Unit 1 part 2
kreb's citric acid cycle
-2 atp via slp -pyruvate acid starts process -nad+ and turns nadh (pick up hydrogen which means they pick up electrons) -
How do you do a dilution series and apply that to plate counts and spectrophotometry
turbidity, light can be measured by using a spectrophotometer, the more bacteria the less light you can see
list and describe 11 chemical methods of microbial control (antiseptics/disinfectants, use 3 types of surface-active agents as part of the 11 & dont include food preservatives) give their mode of action and their applications
1)phenol & phenolics- phenolics- haven been altered to reduce irritating qualities or increase antimicrobial activity when combined w/detergents; injures plasma membranes and may denature protein bisphenols- includes triclosan and hexachlorophene, used in soaps, toothpaste, & in plastic kitchenware 2)Biguanides(chlorhexidine)-disruption of plasma membrane, best for skin disinfection(surgical scrubs) 3)halogens- iodine & chlorine, used alone or as components iodine-inhibits protein function & strong oxidizing agent; effective antiseptic chlorine-forms strong oxidizing agent, hypochlorous acid which alters cellular components; gas can disinfect water & chlorine compounds are used to disinfect dairy equipment, eating utensils, household items & glassware 4)alcohols- protein denaturation & lipid dissolution, more degerming(wiping away w/alcohol swab) of dirt & some microbes than sterilization, not effective on endospores ex: ethly alcohol & isopropanol 5) heavy metals and their compounds-very small amounts can kill or inhibit growth, denatures enzymes & other essential proteins ex: silver nitrate(used on burns or eye drops for newborns) , copper sulfate & mercuric chloride 6)surface active agents- soaps & detergents (removal thru scrubbing) does not kill but removes germs acid-anionic sanitizers- may involve enzyme inactivation & disruption; good for sanitizing dairy & food-processing industries quats- enzyme inhibition, protein denaturation & disruption of plasma membranes; charge gives quats their cleansing ability;most widely used surfactants, kill gram + bacteria, fungi, amoebas & enveloped viruses but does not kill endospores or mycobacteria 7)aldehydes-some of the most effective antimicrobials that will denature proteins ex: formaldehyde(preserves biological specimens) poisonous if consumed glutaraldehyde(less irritating & more effective) used to disinfect hospital equipment 8)chemical sterilization- ethylene oxide(very toxic & explosive with O2) kills all microbes & endospores plasma sterilization-inhibits vital cellular functions; has to be used in closed chamber over several hours; useful for tubular medical instruments (catheters) ex: hydrogen peroxide excited in vacumn by electromagnetic field supercritical fluids- inhibits cellular functions; useful for sterilizing organic medical implants ex: carbon dioxide compressed to a supercritical state 9)peroxygens & other forms of oxygen-works thru oxidation hydrogen peroxide- sporicidal at high concentration peracetic acid- one of most effective liquid chemical sporicide, viruses withing 30 min
Describe the streak plate method for the isolation of bacteria
take broth/sample and put on field #1 -Sterilize loop & wait for it to cool -streak across #1 and drag to #2
Chemical and physical conditions necessary for bacterial growth
temp- most bacteria only grow withing a limited range of temp & grow poorly at high/low extremes ph- grow best in a narrow ph closer to neutral, 6.5-7.5 osmotic pressure- high osmotic pressure can remove necessary water form bacterial cells,causing plasmolysis. low osmotic pressure can cause too much water to enter the cell resulting in swelling & lyses. carbon- all organisms require carbon nitrogen- needed for protein & nucleic acid syntheis oxygen- harmful to some bacteria, but some others require it or tolerate it, others use o2 untill available then switch to energy producing pathways that do not require oxygen other chemicals- sulfur, phosphorus, trace elements & for some microorganisms
glycolysis
the breakdown of glucose by enzymes, releasing energy and pyruvic acid. -creates 2 atp via slp thru energy that is realease -1st step of cellular respiration
disinfection
the process of reducing or inhibiting microbial growth on a nonliving surface ex: bleach
differentiate between antiseptics and disinfectants
antiseptics- CAN be used on skin disinfectants- clean inanimate objects
identify those chemicals that may be used in surgical hand scrubs
biguanides(chlorhexidine)
anabolism
biosynthetic & endergonic -some are dehydration (water output) -we are building/add -water is a product
How do you get actual numbers from indirect methods?
by using a standard curve
What is Pure culture and its significance
culture that contains only one organism -usually obtained by streak plate method
identify the concentration of ethyl alcohol that is the most effective against bacteria. Is ethyl alcohol a good wound antiseptic?
no bc proteins coagulate & form a protective coat around bacteria -70% most effective for denaturation(most market products will be around 70%) -
feedback inhibition
normal mechanism cells use to control amount of product in a complex metabolic pathway -cycle where the end product of an enzymatic chemical reaction becomes the noncompetitive inhibitor of the enzyme that catalyzed the same reaction. -if 1 enzyme changes cant keep going -final product acts as temporary inhibitor -it is reversible/turn off
describe how to kill endospores
autoclave- 15 psi of pressure (121 C) endospores in 15 min.
sterilization
process of destroying all microbial life on an object
antisepsis
process of reducing or inhibiting microorganisms on a living tissue
Difference in cyclic photophosphorylation, non-cyclic photophosphorylation, calvin-benson cycle
- CAlvin-benson cycle- CO2 is fixed (used to synthezise sugars) uses more ATP than photophosphorylation generates, sunlight is an ongoing energy source -cyclic- electrons return to chlorophyll, used in anoxygenic organisms -noncyclic- electrons are used to reduce NADP+, & electrons are returned chlorophyll from H2O or H2S; used in oxygenic organisms
electron transport chain
-electrons are transported by NAD & NADH/FADH2 -carries pass the electrons to one another & use energy released form electrons to pump protons across membrane -protons go down concentration gradient thru membrane channel
How is lipid catabolism and protein catabolism integrated with carbohydrate metabolism?
-lipids and proteins must be broken down to another product before they can enter the krebs cycle -lipids are split into their fatty acid & glycerol components, fatty acids are oxidized thru beta oxidation before entering kreb cycle while glycerol is catabolized & enters glycolysis -proteins must be deaminated, decarboxylated or desulfurizlated before entering glycoysis or krebys cycle
list the 2 general mechanisms that can be used to control microbial agents
-membrane permeability- damage to membrane lipids or proteins, diminished cell growth -damage to proteins(denature by heat & chemicals) and nucleic acids aka DNA/RNA (hingers replication & protein synthesis)
enzyme
-organic protein catalyst(speed up reaction) -lowers activation energy (Less energy is needed in order to drive the reaction) -enzymes lower the energy of activation
enzyme inhibition
-prevent product from being formed
How does a striclty fermentative bacterium produce energy?
-produces it quickly but very little -energy generated from pyruvic acid -thru glycolysis, atp is produced -NAD+ & NADP+ supply is needed to continue glycolysis
5 groups of bacteria based on temperature preference and tolerance
-psychrophiles can grow at 0 C but optimum is about 15 C -phychrotrops-refrigerator temp, can also grow at 0 degrees C but optimum is 20-30 C; important in food spoilage mesophiles-grow best at moderate temperatures around 37 degrees C; many pathogens fall in this category -thermophiles- grow best around 60 degrees -hyperthermophiles- grow best around 80 degrees C or higher (archaea)
substrate-level
-substrate donates phospate group to ADP to form ATP -does NOT produce as much ATPs as oxidative phos. -requires enzyme to transfer phospate form substrate to ADP and end result is product + ATP -enzyme can only create 1 ATP at a time, not very efficient ex: glycolysis, krebs cycle
4 factors that influence enzymatic activity
-temperature: too cold or too hot, cant function, as temp increases the rate of chemical reaction also increases. -ph: enzymes typically have an optimum ph at which they are the most active(just like temp) -substrate concentration- as level increases, so does rate of chemical reaction until enzymes become saturated(all active sites are full) -presence of inhibitors: whether competitive or noncompetitive, decreases rate of chemical reaction *** temp and ph can cause proteins to denature***
active site
-where the substrate go in (bind) -very specific -usually end in (-ase) ex: your house key only works in your house not your neighbors
fermentation
A catabolic process that makes a limited amount of ATP from glucose without an electron transport chain and that produces a characteristic end product, such as ethyl alcohol or lactic acid. -uses energy from sugars or other organic molecules -only substrate level -produces 2 ATP -oxygen is not required but can be used -does NOT use krebs or ETS
colony
A colony is a visible mass of cells formed from the division of one cell over a period of time; often contain over 109 cells ( 1 billion+)
beta oxidation
A metabolic sequence that breaks fatty acids down to two-carbon fragments that enter the citric acid cycle as acetyl CoA. -oxidation to fatty acids used during lipid catabolism
aerobic vs anaerobic respiration (including atp produced)
Aerobic = with Oxygen (36 eukaryotes/ 38 prokaryotes) Anaerobic = without Oxygen (Lactic acid is byproduct) fewer than in aerobic bc no less than 2
activation energy
Energy needed to get a reaction started
chemiosmosis
Process by which a Hydrogen pump pumps protons into the thylakoid membrane. H+ passively flows through the ATP synthase which leads to the creation of ATP. -part where atp is made -
list the chemicals that are used to control microbial growth in foods
SO2, sorbic acid, benzoic acid, & propionic acid -help prevent mold growth -nitrate & nitrate salts can also prevent c. botulinum
substrate
The reactant on which an enzyme works.
How are bacterial growth requirements provided in laboratory cultures
a culture medium (nutrient medium) is prepared for the growth of bacteria in laboratory. Agar(seaweed extract taht most bacteria cant degrade is useful for isolating individual colonies to establish pure cultures and looking at some biochemical properties of the cultures) is a common solidifying agent for a culture medium. Broth is also a common culture medium and is good for growing large batches of organisms.
What is the use of the following equimpent in the bacteriological laboratory?
anaerobe jar- sealed jar used to incubate anaerobic bacteria. Oxygen is chemically removed after culture plates have been introduce and the jar sealed. Used in labs taht only work with few culture plates at a time. anaerobe chamber-air-lock chamber filled with inert gases used to incubate anaerobic bacteria. materials are introduced through small doors in the air-lock chamber & operators work thru arm ports in airtight sleeves. Use in labs that have a large volume of work with anaerobes. candle jar-used as incubators to grow aerobic bacteria that require high CO2 levels. Cultures are placed in a sealed jar contain a lighted candle, which consumes O2. Candle stops burning when the air in the jar has a lowered O2 concentration & a higher CO2 concentration. increase Co2 still leave some O2 for aerobic capnophiles carbon dioxide incubators-used to incubate aerobic bacteria that require CO2 levels that are higher or lower than that found in the atmosphere, desired CO2 levels are maintained by electronic controls.
components of an enzyme system
apoenzyme- the protein portion of an enzyme, inactive by itself cofactors- called a coenzyme if it is an organic( NAD+, FAD++ & CoA)molecule, the nonprotein portion of an enzyme that activates the apoenzyme holoenzyme- whole active enzyme made up of the apoenzyme and the cofactor -Synthesized from vitamins -has to have coenzyme to bind to substrate
what is the preferred method of sterilization if the material to be sterilized is heat and moisture resistant?
autoclave- pressure allows hotter temp to be attained compared to boiling -effective if material to be sterilized isn't damaged by heat or moisture ex: in labs & healthcare facilities to sterilize culture media, solutions & instruments
4 nutritional types of microorganisms and the energy and carbon of each
autotrophs- self feeders, use carbon heterotrophs- feed on others, use organic sources of carbon phototrophs- use light as energy source chemotrophs- use redox reactions of organic or inorganic compounds as energy source
effect of temperature on bacteria
bacteria growth slows as temperature drops below optimum. It also slows as temp raises above optimun, this drop off of bacterial growth is steeper as necessary enzymes denature at the higher than optimum temp.
sepsis
bacterial contamination
describe in detail the filter paper method of antiseptic/disinfectant evaluation
called the disk diffusion method, a disk of filter paper is soaked w/ a chemical & placed on an inoculated agar plate. A clear zone on inhibition indicates effectiveness
Photosynthesis
carried out by plants and many microbes -conversion of light energy from sun into chemical energy -chemical energy is then used to convert CO2 from atmosphere to more reduced carbon compounds, mainly sugars
non-competitive inhibitor
causes protein to change shape and substrate can no longer bind -does not take space of active site but changes shape just enough
2 basic types of media used in the bacteriological laboratory
chemical defined medium- exact chemical composition is know complex medium- chemical composition varies slightly from batch to batch
metabolic pathway
chemical reactions that require enzymes to be present and functional -it takes many different enzymes to catatlyze all the reactions within cells. However, cells do not make large quantities of each
competitive inhibition
competes with substrate for active site -substrate can not attach
list 2 ways that bacteria can be preserved for extended periods of time
deep freezing- done quickly from -50C down to -95C lyophilization(freeze-drying) suck water while freezing in a process called sublimation
catabolism
degradative and exergonic -break things apart & release energy -some are hydrolytic (water input) -water may be a reactant
list & describe the methods for the measurement of bacterial growth generally used in the lab. Which are direct and indirect?
direct- standard plate count- reflects the number of viable microbes & assumes that each bacterium grows into a single colony, reported as the number of colony forming unit (CFU) instead of the # of cells. If the concentration of bacteria is too great, the colonies will grow into each other and the plate will be uncountable. direct microscopic count- microbes in a measured volume of a bacterial suspension are counted with the sue of a specially designed slide. It can be done automatically with an electronic cell counter most probable number (mpv index)-statistical estimation used for microbes that wont grow on solid media or are grown in differential liquid media for identification purposes. A dilution series to no growth is prepared and the combination of positives is used to look up the most probable number in a table. indirect- spectrophotometer- used to determine turbidity(cloudiness) by measuring the amount of light that passes through a suspension of cells. dry weight- measuring dry weight is a convenient method of growth measurement for filamentous organisms such as fungi metabolic activity-measuring the metabolic activity of the population (for example, acid production or oxygen consumption) can help estimate bacterial numbers (all require use of standard curve)
list and describe 3 methods for the evaluation of antiseptics and or disinfectants
disk-diffusion method-disk or filter paper is soaked w/chemical & placed on an inoculated agar plate; phenol coefficient-compares activity of a disinfectant to phenol, was once the standard to evaluate the effectivnesss of a disinfectant american offical analytical chemist use dilution test-standard
give the uses of the following methods of sterilization and or disinfection
dry heat-sterilizes by inoculating loops w/direct flame autoclave- sterilized microbial media, solutions, linens, utensils, dressings, equipment, & other items that can withstand temp. & pressure; most common method of moist heat sterilization in health care environments membrane filtration-sterilize liquids such as enzymes or vaccines that are destroyed by heat pasteurization-used to destroy pathogens & food spoilage organisms in milk, cream & certain alcoholic beverages (beer/wine) hot-air oven-used to sterilize glassware, instruments, needles & glass syringes incineration-burning to ashes, used to sterilize & dispose of paper cups, contaminated dressings, animal carcasses, bags & wipes direct flaming-sterilizes inoculating loops in micro lab
How do enzymes function?
enzyme + substrate (reactant) -> ES complex -> enzyme + product -enzyme is added to substrate which is reactant - this creates the enzyme substrate complex -once enzyme is completed it may change shape and create product -enzyme does not change
list the disinfectants that are use as a gas. what is plasma gas sterilization?
ethylene oxide, chlorine dioxide, plasmas, supercritical fluids (can be gas @ one temp & liquid @ dif temp) plasma- knowns as electromagnetic field, to produce a mixture of nuclei w/various charges & free electrons; good for sterilizing plastic surgical instruments
How is ATP generated?
from adp to atp -through different process
reduction
gain of electrons/hydrogen atom (NAD/FAD reduced to NADH/FADH)
3 processes that are required for the complete catabolism of glucose to carbon dioxide
glycolysis, krebs cycle, electron transport chian & chemiosmosis
commereical sterilization
heat treatment of canned foods to destroy C. botulinun endospores( 121 C for 0.4-3 minutes)
list & describe 7 physical methods of microbial control
heat-boiling, autoclaving, pasteurization(denature proteins) dry heat( directly over flame, like burner, will turn to ashes) can kill endospores; moist heat requires shorter time as compared to dry heat time water conducts heat better than air does filtration-material must be sterilize prior, liquid can pass but membranes can't. Used on heat-sensitive liquids, good for separating bacteria from its suspending liquid cold-more for food preservation; refrigeration(reduces spoilage), deep-freezing(may not kill all organisms) high pressure-denatures proteins in vegetative cells but endospores arent really harmed although can be allowed to grow out & the vegetative form killed w/high pressure ex:fruit juice preservation desiccation-absense of water, inhibits metabolism, microbes can remain viable, but dormant for years, effect varies per microbe osmotic pressure-achieved by hypertonic environment, high salts and sugars causes plasmolysis, inhibits metabolism radiation- either ionizing(destruction) or nonionizing(damage) & not very penetrating can kill microbes depending on wavelength, intensity, & duration
list 2 oxidizing agents used for sterilization.
hydrogen peroxide-sporocidal @ high concentrations & is used in the food industry for aseptic packaging & for contact lens disinfection. peracetic acid- very good
Why is hydrogen peroxide better as a disinfectant for inanimate objects that an antiseptic for wounds?
its better for disinfectant for inanimate objects; it will dissolve tissue for wounds
describe how to sterilize heat labile solutions
labile(destroyed by heat) best method would be filtration ex: you dont want to heat a vaccine
logarithmic growth
is due to reproduction by binary fission (bacteria) or mitosis (yeast) -increase in size of a microbial population in which the number of cells doubles in a fixed interval of time ex: 2, 4, 8, -can grow very quickly
describe the bacterial growth curve identifying the events occurring in each of the four sections
lag phase- few hrs after inoculation, cells preparing for growth, in increase in population size, in Vivo(incubation time after exposure) log phase- logarithmic/exponential growth in population size, # of organisms double every generation time, best time to preform biochemical test, In vivo(symptoms would start) stationary phase- stabilized population, period of equilibrium, death rate= growth rate death phase-population size is decreasing at logarithmic rate, nutrients more depleted
identify the point on the growth curve where bacteria are most susceptible to control mechanisms
log phase?
oxidation
loss of electrons/hydrogens atom (organic molecule oxidized)
explain why chlorine is antimicrobial
needs to be diluted with water to form hypochlorous acid (HOCI) -HOCI is neutral in electrical charge & diffuses as rapidly as water thru the cell wall & when it separates into H+ & hypochlorite, which acts as a strong oxidizing agent that prevents much of the cellular enzyme system from functioning
list 4 conditions that influence microbial control and explain why each has the effect that it does.
number of organisms-determines how long it takes to reduce the # or organisms to a given level at a logarithmic death rate environmental influences-organic matter may interfere with heat treatments and chemical control agents, heat is more effective at lower ph; body fluids can increase killing time time of exposure-chemical agents require longer exposures to control more resistant organisms; longer exposure to lower heat can produce the same effect as shorter time at higher heat microbial characteristics - microbial species and life cycle phases (endospores) have different susceptibilities to physical and chemical controls
5 groups of bacteria based oxygen utilization and tolerance
obligate aerobes- require oxygen to live facultative anaerobes- can grow in aerobic or an aerobic conditions although tend to grow better in aerobic conditions (includes E. coli and some yeasts) obligate anaerobes- unable to use molecular oxygen for energy yielding reactions- most harmed by it aerotolerant anerobes- dont use o2 but tolerate it, have SOD or similar enzyme; many ferment carbohydrate to lactate, which inhibits growth of aerobes microaerophiles- require low o2 concentrations, they are sensitive to oxygen-derived free radicals
oxidative phosphorylation
occurs in plasma membrane of prokaryotes (mitochondria in eukaryotes) -redox reactions produce ATPs via chemiosmosis -electrons(energy carriers) are removed from organic molecules & transferred to ETS(NAD/FAD) -produces a lot more atps than Sl phosphorylation bc it goes thru a series of steps -32-34 atp that can be made during process
binary fission
on parent cell yield 2 daughter cells -reproduction of bacteria bc there is no nucleus -daughter cells are exactly the same as parent cells
define phenol coefficient and use dilution and understand how to interpret data from both kinds of test
phenol coefficient- compares activity of a disinfectant to phenol use-dilution test- bacterial survival is determined in the manufacturer's recommended dilution of a disinfectant
list 5 general principles for choosing of an antiseptic and or disinfectant
properties of disinfectant-how it works determines what it will be effective against concentration of disinfectant-requires proper hydration temperature-increased temp usually enhances the efficacy of disinfectants presence of organic matter-can act as a buffer degree of contact w/microorganisms-if the surface needs cleaning or is porous, microorganisms can escape contact w/ the disinfectant
How can polysaccharides, lipids, amino acids/proteins, and nucleic acids be produced through anabolic reactions?
proteins- use deamination to turn to organic acids, involves dehydration synthesis reactions that require energy. Glycogen is formed from ADPH (ATP + glucose 6 phosphate= adenosine diphosphoglucose) in bacteria & from UDPG in animals lipids-synthesized from fatty acids & glycerol (glycerol derived form dihydroxyacetone phospate & fatty acids from acetyl CoA) both linked thru dehydration synthesis reactions that require energy amino acids- needed form protein biosynthesis. All amino acids can be synthesized directly or indirectly from intermediates od carbohydrate metabolism, particularly form the krebs cycle. nucleic acids- sugars composing nucleotides are derived from either the pentose phosphate pathway or the entner-doudoroff pathway. Carbon & nitrogen atoms from certain amino acids form the backbones of the purine & pyrimidines.
sanitization
purpose is to lower microbial counts on eating and drinking utensils to safe public health levels -either by high- temp washing or use of chemical disinfectant
oxidative decarboxylation of pyruvate
pyruvate converted into acetyl co a -pyruvic acid loses 1 molecule of CO2 & becomes a 2-carbon compound called acetyl group, which then attaches to coenzyme A. -during this reaction, pyruvic acid is also oxidized & NAD+ is reduced to NADH
Define, describe & give ex. of the following media:
reducing media-components that chemically remove molecular O2 taht might interfere w/ the growth of anaerobes ex: thioglycolate combines w/ dissolved O2 to deplete O2 in media selective media-allows growth of only desired microbes in inhibiting unwanted organisms with salts, dyes, or other chemicals ex: bismuth sulfite agar inhibits gram-positive & most gram negative bacteria, used to isolate Salmonella typi. differential media-used to distinguish among different organisms ex: blood agar is used to identify oranisms that lyse red blood cells (streptococcus pyogenes) enrichment media-designed to encourage the growth of a particular microorganism in a mixed culture that is growing in slow levels. It increases the #s of desired bacteria that are present in very small amounts to detectable levels. ex: phenol is lethal to most bacteria but can be used to encourage growth of microbes that can live on phenol
degerming
removal of microbes from a limited area, such as the skin around an injection site w/alcohol swab
Distinguish btw selective & differential media
selective-allow growth for only desired microbes by stopping unwanted organisms w/salts, dyes or other chemicals differential-used to distinguish diff organisms
list the heavy metals used in microbial control
silver, mercury, copper, & zinc (germicidal) -when combined with sulfhydryl groups, proteins are denatured
-stasis
stopping or inhibiting
list 4 ways ATP can be produced in a cell
substrate-level phosphorylation oxidative phosphorylation photophosphorylation electron transport chain & chemiosmosis
Amount of ATP produced directly in following process:
substrate-level phosphorylation-2 oxidative phosphorylation-34 photophosphorylation- varies on how long cell utilizes light energy chemosmosis-34 glycolysis-0 but 2 produced by slp oxidative decarboxylation of pyruvate-0 krebs citric acid cylce-0 but 2 by slp electron transport chain- 0, none is produced until chemiosmosis calvin-benson cycle-0, calvin benson cycle uses ATP but doesnt produce any
metabolism
sum of all chemical reactions that happen in a cell -2 types (anabolism & catabolism)
Roles of superoxide dismutase, catalase and peroxidase in oxygen utilization and/or tolerance
superoxide dismutase, catalase, peroxidase are enzymes that destroy or neutralize toxic forms of oxygen (superoxide radicals & hydrogen peroxide)
What kinds of bacteria have these enzymes(superoxide dismutase & catalase)
superoxide dismutase: obligate aerobes, facultative anaerobes & aerotolerant anaerobes (all bacteria that use oxygen) catalase:obligate aerobes & facultative anaerobes
Why are 38 ATPs produced from oxidation of one molecule of glucose in prokaryotic cell whereas only 36 ATPs are produced from the same process in some eukaryotic cells
there is fewer ATPs in eukaryotes bc energy is lost when pyruvate is shuttled across the mitochondrial membranes, that separate glycolysis from the electron transport chain -this separation does not exist in prokaryotes
explain why salts and sugars can be used to preserve food
there is no liquid due to salts and sugars and prevents growth
generation time
time require for a cell to divide or a population to double -varies per organisms
-cide
to kill
asepsis
without contamination
