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37 Cards in this Set
- Front
- Back
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Agar results of the -pGLO LB |
Lawn of E. Coli (confluent growth) |
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Agar results of the -pGLO LB/amp |
no growth |
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Agar results of +pGLO LB/amp |
E. coli colonies
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Agar results of +pGLO LB/amp/ara
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E. coli colonies- green flourescent under UV light |
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On which plate do you find non-transformed E. coli (i.e., exactly the same as the original bacteria)? Why is there a lawn of bacteria, rather than individual colonies, on this plate? |
-pGLO LB agar Lawn of bacteria because all the bacteria put on the LB nutrient agar were able to grow. |
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Explain why the bacteria from the -pGLO sample were not able to grow on LB/amp agar, but some cells from the +pGLO sample were able to grow on LB/amp agar, resulting in colonies. |
Bacteria from the -pGLO sample were inhibited by the antibiotic ampicillin (amp) in the agar. Some bacteria in the +pGLO sample took up the pGLO plasmid (i.e., were transformed). The plasmid confers resistance to ampicillin (see below), so these cells are able to grow on agar that contains ampicillin. |
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What is the name of the gene on the pGLO plasmid responsible for the growth of some +pGLO cells on LB/amp and LB/amp/ara agar? Describe the activity of the protein encoded by this gene. |
The pGLO has the ß-lactamase (ßLA) gene that codes for ß-lactamase, an enzyme that inactivates ampicillin (outside the cell). |
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What is the name of the gene responsible for the bacteria's fluorescent green color on one of the plates? |
The green fluorescent protein (GFP) gene. The GFP gene was originally isolated from the genome of a jellyfish. |
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Explain how arabinose causes pGLO-transformed bacteria to express the fluorescent green color |
The transformed bacteria make the araC regulatory protein (b/c araC is another gene on the pGLO plasmid). In the presence of arabinose, araC activates RNA polymerase to bind to the promoter that controls the GFP gene and transcribe the gene. |
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For exercise 21, what was the phage titer average results? |
113 plaques |
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What is a bacteriophage? |
A virus that infects bacteria |
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What is the difference between Lytic and Lysogenic? (temperate) |
Lytic- the cell lyses about 30 mins after infection, releasing approximately 100 virus progeny. Lysogenic- The DNA of the phage integrates into the bacterial chromosome and is replicated with the bacterial DNA. |
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To study a virus, you must also grow it's host. How do you do this in lab? Why are the cells mixed in soft agar? (generally know this) |
Phage are too small to be seen in a light microscope, but can be detected on a bacterial lawn. Phage and their host are mixed in a tube of soft agar and then poured on top of an agar base plate. Soft agar contains about half the concentration of standard agar so that the phage can diffuse more easily. During incubation, bacteria multiply and produce a think covering of bacteria, (bacterial lawn) except in those places where phage have infected and killed the bacteria. The infected bacteria lyse, leaving clear areas called plaques. The plaques can be counted, just like colonies. (pfu's) |
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How would an agar plate appear after incubation if you forgot to add the bacteria to the phage/bacteria mixture? |
There would be no growth |
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How would an agar plate appear after incubation if you forgot to add the phage to the phage/bacteria mixture? |
There would be confluent growth of bacteria and no plaques |
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How can you distinguish a lytic phage from a Lysogenic (temperate) phage by observing their plaques? Why do they look like this? |
A lytic phage produces clear plaques and a temperate phage produces cloudy plaques. Temperate phages produce cloudy plaques because many cells within the plaque were lysogenized instead of lysed and thus continue to grow and multiply. |
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Why can a plaque be considered similar to a bacterial colony? |
A bacterial colony can be produced by a single cell and a plaque in a lawn of bacteria can be formed by a single virus particle. |
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If you continue to incubate the plates, will the size of the plates increase? |
Phage cannot replicate in cells that are no longer actively dividing and therefore the size of the plaque does not increase after bacterial growth ceases. |
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Give two reasons why it is very important to correctly diagnose and treat strep throat |
So the disease does not progress to rheumatic fever or glomerulonephritis |
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NAme one genus of Gram negative cocci
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Neisseria or Moraxella |
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If a student had a cold and a sore throat caused by a virus, how would the virus appear on the blood agar plate?
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Viruses do not grow on plates |
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"Normal Streptococci" is alpha hemolytic strep. What does it do to RBC's and how does the blood agar look? |
Partially lyses the RBC's Results in greening of the agar |
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Streptococcus pyogenes, Beta hemolytic What does it do to RBC's and how does blood agar appear? |
It completely lyses RBC's clearing zone where RBC's were killed |
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Gamma What does it do to RBC's and how does blood agar appear? |
No hemolysis Does not mean there are no pathogens |
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Can you tell whether or not an organism can ferment a sugar if it does not grow in the broth? |
If the organism has not grown in thebroth there is no way to know what its fermentation capabilities are. Many organisms can grow in the the base broth without the carbohydrate, but if theycan ferment a sugar that is available, they will. |
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How can an organism have a positive test for acid from glucose in a fermentation tube but have a negative methyl red test, which is also a glucose fermentation test? |
The pH indicator in the fermentation tube will turn red even if slight amount of acid is produced. Methyl red will only turn red if large amounts of acid are produced. |
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Were there any organisms that did not ferment sugar? |
Obligate aerobes do not ferment- among these is Pseudomonas.
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How do you test Methyl Red? What are you testing for? |
Innoculate an MRVP broth. Incubate, then add three drops of Methyl Red. For production of mostly acidic fermentation products, indicating a low pH, broth will turn red Does the bacteria produce acidic products? Negative=yellow |
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Carbohydrate Fermentation test |
Innoculate a carbohydrate broth tube Can the bacteria ferment glucose, lactose, sucrose, mannitol? For fermentation of sugar, broth will turn yellow. Negative = still red. If it did not ferment sugar, it will not produce gas |
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How do you test Voges Proskeur? What are you testing for? |
Innoculate a MRVP broth. Incubate then add 15 drops VPA, 15 drops VPB, 5 drops creatine. Does the bacteria produce non- acidic products? For production of mostly non-acidic fermentation products, broth will turn red at top. Negative=no red |
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Indole production test |
Can the bacteria break down tryptophan? Innoculate a tryptone broth. Incubate then add 10 drops of Kovac's reagent. For tryptone breakdown, broth will turn red on surface. Negative=yellow tryp--(tryptophanase)--> pyruvate + CO2 + indole |
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Catalase test |
Can the bacteria break down hydrogen peroxide? H2O2 --(catalase)--> H2O + O2 Bubbles will appear if positive for hydrogen peroxide breakdown. |
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Casaeinase test |
Innoculate a skim milk agar Can the bacteria break down casein? Is the Caseinase enzyme present? Casein (milk protein) --caseinase--> amino acids If negative, bacterial line will still appear milky and opaque. Positive -clearing around bacteria |
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Oxidase Test |
Does bacteria have oxidase? Add one drop of oxidase. Bacteria that do aerobic respiration will look black. Negative=no change. |
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Citrate Test |
Can bacteria break down citrate? Positive will be blue, green will be negative with a high pH. |
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Urease Test |
Can bacteria break down urea? Positive result will appear hot pink. Negative will stay orange. |
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Explain how fermentation of the carbohydrate causes the color change and name the chemical in the broth that changed the color |
Phenol Red Fermentatiom-more acid lower ph |
