry weight
- quantity of a particular cell constituent

time consuming and not very sensitive
--dump supernate, lose cells, weigh pellet
----lost mass

e.g., protein, DNA, ATP, or chlorophyll
useful if amount of substance in each cell is constant

refers to population growth rather than growth of individual cells

observed when microorganisms are cultivated in batch culture

=closed system
--inoculated media and then closed
--nothing else is out inside

lag, log, stationary, death

cell synthesizing new components
-- to replenish spent materials
--to adapt to new medium or other conditions

rate of growth and division is constant and maximal
--population is most uniform
--population double

closed system (i.e., batch culture)
--population growth eventually ceases

nutrient limitation
--limited oxygen availability
--toxic waste accumulation

cell division stopping but cells aren't dying
-cell division rate = cell death

morphological changes

endospore formation
decreasing in size

assists RNA polymerase in transcribing genes for starvation proteins
**used to stabilize maacromolecules

number of viable cells declines exponentially
--inverse of exponential phase
--dying in discrete time intervals

log or exponential phase, that is when they are most similar to each other
-- once in stationary phase, there is differentiation which adds limits

1: cells are viable but not culturable (VBNC)
2: programmed cell death

cells alive, but dormant, capable of new growth when conditions are right

fraction of the population genetically programmed to die (commit suicide)

cellular constituents manufactured at constant rates relative to each other
--> phase of balanced growth on graph
--> cell has everything it needs to produce molecules at efficient rate

rates of synthesis of cell components vary relative to each other
-- responding specifically to changes in the environment, adapting

change in nutrient levels
demonstrates that microbial grwoth is under precise, coordinated control and that organisms respond quickly to changes in environmental conditions

the time to double the population in the exponential phase
--"doubling phase"

number of generationsnit of time (inverse of the generation time)

the maximum population density and/or amount of cellular material produced by the culture

--time required for the population to double size
--varies depending on species of microorganism and environmental conditions
--10 min for some bacteria to several days for some eukaryotic microorganisms

population is doubling every generation

-growth in an open system-> allowing micro. to come in & out as we grow our micro
-maintains cells in log phase at a constant biomass concentration for extended
-achieved using a continuous culture system

- study of microbial growth at very low nutrient concentrations
- study of interactions of microbes under conditions resembling those in aquatic environments
- food and industrial microbiology

rate of removal of medium from vessel

flows in fresh medium and takes out some old medium / microbes to keep the culture in continuous operation
-- keeps organisms in log phase by keeping one nutrient in limiting quantities, growth stays steady

rate at which medium flows through vessel relative to vessel size

when dilution is too high, cells run

regulates the flow rate of media through vessel to maintain a predetermined turbidity

continuous culture system
- dilution rate varies
-no limiting nutrient
-operates best at high dilution rates

destruction or removal of all viable organisms
sterilants are a chemical agent

killing, inhibition, or removal of disease causing organisms
-does not kill everything, focuses on pathogens

reduction of microbial population

destruction or inhibition of microorganisms on living tissue
-not killing anything but lowering chance of infection

use of chemicals to kill or inhibit growth of microorganisms within host tissue
living tissue

agents that kill microorganisms or inhibit their growth

kill
ex: sporecidal, bacterialcidal

inhibit growth
-do not directly kill but prevent replication

are not killed instantly
population death usually occurs exponentially

decimal-reduction time (D-value) : time to kill 90% of microorganisms and spores in a sample under specific conditions
---lower D value = higher efficiency

population size, population comp. , concentration of agent, contact time, temperature, local environment

larger populations take longer to kill than smaller populations

microorganisms differ markedly in their sensitivity to antimicrobial agents
--easier to kill younfer cells

usually higher concentrations or intensities kill more rapidly
-relationship is not linear

longer exposure - more organisms killed

higher temperatures usually increase amount of killing
--increasing motion so can kill faster

many factors (e.g. pH, viscosity and concentration of organ matter) can profoundly impact effectiveness
--prevents cell interaction

filtration, heat, radiation

physical removal of microbes by filtration
-method for purifying liquids for centuries
-lab applications use membrane nylon / Teflon filters with a pore size of 0.2 or .45um
-viruses can be removed from liquids by ultrafiltration methods

large particles clog filters, ultrafiltration requires high pressure, viscous liquids don't filter well

removes microorganisms from heat sensitive liquids

removes 95% of particles 0.3um or larger

-used in laminar flow biological safety cabinets
-removes 99.97% of particles 0.3 um or larger

used so you do not inhale viruses or microorgs ; uses physical & electrostatic interactions

protein and nucleic acids disrupted
boiling: disinfecting -you do not kill spores
steam sterilizing: steam under pressure kills microorganisms & spores

-can damage cells by forming ice crystals
----use buffer to prevent the formation of crystals -> pop cell membrane

-must be carried out above 100° which requires saturated steam under pressure
-carried out using an autoclave
-effective against all types types of microorganisms including spores

Charles Chamberland - 1884

*plastic that can melt
*metal that can rust
*antibiotics
*protein solution

John Tyndall

intermittent sterilization
-30 to 60 min of steam repeated 3 times with 23-24 hr incubations between
- used to kill spores: spores germinate and are then killed by the steam treatment

controlled heating at temperatures well below boiling (55 - 60 C)
-used for milk, beer, and other beverages
-process does not sterilize but does kill pathogens present and slows spoilage by reducing the total load of organisms present
-introduced to US by 1889

less effective than moist heat sterilization, requiring higher temperatures, and longer exposure items
-oxidizes cell constituents and denatures proteins
--could put metal in autoclave, but it may rush and get damaged

bench top incinerators are used to sterilize inoculating loops used in microbiology laboratories

using electromagnetic radiation to control microbes
- UV and GAMMA differ in energy they are projecting
- UV -alpha & beta rays, lower energy
- UV is disinfection, does not kill, not sterilization

260 to 280 nm

gamma radiation penetrates deep into objects (sterilizing, spores cannot stand up to it, kills everything)
-destroys bacterial endospores; not always effective against viruses
-used for sterilization and pasteurization of antibiotics, hormones, sutures, plastic disposable supplies, and food

when using food, cobalt 60 not a lot of neutrons so won't make food radioactive
--- put on convayer belt and pass it under radiation

disinfection, antisepsis, sterilization

chemical used on non-living surfaces to kill pathogenic microbes

chemicals that can be used on living tissue to kill pathogenic

-should kill a wide-range of microbes
-shouldn't be corrosive or overly toxic
- shouldn't leave a residue
- shouldn't emit fumes or smell TOO bad
- should be cheap
- should be temp stable

routinely used in lab setting ; also used present in most hand sanitizers

added to numerous products, including some soaps, deodorants, and cosmetics

commonly added to swimming pools and hot tubs to inhibit microbial growth
"major ingredient in Lysol"

no

phenol was 1st widely used antiseptic and disinfectant, used in 1867
-commonly used as laboratory and hospital disinfectants
-act by denaturing proteins and disrupting cell membrane
-tuberculocidal, effective in presence or organic material, and long lasting
- disagreeable odor and can cause skin irritation

-commonly used agents are formaldehyde and glutaraldehyde
-highly reactive molecules
-sporicidal and can be used as chemical sterilants
- combines with and inactive nucleic acids and protein

among the most widely used disinfectants and antiseptics
-two common are ethanol and isopropanol (60-80%)
- not sporicidal
- inactivate some viruses
- denature proteins and possibly dissolve membrane lipids (proteins lose structure, lose function)

-skin antiseptic
-oxidizes cell constituents and iodinates proteins
-at high concentrations kills spores
-skin damage, staining, and allergies can be a problem
-iodophore

-oxidizes cell constituents
-destroys vegetative bacteria and fungi
-chlorine gas is sporicidal
- can react with organic matter to form carcinogenic compounds

a disinfectant

effective but usually toxic
- combine with and inactivate proteins ; may also precipitate proteins

-amphipathic organic cleansing agents
-act as wettings agents and emulsifiers
-disrupt membranes and denature proteins
-kill most bacteria, but not Mycobacterium tuberculosis or endospores
-safe and easy to use, but inactivated by hard water and soap

-easier to get things into gram-positive than it is to get into gram-negative
--look for zone of inhibition

-used to sterilize heat-sensitive materials like plastics
-microbiocidal and spoicidal
-ethylene oxide sterilization is carried out in equipment resembling an autoclave
-EtO is a very strong alkylating agent
-betapropiolactone and vaporized hydrogen peroxide