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83 Cards in this Set
- Front
- Back
Digital Pipettor |
- Used to accurately transfer liquids for quantitative results - Work by air displacement |
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Sterile, disposable plastic tip |
- Placed on the end of the pipettor to prevent cross contamination of samples and contamination of the instrument |
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Metal shaft |
- Ejects the tip when pressed, so user doesn't touch tips - Important when transferring microbial agents, or hazardous chemicals |
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Cotton/Fiber Plugs |
- Sometimes with the tips to prevent possible contamination of the pipettor when transferring solutions containing these components |
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0.1 -10 micrometers uL 10 - 100 micrometers uL 100 - 1000 micrometers uL |
3 Volume Ranges of the Digital Pipettors used in Lab |
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1000
880 540 |
Indicate the volume that would be dispensed on a 1000 uL unit - 100 - 88 - 54 |
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Piston
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- When specific volume is set, the distance that this will move is determined, and therefore, the volume that will be transferred
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First Stop/Loading Stop |
- First point of resistance on plunger |
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Second Stop/ Blow Out Stop |
- Needs to be reached to dispense all the liquid, since capillary action of the tip will actually retain some of the sample if only gone to the First Stop |
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- Splattering - Loss of reagents - Incorrect volume to be dosed |
Pushing and releasing the button too quickly can cause what 3 possible sources of error? |
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Third Stop/ Tip Eject Stop |
- Some pipettors also have this to pop the disposable tip off the end of the barrel |
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20 Drops 1000 microliters uL |
1 mL contains how many drops? How many microliters? |
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50 microliters uL |
1 Drop is about how many microliters? |
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Psychrophiles |
- Grow below 10 C |
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Mesophiles |
- Grow in a range of 20-40 C - Found on body |
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Thermophiles |
- Grow in a range of 50 - 75 C - Found in hot springs |
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Hyperthermophiles |
- Grow in a range above 80 C |
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Optimum temperatures |
- Indicate the fragility or stability of their metabolic enzymes |
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Pseudomonas fragi Staphylococcus epidermidis Bacillus stearothermophilus |
Give example of psychrophile, mesophile and thermophile. |
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- Pseudomonas fragi - Pseudomonas fragi; Staphylococcus Epidermidis - Staphylococcus Epidermidis - Bacillus stearothermophilus |
What bacteris would you expect to see at 4C? 25C? 37C? 55C? |
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Bacillus stearothermophilus |
What bacteria had growth at 55 C? |
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Pseudomonas fragi and Staphylococcus epidermidis |
What bacteria had growth at 25C? |
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Pseudomonas fragi |
What bacteria had growth at 4C? |
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Staphylococcus Epidermidis |
Which bacteria had growth at 37 C? |
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Proteases Lipases Amylases |
3 Types of hydrolytic enzymes |
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Amylase |
- Digest starch for they can absorb glucose for energy production |
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Hydrolytic Enzymes |
- Some bacteria excrete this to digest nutrients around them so they can absorb the products for sources of energy, carbon and nitrogen - Many digest starch for they can absorb glucose for energy production |
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Bacillus subtilis - Iodine binds to starch and gives black coloration, so if starch is consumed, halo appears |
Which bacteria is the one with the 'halo' around its growth? Does this indicate presence or absence of amylase enzyme? Explain. |
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Catalase |
- Enzyme that converts toxic peroxide H2O2 to H2O and 1/2 O2 - Reaction shown by bubbles |
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- Staphylococcus epidermidis has catalase - bubbles - Enterococcus faecalis |
Identify the bacteria with bubbles. The one without. Which one is catalase present in? |
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Enterococcus Faecalis |
- Gram positive, does not secrete catalase, present in gut (gastrointestinal tract) |
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Cytochromes |
- Electron transport system of aerobic organisms contain many iron containing proteins called? - High energy electrons from NADH and FADH2 are passed from these to the next and energy is extracted from them to make ATP |
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Cytochrome C Oxidase |
- Last cytochrome in the chain - Uses 2 protons (H+) to add to oxygen to make water (H2O) - If this is present, it means all previous cytochromes are present - Aids in the classification of bacteria's aerotolerance and identification of Gram negative organisms |
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- Do not have cytochrome C oxidase
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Obligate Anaerobes and Enterobacteriaceae (enterics) which are Facultative anaerobes (have/do not have) cytochrome C Oxidase
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Oxidase Test |
- This test can be used to differentiate between Pseudomonads and Enterobacteriaceae |
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Square with oxidase reagent will turn blue if chemical is oxidized, indicating presence of cytochrome C oxidase within 5-10 seconds
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What is positive result for oxidase? |
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Pseudomonas aeruginosa; Cytochrome C Oxidase is present |
Identify the bacteria where color of square is blue. What cytochrome is present? |
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Facultative Anaerobic
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- Grows in the presence or absence of O2, but prefers because aerobic cellular respiration is more energy efficient (> ATP)
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Strictly Anaerobic |
- O2 is toxic (Doesn't grow, or sometimes dies) |
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Aerotolerance |
- Describes the ability/inability of organism to grow in presence of molecular oxygen |
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Obligate Aerobe Facultative Anaerobe Obligate Anaerobe Aerotolerant Anaerobe Microaerophile |
5 Classifications of Aerotolerance |
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Pseudomonas aeruginosa Aerobic Growth on top. |
Identify bacteria and its aerotolerance. Describe growth. |
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Escherichia coli Facultative Anaerobic Growth through agar. |
Identify bacteria and its aerotolerance. Describe growth. |
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Clostridium sporogenes Strictly Anaerobe |
A test tube inoculated with a needle through agar deep has growth only on the bottom. This describes which bacteria and what kind of aerotolerance? |
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Thioglycollate Media |
- Referred to as reducing media and is designed for cultivating anaerobes without an anaerobe jar - Binds molecular oxygen and removes it - Must be boiled and autoclaved to remove all dissolved gases from medium including O2 - Sometimes above 1 cm red layer is observed because of contact with air |
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Resazurin (pink/red) |
- Dye that is included in medium to indicate presence of O2 by turning ____________, otherwise it is colorless. |
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Pseudomonas aeruginosa Aerobic Growth on top. |
Identify bacteria and its aerotolerance. Describe growth. |
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Escherichia coli Facultative Anaerobic Uneven growth through thioglycollate medium. |
Identify bacteria and its aerotolerance. Describe growth. |
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Clostridium sporogenes Strictly Anaerobe No growth on top |
Identify bacteria and its aerotolerance. Describe growth. |
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Anaerobe Jar |
- Provides an anaerobic environment by using a hydrogen generator to produce hydrogen gas to react with oxygen in the presence of a metal catalyst to produce water |
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Methylene Blue Resazurin |
2 Types of Oxygen Indicators - In its absence, colorless |
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Direct Method |
- Basic mean to follow bacteria growth that produces actual numbers of cells - Includes counting chambers and viable plate count |
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Counting Chambers Viable Plate Count |
2 Types of Direct Methods for following bacterial growth |
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Indirect Method |
- Basic mean to follow bacterial growth that does not produce real numbers but relative amounts compared to the beginning of the experiment - Include absorbance (spectrophotometer) and weighing dried samples of cells collected by centrifugation |
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- Absorbance (spectrophotometer) - Weighing dried samples of cells collected by centrifugation |
2 Types of Indirect Methods of following bacterial growth |
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Viable Plate Count |
- Take sample from culture, dilute it, then plate samples to grow - Colonies can be counted, then multiplied by the inverse of the dilution to attain original number of colony forming units in broth culture - Only living cells are counted |
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Counting Chamber Absorbance
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2 Methods that count both living and dead cells
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Important to tell if one is sick or something is contaminated |
Counting Bacteria is important because? |
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TNTC Too Numerous to Count |
- 10^9 Cells/mL - Lawn (Confluent Growth) observable on plate - Error of margin becomes too drastic |
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Serial Dilution |
- Solves problem of TNTC - Dilutes original sample into series of sterile water or broth tubes - Assumed there will be plated dilution "just right" Goldilocks Principle (30-300) |
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Amount Transferred Divided by Total Volume
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Equation for Dilution Factor |
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Colony Count on Plate divided by Dilution Factor |
How to calculate Colony Forming Units by mL? |
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Short Wavelength (260-280 nm) UV Radiation |
- Mutagenic because it excited nucleic acid bases in DNA and causes formation of thymine dimers - Considered bactericidal since mutations can be lethal to bacteria What is the range? |
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Thymine Dimer |
- AKA pyrimidine dimer - Forms double bonds with each other, because its nucleotide is most susceptible to UV radiation |
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Cystic Fibrosis Multiple Sclerosis |
- DNA polymerase can bind up to 1 million bases per second but gets stuck at T = T; asks it self, should I halt? Or let cell die. - Thus it keeps going with mutation, eventually binding G to T, then later base C - These thymine dimers can cause what 2 diseases? |
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Serratia marcescens |
- Environmental organisms that produced red pigment called prodigiosin when grown at 25C or does not produce the pigment and appears white at 37 C |
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Blood Poisoning |
- Fatal, kills within 8 hours |
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Mouth |
Streptococci Staphylococcus Influenzae Examples of bacteria in the? |
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Opportunistic Pathogens |
- Microorganisms are evolving new mechanisms of pathogenicity and resistance to chemotherapeutic agents
Ex: E. coli, Staphylococcus aureus, Mycobacterium tuberculosis, Neisseria gonorrhoeae |
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Escherichia Coli |
Important part of our natural intestinal flora, that picked up plasmids from other bacteria to produce Shiga-like toxin and become more invasive as seen in the deadly O157:H7 strain
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Neisseria gonorrhoeae |
- Now become resistant to all drugs except one class of cephalosporins |
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Normal Flora |
- On skin that is in competition with transient microbes, and easily transfer them to one another |
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Hand Washing |
- Most effective practice to limit transmission of pathogens from one person to another - Best and easiest method to cut nosocomial infections (hospitals and clinics) which kill thousands of patients every year - Removes all transient microbes and part of your normal flora |
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More More |
The higher the treatment (soap+ scrub+ water) the (less/more) the number of colonies and there are (less/more) number of different colony forms. |
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Antibiotics |
- Not effective against Gram + - More effective against Gram - |
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Susceptibility |
- Means effective disinfectant |
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- Concentration and rate of diffusion of the chemotherapeutic agent on each disk - Density and age of bacterial growth - Thickness and viscosity of medium - Temperature and time of incubation |
Variables of Kirby-Bauer assay |
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Mueller-Hinton Agar |
Appropriately enriched medium to support most microorganisms studied - Prepared to standardized recipe to guarantee global ingredient and viscosity consistency and a precise volume (20 mL) so medium thickness is consistent |
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McFarland No. 0.5 Turbidity standard |
2-3 Drops insures standard turbidity, must be from suspension of inoculum prepared from 24 hour broth culture |
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1.5 * 10^8 bacteria/mL |
McFarland No. 0.5 Turbidity standard yields bacterial counts of about? |
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-Inanimate objects or living tissue
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Difference between disinfectant and antiseptics? |
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Disinfectants |
- Less stable than antibiotics or are volatile and therefore standardization is more difficult - So evaluation is a little more difficult |
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- Clorox Bleach
- Peroxide, betadine - Ethanol |
Example of Disinfectants 2 Examples of Antiseptics Example that can be both |