Bmsc 240

Front 1

Describe the characteristics of a macConkey Agar plate:

Back 1

Selective: gram positive bacteria can not survive because it contains bile salts and crystal violete. Differential; media contains lactose and ph indicators: bacteria with the enzyme to break down lactose will change to pink and those without the enzyme will not change color. The pink color will spread due to acid diffusion. Neutral red is the Ph indicator and will turn red at ph's less then 6.8 and clear at greater than 6.8.

Front 2

Describe the characteristics of a Mannitol Salt agar plate:

Back 2

Selective: only members of the staphylococcus genus can survive at such high salinity's ( 10%). Differential: phenal red is the ph indicator: if the specimen can ferment the sugar alcohol its by product will cause the agar to change to yellow green color. If the specimen can not ferment it will remain red.

Front 3

Describe the characteristics of a blood agar plate:

Back 3

Enrichment: contains general nutrients and 5-10% sheeps blood. Differntial; test for hemolytic activity: Cells with the exoenzymes that burst red blood cells will change the color of the media. Beta-hemolysis- creates a clearing - see through, Alpha hemolysis - creates a greenish color = partial lysis and Gamma hemolysis- nothing occurs. Hemolytic activity is enhanced in anaerobic coniditons - candel jar

Front 4

Describe the characteristics of a saboraud Dextrose Agar plate:

Back 4

Similar to the nutrient agar but contains a high concentration of glucose (2%). Bacteria will not grow well in this plate; mostly used for growing molds fungi, yeast, things get fuzzy. Selective for yeast and molds due to the high sugar and acidic PH.

Front 5

Describe the 3 differential test achieved by enuculating SIM deep:

Back 5

1. Sulfur Reduction:good for identifying enteric bacteria ( live in your intestines); SIM media contians nutrients. If the organism is able to reduce sulfar to hygrogen sulfide, the hydrogen sulfide will combine with the iron = ferric = black percipitate. Indole production: one of the nutrients in the SIM deep media is tryptophan, some bacteria posse the enzyme trytophanase which hydrolyzes tryptophan. The end products are ammonia, pyruvic acid adn indole. Using a Kovak reagent it will turn red if indole is present. Motility: If cells grow away from the stab - cells are motile.

Front 6

What is the purpose of the oxidase test?

Back 6

The oxidase test identifies organisms with the enzyme cytochrome oxidase present which is part of the electron transport chain. The reagent will change color if cytochrome 3 oxidase is present.

Front 7

What is the purpose of using a dichotomous key?

Back 7

It is a series of yes and no questions that allow you to narrow the field of bacterial species to identify an unkown.

Front 8

What are other useful identification systems of bacteria in a medical setting?

Back 8

Enterotube II: it is designed for oxidase(-), gram(-) bacilli. Several metabolic tests are all shoved into one tube. + and - scores are compared to a predetermined listing.
API 20E: Used for enteric gram (-) species ( have to innuculate each tube)

Front 9

Identification systems: DNA testing and antibody testing?

Back 9

DNA testing: extractin DNA and testing it using various procedures: more powerful tool for identification but may be limiting
Antibody testing: immune response: assaying if specifc antibodies bind to unkown- ie rapid strep test to determine if throat infection is a group A strep.

Front 10

Describe the steps of Binary Fission:

Back 10

1. cell elongates and DNA is replicated - need lots of NRG and nutrients. 2. Cell wall and plasma membrane begin to grow inwards. 3. Cross-wall forms completely around divided DNA. 4. Cells seperate.

Front 11

What are the different phases of bacterial growth?

Back 11

1.Lag phase 2. exponential growth 3. stationary phase 4. logarithmic decline

Front 12

What is the difference between a viability and whole count?

Back 12

You can see that yes this grow or no it did not verus a visual count you are counting dead and alife cells = whole count

Front 13

What is an example of a method of counting bacteria (indirect, total)

Back 13

Turbidity Estimates: spectrophotometer; there is a factor used for e.coli .1A600= 1*10^8CFU/ml

Front 14

What is an example of a method of counting bacteria that is (direct, and total)?

Back 14

Counting chamber:these cells that are counted are dead because they have to be stained.

Front 15

What is an example of a method of counting that is indirect and viable?

Back 15

Serial dilutions; there is room for error however bacteria is a live and when counting you are only counting those that grew in the selected media.

Front 16

Terms: Antibiotic, Bactericidal/Bacterlytic, bacteriostatic, mechanism of actions, spetrum of activity, effective dose and toxic dose?

Back 16

Antibiotic: has selective toxity ie kills the bacteria but not the host. Bactericidal - kills bacteria, lytic causes the cell to lyse and die. -Bacteriostatic - inhibits further growth. Mecahnism of action: what is the target site of the antibiotic. Spectrum of activity ( narrow - slect for a few target species, broad - works for many species). Effective dose: is the dose requried to achieve the desired action - will depend on the drugs 1/2 life, distribution, etc... Toxic dose: the dose that causes adverse effects.

Front 17

Turbidity Estimates:

Back 17

Use of the spectrophotometer; an indirect total measurement of the number of bacterial cells present in the cuvette are measure. The absorbance values relates to the light that was not scattered by the bacterial molecules.

Front 18

Counting Chamber:

Back 18

A grid with each square having a preddefined volume is washed with the bacterial suspension. This is a direct method of counting the total amount of bacterial cells present. May need to stain the cells - death

Front 19

Serial dilutions and plate counts:

Back 19

using dilution, an indirect viable count of bacterial cells can be made. The sample is diluted so that the resulting plate colony is wihtin 25-300 CFU/ml

Front 20

What are some antibiotic sources?

Back 20

Various bacteria and fungi, the best sources are soil dwellers

Front 21

What is meant by narrow and broad spectrum of antibiotic activity? give an example of an narrow and broad antibiotic.

Back 21

Narrow spectum - limited activity to a specific microbial. Braod spectrum anitbiotic is indescrimint in is application to various microbials. Narrow antibiotics are found mostly in eukaryotics such as fungi because they are closer organism to us it is harder to treat as the drug has to be very specific = malaria. Tetracycline- bacteria has a broad spectrum of application. Viruses - host cell = acyclovir

Front 22

Antimicrobial targets: discuss the obvious targets

Back 22

1. Cell wall syntheis
2. Transcription -inhibition of nucleic acid replication 3. Translation - inhibtion of protein synthesis 4. Enzymatic activity, synthesis of essential metabolites 5. Replication 6. injury to the periplasm

Front 23

What are the characterisitcs of cell wall inhibitors:

Back 23

Penicillian, cephalosporins ( lytic activity). The antibiotics are natural and semi-synthetic. All contain a beta lactam ring- which targets the transpeptidase ( formation of peptide bonds in the cross links). Naturally occuring penicillin works only against gram positive bacteria. Mode of action: binds irreversibly in the periplasmic space and inhibt the action of the transpeptidases thus decreasing the integretity of the peptidoglycan layer - autolysis

Front 24

Whata re the characteristics of protein synthesis inhibitors:

Back 24

They have a static action - halt growth by interfereing with ribosome, mRNA, and tRNA complexes. Some common drugs: chloramphenicol- broad causes anemia, Streptomycin can cause deafness, neomycin - topical of streptomyocin, tretracyline, broad, discolours teeth and sun sensitivity

Front 25

Reasons for modifying penicillins:

Back 25

1. To make it resistant to acids when digested in the stomach
2. To make it more stable - longer 1/2 life
3. broaden its spectrum of activity.

Front 26

What are some characteristics of plasma membrame damaging antibiotics?

Back 26

This antibiotic tagets plasma memebranse which is not a good target. Polymyxins damage gram - membranes; it is poorly absorded and toxic to the kidneys. Daptomycin - depolarizes gram + membranes. If polymyxins are mixed with bacitracin = gram + cell wall inhibitor= topical antibiotic low usefulness as systemic drug = polysporin

Front 27

Dicuss the characteristics of nucleic acid syntheiss inhibitors:

Back 27

Rifamycin/rifampin inhibt the synthsis of mRNA-STATIC. They can pass into the glycolic wall and thus are good for the treatment of TB and leprosy = highly permeable. Quinilones: have a speicifc target = DNA gyrase which elivates the tension or torque that the replication forque causes in bacterial cells only. When DNA gyrase is inhibited replication is inhibited.Well known quinoline = Ciprofloxin = antrax

Front 28

Discuss metabolic inhibitors:

Back 28

Are synthetic sulfa drugs used in WWII originally used as dyes. Solders used it to put on wounds to prevent gangrene. Sulf drugs block the folic acid enzyme needed to create thymine and uracil - humans are unable to synthesize folate - get through diet. Sulfa drugs have no effect on us because we don't make folic acids

Front 29

Oxidase test;

Back 29

Test for the presence of cytrochrom oxidase required in the electron transport chain. the oxidase reagent will change color in the presence of the cytochrome oxidase enzyme.

Front 30

Catalase test:

Back 30

Catalase is the enzyme that breaks hydrogen peroxide (H2O2) into H2O and O2. Hydrogen peroxide is often used as a topical disinfectant in wounds, and the bubbling that is seen is due to the evolution of O2 gas. H2O2 is a potent oxidizing agent that can wreak havoc in a cell; because of this, any cell that uses O2 or can live in the presence of O2 must have a way to get rid of the peroxide. One of those ways is to make catalase.

Front 31

What is the purpose of sensitvity testing?

Back 31

To determine what antibiotic should be used and in what concentration.

Front 32

What is a disk diffusion assay?

Back 32

It is used to determine what drugs are useful against a bacteria. It provides non quantitative data as its more of a visual queu. The distance from the disk provides ranges for resistance and sensitivity.

Front 33

What is an Epsilometer tests (Etest)?

Back 33

It ustilizes an strip of an antibiotiv to determine the therapeutic dose. The antibiotic strip provides quantitative data. Also provides information regarding the minimal inhibitory concentraction required.

Front 34

List some of the things people do that cuase the accerlaration of the antibiotic resistance?

Back 34

1. discontinue antibiotics before the pathogen is eradicated;2. Using poorly chosen antibiotic; 3. Use of antibiotics against viral infections; 4. preventative use of antibiotics in animal feed.

Front 35

Modes of resistance transfer include:

Back 35

1. drug modificaton/destruction - the drug is inhbited by the bacteria 2. Pathway protection: synthesis of false targets; 3. Target alteration; mutations in a ribosome change the confirmation of a binding site; 4. Rapid efflux: the organism is capable of actively pumping out the antibiotic via heavy metal pumps- no specificty. 5. Alternative pathways; essential pathway becomes none essential - scavenge for folate whith out using the pathway

Front 36

Spreading of antibiotic resistance: How is this achieved?

Back 36

Plasmids are a smll extra-chromosomal ring of DNA that can be transfered to toher individuals or species. Natural selection will proomtoe these rare occurances.

Front 37

Antibiotic resistance how can this be combated?

Back 37

1. using a combination of synergistic antibiotics; decreases the bacteria's ability to develop resistance. 2. withholding newer antibiotics to reduce bacterial exposure until necessary.

Front 38

What are proteins? what is their basic structure?

Back 38

Proteins are structural components of cells, enzymes etc....They are made of an amino, carboxyl and R group. There are 20 different R groups. R group interactions govern the secondary and tertiary structures. The charge of some the R groups are ph dependent. Hydrophobic non polar proteins tend to fold internally- reducing their exposure to water. Hydrophillic polar proteins tend to fold externally. The specific AA composition and order will be exploited when purifying one from a mixture.

Front 39

What is an enzyme/

Back 39

It is a protein or RNA that acts as a catalyst for a chemical reaction: makes the reaction more favroable at physiological conditions: takes an impossible reaction and makes it possible.

Front 40

What is the purpose of enzyme specificity?

Back 40

Enzyme specifity is a product of : 1) haveing the right shape to hold the substrate in place; 2) catalytic center to facilitate the reaction ; may need cofactors- organic molecules of metal ions.

Front 41

List the 4 types of reactions?

Back 41

1. A goes to B = isomerization
2. A + B goes to C = condensation
3. A goes to B + C = lysis
4. A + B goes to C+ D = complicated

Front 42

What is the action of Enzymes on activation energy?

Back 42

Enzymes lower the activation energy making the reaction more favorable.

Front 43

How are reactions measured?

Back 43

Rate of reaction = velocity
Reaction rate (v) = how fast the substrate is converted to product or how fast product is accumulated. v = -Change S/ Change in Time or v = + change in product/ change in Time

Front 44

When investigating the basics of enzymatics what things might a researcher be interested in looking at?

Back 44

1. First they need a assay that can be quantified. 2. determine what the optimal conditions are for that enzyme. 3. Determine how quickly that enzyme can catalyze the reaciton. 4. how the speed varies when the substate concentrations vary.

Front 45

What 2 things can limit the rate of the enzymatic reaction?

Back 45

1. The rate can be limited by the amount of available substrate. 2. the rate can be limited by the production of product.

Front 46

What is the Michaelis-Menten Equation? When do the Michaelis kinetics hold true? What is KM?

Back 46

vo= [S]*vmax/[S] + km ; the kinetics hold true when the substrate concentration is in excess of the enzyme. KM = michaelis constant which is experimentally determined Km= [s] when V0 =1/2 vmax ( max change in substrate over time). KM is the enzymes affinity for a substrate; low Km ES complex will not break appart easily = High affinity.

Front 47

What is competitive inhibition?

Back 47

This occurs when a agonist compound binds to the active site of the enzyme preventing the ES complex formation. This typ of inhibition can be over come by increasing the concentration of the substrate = reversible, Vmax remains constant however the substrate concentration to reach 1/2 vmax increases as does Km ( more enzymes are occupied with the substrate ; decrease in affinity)

Front 48

what is uncompetitive inhbition?

Back 48

A compound binds to the ES complex and slows down ht transition to EP; but does not bind to the enzyme itself. ( iced up lock). Vmax decreases, and becuase the enzyme itself is not inhibited,Km is lowered increases the substrates affinity

Front 49

What is mixed/noncompetitive inhibition?

Back 49

A compound binds to Enzyme and changes its shape of the active site = antagonistic action. There is less free enzymes available, and vmax decreases while KM remains constant as any free E will have the same affinity. This is irreversible inhibition as adding more substrate will not overcome the inhibition

Front 50

Why do we use the lineweaver-burk plot?

Back 50

It uses the double reciprocal of rate and concentrations to place Vmax and Km o the X and Y intercepts. With a slop = Km/vmax. X axis = 1/[s] and y axis = 1/vo; x intercept = -1/km and y intercept = 1/vmax

Front 51

How is enzyme standardization achieved?

Back 51

This is achieved by comparing the amount of enzyme needed to complete a set amount of reactions in a set period of time. 1 unit (u) of enzyme is the amount needed to catalyze 1 umole of substrate in 1 minute,

Front 52

What is enzyme activity?

Back 52

= the number of units per enzyme volume
Activity = Change [] * rxn volume/ enzyme volume
units = U/ml or U/ul

Front 53

What is specific activity?

Back 53

= activity/ [protein] units U/mg or U/ug

Front 54

What should be monitored to ensure that the extract is stable?

Back 54

Ph - chanages want to keep in a buffered solution. Heat denaturation- keep sampple cold
Oxidation: free thiol groups from cysteines may react to form disuldies - use DTT of BME to prevent accidental cross linking
Proteolysis - enzymes that break down protiens - keep at lower temps or add proteas inhibitors

Front 55

When doing a purification of a an assay what things should the assay be?

Back 55

Sensitive, specific, rapid, and quantitative

Front 56

What things should be tracked during purification?

Back 56

Yield= how much is recovered after each step % recovery activity = measure of rate of enzymatic reaction and specific activity or purity - which should be increasing

Front 57

List some of the means of purification by properties?

Back 57

Solubility - salting out,; Ion charge : ion exchange chromophotography; Polarity: revers pahse
Size: dialysis
Binding specificity: affinity chromatogrpahy

Front 58

Before perfoming an purification what should be done/

Back 58

Initial purification should include the centrifuge to get rid of the big chunks which will settle at the bottom

Front 59

What is differential precipitation?

Back 59

The supernatant = fluif at the top of the centrifuge tube can be further treated to make some of the proteins in the mixture less soluable. - alter temperature, salinity or ph

Front 60

What is salting out?

Back 60

It is a common reversible treatment: as salt concetrationincrease the + and - ions will compete with the hydrophillic surface AA for water. Less water available some proteins will loss out. The protein moelcules with insufficient hydraption will aggreagate and lose soluability

Front 61

What is Dialysis- De-Salting?

Back 61

The excess salt must be removed or it could further impede subsequent isolation steps. To remove the salt a dialysis tube used for( with high protien and salt ) with pores with a specific molecular weight can pass through. the tube is sitting in a buffer solution and this needs to be performed 3 times and the salts will move out.

Front 62

What is chroatography?

Back 62

It is a technique used to seperate mixtures based on physical proterties such as size and charge

Front 63

What are the 2 phases that are looked at in chromatography>

Back 63

Stationary phase- a substance that the compounds to be seperated pass by or interact with. Verus the mobile phase the carrier for the coupounds to be seperated - water

Front 64

What is a mixed sample as it relates to chromatography?

Back 64

A mixed sample will have some items with a higher affinity to the mobile phase were as others have a high affinity for the stationary phase. different affinities = different rates - seperation of molecules

Front 65

Size exclusion Chromatography: Large, medium, and small beads?

Back 65

Large proteins can not enter the beads and those they will migrate around them - travel only in Vo and travel the fastest. Medium sized proteins travel both in the beads and outside the beads. there travel pathway includes both Vo and Vi travel slower then the large proteins. small proteins travel throug the beads - there is no Vo or Vi just Vt - move the slowest.

Front 66

What is the partition coefficient?

Back 66

used as a way to reproduce the reuslt from variable columnes- way to standardize elution measurements ( every colume will vary in volume). the particition coefficient is the fraction of the volume of the beads available to the sample - Kav =( Ve-Vo)/Vt-V0

Front 67

What is ion exchaneg chromatography?

Back 67

Things can get stuck in the coluem: A colume of positively or negatively charged beads forms the stationary phase. Anion exchange- colume is + ve and target is -ves. An Cation exchange- colume is - ve and the targe is +

Front 68

How does the ion exhange work>

Back 68

Protiens in solution are carried down by gravity and will get slowed by the beads proportionally based on charge. Specific combination of mobile and stationary phases are chosen to retain protein of interest. Nothing will flow through until the void volume is excreted.

Front 69

What is electrophoresis? How does size, charge difference, and shape impact movement?

Back 69

Electrophoresis is the seperation of ions in a electric field. Velocity of movement is directly proportional to charge and inversely proportional to shape and size. As the shape and size increased velocity decreases, as the diffference in charge increases the velocity increases

Front 70

What is Gel electrophoresis? What type of gel is used? Why is it used?

Back 70

Gel electrophoresis utilizes a gel to slow down the movement of the proteins to create seperation. The gel is used to coat all the proteins given them all a consisten charge. A type of gel used is SDS-PAGE= deterfent that will denature the proteins and give them a negative charge.

Front 71

How is cross linking mesh work in the gel acheived?

Back 71

Acrylamide ( found in gels) used as a cross link to create pores for which the protiens will not be able to pass through. Adding acrylamide and methlene bis acrylamide in a gel does not ensure cross linking. The use of ammonium persulfate TEMED catalyze the polymerization of the double bonds to form the cross linking due to the free radical formation

Front 72

What is a ladder?

Back 72

It is a collection of known sized proteins

Front 73

What types of visulalization dyes are use in electrophoresis?

Back 73

1. Croomasie blue: stain 10 min soak in buffer 10 min *2-3 times
2. silver nitrate stainning is more complicated more sensitive applications
3. Fluorescent dye staining: requires specialized equipment