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170 Cards in this Set
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Streak Plate
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Isolation technique of a mixed sample
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How to do a streak plate?
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1. streak over a plated agar medium
2. 4 streaks (flame the loop after each streak) |
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Purpose of Streak plate
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during streaking, the cell density decreases
colonies --> individuals (isolation) |
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Streak plate success?
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Cells that have been sufficiently isolated will grow into colonies of ONLY the original cell type
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Colony-forming unit
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CFU
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CFU?
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colonies form from individual cells & others from pairs, chains or clusters
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Streak plate method form what?
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individual colonies on plates
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Why use Streak plate method?
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CFU transferred to a sterile medium to start a pure culture
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Zigzag inoculation of Agar plates with cotton swab when?
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have pure cultures.
when sample does not have high cell density |
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Innoculation of agar plates with cotton swab in prep for Quadrant Streak plate:
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1. preformed as initial streak for isolation (streak 1).
2. done with mixed cell culture with high cell density 3. streak Q1 with swab; use loop Q2-4 |
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What is Steam sterilization?
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Autoclave
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Why is Steam Sterilization used?
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most effective and most common way to sterilize contaminated items
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Increase the atmospheric pressure and what happens to the boiling temperature?
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It increased, but the cook time shortens
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Autoclave specifics
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121*C
15 psi (pressure/square inch) 15 min super heated steam under pressure to kill heat-resistant organisms |
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SDA
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sabourand dextrose agar
good media for fungi acidic pH 5.6 lower pH = lower bacteria |
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TSA
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tryptic soy agar
enriched media pH 7 good for bacteria growth |
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Heat resistant organisms
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spore producing (in protective form) Bacillus, Geobacillus & clostridium
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Sterilizing Temp.
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121-127*C (250-260*F)
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Sterilizing time
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Vary to size & consistency of material
Items must reach optimum T*C for 15 min. |
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What is used as good indicators that sterilization is complete?
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special thermometers
colored-coded autoclave tape |
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What is a Biological indicators?
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determine sterilization
device containing a known pop. of viable organisms (significant & quantified resistance to sterilization) used to validate autoclave |
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What is used as a typical biological indicators
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bacterial spores
Dormant form of an organism, highly resistant to chemical & physical changes If not killed, they will germinate & ferment => lowers pH (acidic) purple --> yellow |
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Standard Plate Count
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Viable Count
Indirect Count Live cells ONLY Estimate of actual living cells in a sample |
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Standard Plate Count allows?
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An estimate of pop. density in a liquid sample by plating dilute portions & counting # of colonies formed (CFU)
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CFU
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Number of Colonies Formed
# of cells |
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Serial dilution
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begin with unknown concentration (density) of cells-> dilute with a few cells
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Countable plates
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30 - 300 colonies (CFU)
Above 300 (too many too count; too many to be viewed as individual colonies) Below 30 (too few to count; statistically unreliable) |
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In dilutions
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V1D2 = V2D2
D2 = (V1xD1) / V2 = (1.0 mL x 10^-1) / 10 mL |
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Plate Spread
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Known volume of dilution
Count colonies |
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OCD
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original cell density
OCD=CFU/Dil'n x Vol.transfered to plate |
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OCD express in what units?
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CFU/mL
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Volume of original sample
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is the product of vol. transferred & the dilution of the tube it came from.
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Direct Counts
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take a small portion of a sample & use the data gathered from it to calc. overall pop. cell density
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Petroff-Hausser counting chamber
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-microscopic slide with 0.02mm deep chamber ("well") in center with grid
-25 large squares (16 small squares in each) = 400 -1 square mm well (when covered with cover glass & filled with suspension cells) vol. =5 x 10^-8 mL -each small square = 50,000 avg. size cocci cells |
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OCD
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original cell density = cell density
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OCD used for?
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counting cells found in a predetermined group of small squares & dividing by (# of squares)(Dil'n)(Vol)
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OCD calculation
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(Cells counted)
__________________________ (# of Squares) (Dil'n) (Vol.) |
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Optimum density
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5-15 cells/small square
*For accuracy recommend min. overall count of 600 cells |
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Indirect Count Advantages
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1.Works well for cells that separate in a short amt of time after they divide
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Indirect Count Disadvantage
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1. Doesn't work well for cells that stick together after cell division
2. Mixed samples have diff. nutritional needs 3. Sample error = uneven distribution of the sample on the agar |
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Direct Count Advantages
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Faster
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Direct Count Disadvantages
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There must be a certain # of organisms b/f there are enough to be seen (both viable & nonviable count)
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Spread plate technique
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method of isolation in which a diluted microbial sample is deposited on an agar plate & spread uniformly across the surface with a glass rod
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How to tell if a sample it properly diluted?
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With a properly diluted sample, cells (CFUs) will be deposited far enough apart on the agar to grow individual colonies
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Consequences of not spreading the inoculum adequately?
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*uneven distribution, so
what try to grow/inhibit = no isolated colonies |
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Separate a mixed culture
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separate, isolate, & harvest out = streak method
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Optical Density
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OD
*Used to measure the bacteria concentration in a suspension by measuring the amt of light scatter |
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What is the correlation b/w the OD & cell # for your culture?
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*Greater the scatter = more bacteria present
*Greater the dilution = the smaller amt of light scatter occurs |
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Why do you plate count (viable) & OD measure?
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data will give us exact measurements of bacteria concentration in our culture for graphing
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Determining the Growth by Optical Density uses what? quantify what?
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Spectrophotometer
Quantify the microbial population in a sample |
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Bacteriophages
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Phages
Viruses that attack bacteria |
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How do phages kill bacteria?
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1. Some viruses attach to the bacterial cell wall
2. inject viral DNA into the cytoplasm 3. Viral genome commands the cell to produce more viral DNA & viral proteins, used to assemble more phages 4. Phage assembly completed, the cell lyses & releases the phages 5. Phages attack other bac. cells |
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Lytic cycle
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1. Some viruses attach to the bacterial cell wall
2. inject viral DNA into the cytoplasm 3. Viral genome commands the cell to produce more viral DNA & viral proteins, used to assemble more phages 4. Phage assembly completed, the cell lyses & releases the phages 5. Phages attack other bac. cells |
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Lysis of bacterial cells on agar plate produce what?
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A clearing
Plaques |
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Plaques
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Clearing
Areas where virus has killed bacteria |
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Plaque assay used for what?
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Means of calculating the phage concentration in a given sample
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Plaque assay
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1. Sample of bacteriophage (diluted by serial dil'n) added to a plate inoculated with bacterial host to produce a lawn of growth
2. # of plaques formed used to calculate the original phage titer (density) |
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Titer
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Density
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Phage titer
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phage density
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Plaque assay technique
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1. serial dil'n to produce countable plates
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Plaque assay uses what technique
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Pour-plate technique
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Pour-plate technique
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bacterial cells and viruses are first added to molten agar and then poured into the plate
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What does the pour-plate technique do for the plaque assay?
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*diluted phages added to exposure time to bacteria = preadsporption period
*Then this phage-host mix is added to a tube of soft agar, mixed, & poured onto prep Nutrient Agar plates as an agar overlay |
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Preadsporption period allows for what?
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allows the virus to attach to the bacterial cells
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Agar overlay
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This technique allows you to produce a homogeneous lawn of bacteria within a thin layer of agar across the surface of a plate. Bacteria are added to a soft top agar (0.75% agar, as opposed to the usual 1.5% for agar plates) which has been melted at 100°C and cooled to 45°C. This is warm enough so the agar remains liquid, but cool enough so that the bacteria are not killed (for a period of time). The melted agar/bacterial suspension is mixed and poured evenly across the top of an agar plate and allowed to solidify.
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Why is agar overlay used in plaque assay?
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To produce a homogenous mixture of phages and bacteria;
Allow bacteria exposure to phages to occur *when solidifies, the agar immobliizes the bacteria while allowing the smaller phages to diffuse short distances & infect surrounding cells |
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Plaques appearing on a plate is an indication of what?
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Bacteria infected with viruses;
the bacteria cells have been lysed by the virus |
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Countable plates
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30 -300 plaques
Same as standard plate count |
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PFU
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plaque forming units
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Phage titer
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original phage density
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Phage titer calculation
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Phage titer = (PFU) / (Volume plated x Dilution)
*PFU / Original sample volume PFU/mL |
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What bacteriophage was used to kill Escherichia coli?
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T4 coli-phage
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T4 phage lytic cycle
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1. Attachment
2. Penetration 3. Uncoating 4. Synthesis 5. Assembly 6. Generalized Transduction 7. Release Cycle can take 25 minutes Can release a few hundred phage progeny |
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T4 phage lytic cycle
Attachment |
Phage attaches by its legs to specific receptors on E. coli
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T4 phage lytic cycle
Penetration |
T4 process of injecting its DNA into E.coli
*Virus condenses Entire phage genome is in the host |
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T4 phage lytic cycle
Uncoating |
Removal of virus genome from its capsid
Phage DNA is being replicated |
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T4 phage lytic cycle
Synthesis |
Phage DNA replicated in bacteria host
Synthesis of Virus proteins (to form virus parts) Degrades host DNA (sometimes host DNA incorporated into phage DNA) |
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T4 phage lytic cycle
Assembly |
Synthesis leads to assembly of the phage progeny, where capsid subunits come together to form the capsid into which the genome is inserted
& tail comes together & attaches to the capsid |
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T4 phage lytic cycle
Generalized Transduction |
Host DNA is incorporated into new T4 phage DNA, giving it new genes
Genetic diversity increased |
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T4 phage lytic cycle
Release |
Fully assembled phages are released as the cell bursts
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Why must there be enough bacteria inoculated to produce a lawn of growth?
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Plaque assays ID the presence of viruses by the absence of growing bacteria.
If not enough bac. on plate then there will be regions of no growth = false phage assay |
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How might results be altered if skip preadsorption phase?
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False absence of phage, due to low plaque count.
Preadsorption allows time for the phages to attach to the bacterial cells (not time to kill them, just start the process) |
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Why is the water bath set at 50*C?
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50*C = 122* F (shower, hot food)
Over 130*F (tissue damage) Lower Temp. might be to cold for growth or slow growth (chemical rxns slower) High temp. will denature proteins & kills speciment |
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Zone of inhibition
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Phage kill zone of bacteria
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Cardinal Temperatures
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Each species is characterized by a minimum, maximum, and optimum temperature of growth
*Below min. the organism will not survive |
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Optimum Temperature
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Temperature an organism shows the greatest growth over time
Highest growth rate |
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Psychrophiles
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Grow only below 20*C
Common in ocean, Arctic, Antarctic habitats (permeant cold) No fluctuation |
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Psychrotrophs
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Adapted to cold habitats that fluctuate
0* - 30*C |
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Mesophiles
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15* - 45*C
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Thermophiles
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Above 40*C
Composting organic material & in hot springs |
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Obligate Thermophiles
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Will not grow below 40*C
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Facultative thermophiles
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Grow below 40*C
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Extreme thermophiles
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65* - 110*C
Bacteria isolated from hot ocean floor ridges Grow best above 80*C |
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Optimal Temperature of
Serratia marcescens |
37*C
(Mesophiles range 25-45) At optimal Temp. produces a red pigment |
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At what temperature will Serratia marcescens stop producing a red pigment?
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42*C (off-white)
The enzyme is beginning to b/m denatured and no longer works |
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Why do different temperatures produce different growth rates?
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Enzyme activities are temp. dependent.
Higher temp. means higher activity (dentures/unravels the protein) Lower temp. means lower activity (stops synthesis) |
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pH
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concentration of hydrogen ions in a solution
*"pondus hydrogenii" = hydrogen power or hydrogen potential |
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Soren Peter Lauritz Sorensen
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Danish biochemist
1909 invented pH range (0-14) based on logarithmic scale |
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pH calculation
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pH = -log [H+]
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Pure water pH
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7
10^-7 moles of hydrogen ions per liter |
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Acidophiles
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organisms adapted to grow well in environments below pH 5.5
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Neutrophiles
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pH 5.5-8.5
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Alkaliphiles
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pH 8.5
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Bacteria under normal circumstances
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maintain a near-neutral internal environment regardless of their habitat
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pH outside the normal range of the organism, what happens?
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destroy necessary membrane potential (in the production of ATP) and damage vital enzymes beyond repair
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Denaturing proteins/enzymes
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*conformational changes in the proteins' tertiary structure
Lethal to the ell |
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Acids form from what fermentation?
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carbohydrate fermentation
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Alkaline products from from what?
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protein metabolism
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Hydrogen phosphate
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buffer
In solution can alternate b/w WA (H2PO4-) & conjugate base (HPO4-2) Maintains H+/OH- equilibrium |
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Why should you not connect data points on a graph?
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Indicates a continuous and that there is data inbetween the points are valid; they may not be
*only connect data points if the data is continuous, but it is changing all the time |
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Water is the principal component of what part of the cell? Essential for what
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cellular cytoplasm
* Essential source of E+ & H+ions |
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Turgor pressure
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Internal cellular pressure
Important in prokaryotes, like plants |
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Plasmolysis
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Separation from the cell wall
*Prokaryotes require water to prevent shrinking of cell membrane resulting in separation from the cell wall |
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Bacteria control their turgor pressure how?
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transporting in & maintaining high K+ & Na+ ion concentration
creating a [ ] gradient that promotes diffusion of water into cell |
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Osmosis
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movement of water from an are of low solute concentration to an area of higher solute concentration
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Osmotic pressure
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the pressure which needs to be applied to a solution to prevent the inward flow of water across a semipermeable membrane.[1] It is also defined as the minimum pressure needed to nullify osmosis.
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Osmotic pressure refers to what?
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The ability of a solution to pull water toward itself through a semipermeable membrane
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Hyposmotic
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solution having low osmotic pressure
low solute concentration |
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hyperosmotic solution
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solution having high osmotic pressure
high solute concentration |
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Isosmotic
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a solution having osmotic pressure equal to that of the cell
Water will tend to move in both directions |
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Halophiles
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grow optimally in [NaCl] of 3% or higher
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Extreme halophiles
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specialized cell membranes and enzymes that require salt concentrations from
15% - 25% Will not survive in lower salinity |
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Osmotolerant
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grow over wide range of salinities
Most bacteria live in [NaCl] less than 3% |
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Germicides
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Substances/systems used to prevent the spread of pathogens
*specific in nature & typically include the name of the target pathogen ("tuberculocide," "virucide," "sporocide") Most germicides are broad-spectrum 2 categories (decontamination, disinfection, sterilization) |
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Decontamination
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germicides lowest level of control
"reduction of pathogenic microorganisms to a level at which items are safe to handle without protective attire" *physical agents used, removal, cleaning *sterlize must come in direct contact with pathogens |
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Disinfection
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Next level of control
3 sublevels (low, medium, high) Based on effectiveness against specific control pathogens (kill targeted pathogens, not spores) *chemical sterilants (kill all vegetative cells & spores) *Methods: dry heat, moist heat, & UV *reduce/eliminate pathogens on/in living tissue = antiseptics |
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Sterilization
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*Complete elimination of viable organisms, including spores
*highest level of pathogen control *chemicals, gases, incineration, dry heat, moist heat, ethylene oxide gas, radiation, low-temperature plasm |
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Electromagnetic energy
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X-rays, UV, Visible light, Infrared
Wavelengths of light |
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UV light
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UV-A longest wavelength 315-400 nm
UV-B 280-315 nm UV-C 100-280 nm |
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Wavelengths
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the distance b/w adjacent wave crests and is typically measured in nanometers
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Which UV is detrimental to bacteria? why?
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UV-C (100-280nm)
Results in irreparable DNA damage & death of the organism *Mutagenic effects of UV & DNA repair Shortest wavelength = highest energy |
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Why do endospores survive UV exposure?
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Spore-forming bacteria such as Bacillus are able to survive much higher doses of UV and other types of radiation. Spores can survive very harsh conditions (high/low temperature, low water, radiation, antibiotics, osmotic pressure, etc.) for extended periods of time before becoming active again when conditions improve.
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Endospores
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dormant, tough, and non-reproductive structure
It is a stripped-down, dormant form to which the bacterium can reduce itself. Endospore formation is usually triggered by a lack of nutrients, and usually occurs in Gram-positive bacteria. In endospore formation, the bacterium divides within its cell wall. Endospores enable bacteria to lie dormant for extended periods, even centuries. Revival of spores millions of years old has been claimed.[2] When the environment becomes more favorable, the endospore can reactivate itself to the vegetative state resistant to ultraviolet radiation, desiccation, high temperature, extreme freezing and chemical disinfectants. The core contains the spore chromosomal DNA which is encased in chromatin-like proteins known as SASPs (small acid-soluble spore proteins), that protect the spore DNA from UV radiation and heat. |
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Antibiotics
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natural antimicrobal agents produced by microorganisms
Penicillium notatum *treat bacteria infections |
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Antimcrobials/antimicrobics
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all substances used to treat bacterial infections that are synthetic
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Kirby-Bauer Test
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disk diffusion test
*valuable standart tool for measuring the effectiveness of antimicrobics against pathogenic microorganims *Ab impregnated paper disks placed on plate inoculated to form bacterial lawn, incubated (to allow bac growth & time for Ab to diffuse into agar). *as the drug moves through the agar, it established a [ ] gradient *if an organism is susceptible to it = clear zone (around disk) of inhibited growth |
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Zone of inhibition
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clear zone
If an organism is susceptible to it, growth will be inhibited *depends on the sensitivity of the bacteria to specific Ab agent & MIC |
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Minimum inhibitory concentration (MIC)
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*important to confirm resistance
the min. amt. of Ab to get results |
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Bactericidal
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kill organism
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bacteriostatic
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stop growth
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Muller-Hinton agar
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pH 7.2-7.4
4mm depth (important b/c its effect on diffusion) low levels of thymine & thymidine & controlled levels of Ca & Mg = required to produce a correct zone diameters with the Abs used Store @ 2-8*C |
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Factors that influence disk diffusion susceptibility tests
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Medium
Excess surface moisture on medium Agar depth Disc potency Inoculum concentration pH B-lactamase production by test organisms |
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Do not use plates if they show:
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Microbe contamination
Discoloration Drying Cracking Deterioration Excess shrinkage (desiccation) may lead to false suseptibility results |
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How does depth affect diffusion of Ab?
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Thick agar = slows lateral diffusion & produces smaller zones than plates held to the 4mm standard
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Inoculation for Kirby-Bauer test is made with a broth culture diluted to match what?
|
A 0.5 McFarland turbidity standard
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McFarland Turbidity Standard
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comparison of McFarland turbidity
Has a specific concentration of precipitated barium sulfate to produce turbidity Reached b/f inoculating the plate |
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McFarland standard
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0.5 McFarland
1.5 x 10^8 cells/mL |
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Disk dispenser
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contains a specified amt of Ab agent (printed on disk) dispensed into plate
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Kirby-Bauer test Advantages
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Allows determination of the most effective Ab to disease
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Kirby-Bauer test Disadvantages
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takes 24 hr turn around
doesn't indicated the dose needed to kill/inhibit the pathogen long enough for the immune system to work |
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Resistance mechanisms
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Break down in 3 main categories:
1. altered target such that the Ab no longer can interact with the cellular process 2. alteration in how the drug is taken into the cell 3. enzymatic destruction of the drug |
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Antibiotics affect on cells
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1. attack cell wall
3. interfere with biosynthesis rxns |
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Chloramphenicol
Cellular target |
prevents peptide bond formation during translation
|
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Chloramphenicol
Resistance mechanism |
1. poor uptake of drug
2. inactivation of drug |
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Chloramphenicol
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bacteriostatic
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Ciprofloxacin
Cellular target |
interferes with DNA replication
|
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Ciprofloxacin
Resistance Mechanism |
1. Altered target
2. Poor uptake of drug |
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Ciprofloxacin
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bacteriocidal
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Trimethoprim
Cellular Target |
inhibits purine and pyrimidine synthesis
|
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Trimethoprim
Resistance mechanism |
1. altered target
|
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Trimethoprim
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Bacteriostatic
S >16 I 11-15 R <10 |
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Penicillin
Cellular target |
inhibits cross-linking of the cell wall's peptidoglycan (b-lactam Ab)
Narrow spectrum |
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Penicillin
Resistance mechanism |
1.altered target
2.poor uptake of drug 3.production of B-lactamases |
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Penicillin
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Bacteriocidal
S >29 R < 28 |
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Chemical germicides
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reduce # of pathogens on a surface, in a liquid, or on/in living tissue
|
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Germicides
|
designed for use on surfaces
|
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Disinfectants
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liquids
germicides designed for use on surfaces |
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Antiseptics
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germicides designed for use on/in living tissue
|
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Use-Dilution Test
|
standard procedure used to measure the effectiveness of disinfectants specifically against Staphylococcus aureus, Salmonella enteric serovar Cholerasuis, & Pseidomonas aeruginosa
*Standard procedure: glass beads exposed to bacteria, then germicides and medium to grow (test effectiveness of germicide) |
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Sufficient germicide
|
prevents 95% microbial growth
*meets required standard *considered usable dilution of that germicide for a specific application if 95% |