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182 Cards in this Set
- Front
- Back
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How do you obtain a template for PCR?
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Template is made from adding cells to Tris Buffer, using glass beads to help break up during vortex. Then heat is used by boiling on float in locked PCR tube, Vortex again for 10 Mins.
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What are the names of the Primers we used in PCR? |
Bacteria 8 Forward, Reverse 1492-r or 1530-r |
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What is the purpose of PCR primer? |
They are designed to stick to the beginning portion and the ending portion of a gene. It gets us the section we want to analyze |
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What is the name of the gene that our primers can stick to?
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16S rDNA |
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Approximately how long should your PCR product be? |
1500 bp nucleotides long |
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What is the name of the special nucleotides that are required for the sequencing method at UC Davis? |
ddNTPs- dideoxynucleotide triphosphates |
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What mades ddNTPs different from regular nucleotides? |
They have no OH- group at 3' carbon which causes termination and chromofluor @ base |
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What website is used to identifiy unknown organisms using DNA
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NCBI |
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What program is used to compare the contiguous sequence?
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BLAST |
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What is PCR?
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It is directed replication technique. Directed by primer sequences |
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Who invented PCR and When?
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Kerry Mullis 1986 |
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What are the ingredients for PCR? |
Tris Buffer, Primer Mix, |
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What is the first step to PCR?
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Denaturation- 94-95 degrees0 heat will break H-Bonds to single strands from double |
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What is the 2nd step to PCR?
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Annealing (Priming)- 45-60 Degrees- cool- allow HBonds between primer and template. dNTPs also HBond to template and Taq polymerase attaches |
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What is the 3rd step to PCR?
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Extension- warm up to 72 degrees- optimum for Taq polymerase to make H bonds @ 1000 bonds/min |
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What does PCR mean? |
Polymerase chain reaction |
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Describe PCR Process
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2. Vortex 3. Boil on float for 10 mins 4. Vortex 10 mins (End with Template DNA) 5. Add Water, Primer mix, Template DNA, Taq master mix>> Place on ice |
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What is the dideoxy chain termination method of DNA Sequencing?
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Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.
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What are the ingredients for the Sanger method?
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* Template DNA *Heat *dNTPs * Primer *Taq DNA polymerase *ddNTPs |
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What is the product of the PCR reaction?
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Milions of pieces of genes (16S rDNA) * Many are different lengths and not full lengths * Thousands of copies of each length, every molecule ends with ddNTP. |
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How do you understand the sequence? |
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What is an electropherogram? |
A 4 color record of DNA sequence generated by electrophoresis |
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How do you make a contigious sequence? |
Use multiple electropherograms and delete overlaps to prevent missing data |
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What does NCBI Stand for? |
National center for biotechnology information
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What does BLAST stand for? |
Basic local alignment search tool |
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What is a plasmid?
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a genetic structure in a cell that can replicate independently of the chromosomes, typically a small circular DNA strand in the cytoplasm of a bacterium or protozoan. Plasmids are much used in the laboratory manipulation of genes |
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What is a copy number?
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# of plasmids a cell will maintain |
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How is the copy number determined? |
Low copy =< 10/cell High copy = 20-200/ cell |
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What are marker genes?
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allows for selection of cells within a plasmid
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Which marker gene did we use? |
bla- Beta-lactamase (Amp R) * amp -resistant * Very small in size |
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What enzyme does the marker gene code for? |
Beta lactamase- the enzyme inactivates penicillin- family antibiotics |
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Why would you want to isolate the plasmid DNA from bacteria?
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to change characteristics about the bacteria, make it resistant or glow green, etc.
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What is a cloning vector? |
It has the DNA needs to replicate a gene in vivo. * It must have an ori, cloning site, marker gene. |
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What is an expression Vector? |
An expression vector has all the DNA necessary to replicate and express ( transcription and translation) a gene in vivo
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What is gel electrophoresis?
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Gel electrophoresis is a technique using electricity to separate DNA of diferent sizes.
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How does gel electrophoresis work? |
DNA has a negative charge and moves toward the positive pole. Smaller pieces move faster than larger.
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What is a restriction enzyme? |
Bacterial enzymes that cut DNA- It breaks phosphodiester bonds- Only will cut at a specific letter sequence (recognition sequence- restriction site)-These enzymes DO NOT cut the host cell DNA but instead have evolved as protection against viral attack- e.g. restriction endonuclease cut bacteriophage genome DNA if it gets into the cell.
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How are restriction enzymes named? |
They are in Italics. * First letter is Genus - CAPS * Next are the abbreviation for specific epithet- lower case * Strain Letter- CAPS * Roman numeral 1- I- means first enzyme found |
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What does sequence Alu1 recognize?
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AG|CT
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Is Alu1 a blunt cutter or does it leave sticky ends? |
Blunt ends
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What is RFLP? |
Restriction Fragment length polymorphism * pieces of DNA that results from mixing restriction endonucleases + DNA * Many different lengths of DNA results depending on sequence of DNA molecule |
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How is RFLP Generated? |
. |
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How does RFLP relate to DNA fingerprinting? |
It can be used to compare two DNA samples, it would show you if two sequences were similar
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What is transformation? |
Adding a plasmid to bacterial chromosome and adding gene to bacteria to change a characteristic
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What is a competent cell?? |
A cell that is capable of taking in DNA
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Describe the transformation experiment
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inserting pGLO into bacterial chromosome
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What organism did we use for the transformation experiement? |
E Coli
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Re: Transformation experiment How did we make it competent? What does that mean? |
1. Grow culture to mid-log phase 2. Centrifuge and pour off supernatant 3. resuspend in cold calcium chloride * inactivates the enzyme to repair cell walls. This allows for the gene to make it into the cell. 4. No Air bubbles 5. Mix cells and plasmids 6. Heat shock 42*C. Add TSB and incubate for hour at 37* |
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Re: Transformation experiment What plasmids did we use? |
..pU19, pGEM, pGLO |
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Re: Transformation experiment How come transformed bacteria were not killed on ampicillin plates? |
.because they were transformed, they acquired the ampicillin resistance. |
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Re: Transformation experiment What do the bacteria make that makes them resistant to ampicillin? |
.-
B-lactamase- bla gene |
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Re: Transformation experiment How does the gene that makes the bacteria amp resistant work? |
. |
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Re: Transformation experiment Why did some colonies glow green on the TSA+AMP+Arabinose plate? |
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Because they acquired pGLO |
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Re: Transformation experiment Why did some of the colonies NOT Glow? |
Because some did not acquire pGLO
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1. Which type of hemolysis reaction is shown here and how do you know? 2. What other categories of hemolysis were observed in lab? |
*Clearing or Yellowing tint around colonies- Betahemolysis- Complete break down of blood cells |
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What is the test and what are results for B? |
Blood Agar *Green medium, slighty changed- Alphahemolysis- Partial break down |
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What is the test and what are results for A? |
Blood Agar- Betahemolysis- Clearing or Yellowing tint around colonies- Complete break down of blood cells |
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What is the test and what are results for C? |
Blood Agar *Medium unchanged- Gammahemolysis_ Organism cannot break down blood cells |
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What is the test? explain results for positive |
Bile Esculin (Esculin Hydrolysis)
(+/-) Growth but medium is not black- not Resistances to bile salts, but cannot break down esculin B- +/+- Medium Black-not Resistant to bile salts and break down esculin |
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What is the test? explain results for negative |
Bile Esculin A- Negative- no Growth (Organism sensitive to bile salts) |
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What is the name of the test? Which of these tubes contains organisms that are capable of fermenting the carbohydrate present? |
Carb Deeps, Right- Shows Bubble- which means ferment into gas, and because it is yellow, it can also ferment to acid. |
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What is the name of the test? Which one shows negative results? |
Carb deep- Left- No change in color from pink medium- no acid or gas production |
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What is the name of the pH indicator in this medium? What is the name of the test?? |
Phenol Red- Yellow Acid- red basic. Carb deep |
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Which of these tubes contains organisms that are capable of taking in and utilizing citrate? |
Left- Medium is blue- Shows positive result- uses citrate as carbon source (has transport protein- citrate permease) |
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What is the name of the pH indicator in this medium?
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Bromothymol Blue |
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What is the name of this test and what is the results for the Right tube? |
Citrate Test-Simmon's Citrate- Negative - Cannot use citrate as sole carbon source- doesn't have citrate permease |
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Which of these tubes contains organisms that are Methyl Red positive?
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Left- Organism can perform mixed acid fermentation |
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The Methyl Red test is a quantitative test used to determine the amount of what end product of glucose fermentation?
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* ask professor |
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What is the name of the pH indicator used for this test?
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Methyl red |
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Which tube set contains organisms that are fermentative? What are the fermentation products present, and how are these products indicated, i.e., what evidence of fermentation is visible? |
Left Set- Yellow (Ferments glucose to Acid and produces Gas (Bubbles)) |
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Which tube set contains organisms that are using a respiratory (oxidative) type of metabolism? |
Right side |
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What can be assumed about the gas requirements of the respiratory organisms on the right? |
aerobic |
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What microbial activity is responsible for the color change observed on the surface of the unsealed tube in tube set on right? |
** Ask Professor- Isn't it from contamination or getting dried out? |
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What is the name of the pH indicator in this medium? |
bromothymol blue (Starts green, turns yellow for acid production. |
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Which of these tubes contains organisms that are capable of producing hydrogen sulfide?
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Right Tube with black precipitate |
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What type of organic compound is being degraded by the bacteria producing the hydrogen sulfide?
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Amino Acids |
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What is the name of the black precipitate present in tube C? |
Iron Sulfate |
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The liquid (reagent) visible on the surface of tubes B and C was used to test for a nitrogen containing compound called what? Which tube contains organisms that can form this substance?
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Kovacs Reagent- does organism produce Indole from tryptophan |
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What is the name of the test reagent (liquid) used in tubes B and C? Organisms that test positive with this reagent are capable of catabolizing what?
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Kovacs Reagent- Positive shows Indole Production- Turns pink. A yellow color denotes a negative indole test after addition of Kovacs Reagent. |
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Which of the three tubes contains organisms that are motile? How do you know? |
Middle one- You can see growth out from the stab |
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What is this test? |
Sim Motility |
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Both of these tubes contain a black precipitate called what?
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Iron Sulfate |
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What metabolic end product is reacting with the iron in the SIM medium to form this black precipitate?
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Hydrogen Sulfate |
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What type of organic compound is being degraded by the bacteria producing the black precipitate? |
SIM test- Bacteria degrades sulfur-containing amino acids to produce hydrogen sulfide, that mixes with the Iron in the medium to create Iron Sulfide |
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Which tube contains organisms that cannot ferment lactose? How do you know? |
Left, The Slant is Pink |
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What is the name of the pH indicator in the TSI medium?
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Phenol Red- Yellow is acid, Red is Basic |
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Are the organisms growing in these tubes capable of forming hydrogen sulfide? How do you know?
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No, Because there is no black precipitate |
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Which tube contains organisms that can ferment glucose and probably sucrose with the production of acid and gas? How do you know?
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?? The right one, it shows all yellow from fermenting to acid and gas formation at the bottom. |
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Which tube contains organisms that cannot ferment lactose? How do you know? |
The left one, because it is not pink towards the top of the test. If the bottom 2/3 is yellow and the top is pink, you know that it cannot ferment lactose, but can ferment glucose and fructose |
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What is the name of the pH indicator in the TSI medium?
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Phenol Red |
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Which of these tubes contains organisms that are capable of urea hydrolysis?
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The far Right |
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What is the name of the enzyme involved in this process?
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Urease- Urea Hydrolysis test |
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What is the name of the pH indicator in this medium?
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Phenol Red- Urea Hydrolysis test |
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What change in pH is indicated by the color change in the positive tube, and what metabolic end product is responsible?
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CO2+NH3+H2O>NH4+ + OH- (Causes pH increase) hydrolysis of the urea- Urea Hydrolysis test |
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Which of these tubes contains organisms that test positive for the Voges Proskauer test?
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Right one- Red |
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What end product of glucose fermentation is being tested for here? What is the name of this test? |
Butanedoil fermentation- VP - Vogues Praskuer |
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The completion of this test requires that a 1ml sample of MR-VP broth containing a 48 hour culture be transferred into a clean screw-top tube, and that 18 drops of two reagents be added. What are these reagents called?
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Barritts A& Barritts B |
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What action must be completed (twice) in order to obtain accurate results for the VP test?
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shake vigorously |
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Replica Master Plate. The replica master plate contains nutrient agar that was inoculated with five different types of bacteria. The three colonies at the top of the plate areStaphylococcus aureus, those at the bottom of the plate are Staphylococcus epidermidis, those on the left are Escherichia coli, those at the right areSalmonella choleraesuis, and those in the center of the plate are Bacillus cereus.A velvet covered block was used to transfer the colony pattern from this plate onto a series of plates containing selective and differential media. The last plate in the series was again nutrient agar. |
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What is the name of this plate? |
Manitol Salt Agar
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What is the name of this plate?
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Tergitol-7 Agar Tergitol-7 agar or T-7 contains chemicals that inhibit the growth of Gram-positive bacteria, but will generally support the growth of (will select for) Gram-negative organisms. The pH indicator in this medium is bromothymal blue, and the carbohydrate present is lactose.The Escherichia coli colonies on the left side of the plate have produced acid through the fermentation of lactose, and have caused the pH indicator to change from green (neutral) to yellow. The Salmonella colonies on the right side of the plate have produced alkaline end products by catabolizing the peptone in the medium, and have caused the pH indicator to change from green (neutral) to blue.Tergitol-7 agar is a good selective medium in that it fully inhibited the growth of the Gram-positive organisms deposited on its surface.
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MacConkey's AgarMacConkey's agar or MAC contains neutral red, crystal violet, and lactose. The crystal violet (a basic dye) inhibits the growth of Gram-positive bacteria, making the MacConkey's agar selective for Gram-negative bacteria. The Escherichia coli colonies on the left have produced acid by fermenting the lactose present, and have caused the neutral red to accumulate within and around their colonies. TheSalmonella choleraesuiscolonies on the right have caused the medium to fade from pink to tan, and are much less colorful. Non-fermenting colonies likeSalmonella tend to be pale and translucent on this medium, while fermenting colonies tend to be bright pink and more opaque.MacConkey's agar is a good selective medium in that it fully inhibited the growth of the Gram-positive organisms deposited on its surface.
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Eosin Methylene Blue AgarEosin Methylene Blue agar or EMB contains lactose, eosin and methylene blue. Like Tergitol-7 and MacConkey's agar it contains lactose, and tends to inhibit the growth of Gram-positive bacteria. It is less effective at inhibiting the growth of Gram-positive organisms than are the other two media.The Escherichia coli colonies on the left side of the plate have produced acid by fermenting the lactose present and have formed dark colonies with a metalic green sheen (irridescent color). The Salmonella choleraesuis colonies on the right are unable to ferment the lactose, and appear more translucent and pale pink in color.Although their growth is poor, all three of the Gram-positive cultures transferred onto the surface of this medium have grown some.
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Positive Control-The nutrient agar plate used at the end of the replica series demonstrates the effectiveness of the replica plating method. All five types of bacteria present on the replica master plate are also present on this plate. The colonies of Staphylococcus epidermidisat the plate bottom are partially obscured by bubbles in the medium.The large colonies at the center of the plate areBacillus cereus. Although these organisms grow well on nutrient agar, they are not halophiles so will not grow on mannitol salt agar.These organisms are Gram-positive, so their growth is effectively inhibited by the chemicals in Tergitol-7 and MacConkey's agar. Their growth on Eosin Methylene Blue is minimal
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What is the name of the test and what does positive results mean? |
Mueller-Hinton Agar- Shows if organism produces a diffusable pigment. Green Section shows positive results for producing a diffusable pigment. |
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What is the name of the test, what is the result for the left, what do results means? |
Oxidase Test- left result is negative, shows organism doesn't have Cytochrome C in ETC |
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Name of test and what is the results for right? |
Lysine Decarboxylase- can organism break down decarboxylase-? Lysine * Results= can ferment, can debarcoxylate lysine |
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What is the name of the pH indicator? |
Bremcresol Purple |
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What is the name of the test, which one is positive? |
Lysine Decarboxylase Test, The far right is positive, it turned purple. |
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Catalase Test:1.Question asked?2.Key ingredients?3.Positive result? (conclusion)4.Negative result? (conclusion)
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1. Does the organism have catalase?2. H2O2 (Hydrogen peroxide)3. Bubbles (organism has catalase4. No bubbles(organism does not have catalase)
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pH indicator bromothymal blue: What color is it in acid? neutral? base?
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Acid: yellow
Neutral: green Base: blue |
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pH indicator methyl red: What color is it in acid? neutral? base?
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Acid: Red/Pink
Neutral: yellow Base: yellow |
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pH indicator phenol red: What color is it in acid? neutral? base?
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Acid: yellow
Neutral: orange Base: red Really Basic: hot pink |
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Top- +- clot, coagulase positive, bacteria tested produces coagulase
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Mannitol Slant Agar- Uses Phenol red as pH Indicator- Left is positive- produces acid from mannitol. |
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Basophil- dark blue granules |
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Eosininophil- red/orange granules in cytoplasm- eosin- stained |
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lymphocyte- Small, mostly nucleus |
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Monocyte- always bigger than RBC |
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Neutrophil- Segmented Nucleus |
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What is selective media? |
Media providing a means of isolating a particular species or type or microorganism, while inhibiting growth of others |
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What is differential media? |
Media supporting the growth of various types of microorganisms while providing an environment that makes it easy to distinguish various different forms (e.g., EMB, MAC, T-7, MSA, etc.). |
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Enriched media?
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Media containing cells, tissue or tissue fractions (e.g., Blood agar and Chocolate agar).
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What type of Medium is Mannitol Salt Agar? |
Selective for Halophiles, MSA also acts as a differential medium for the differentiation of organisms that can ferment mannitol from those that cannot. Yellow means its can ferment acid, |
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What is this media and what can grow on it? |
Mannitol salt, If it is growing it can tolerate the 7.5% sodium chloride |
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What does the growth on the top colonies show? |
It can grow in high salt content and can ferment mannitol to acid (Yellow) |
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What is the pH indicator for the Mannitol Salt Agar? |
Phenol Red- High pH =Yellow, basic=pink |
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What is the name of this test? |
Tergitol-7 Agar- inhibits growth of gram+ organisms |
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What is the pH indicator for the Tergitol-7 Agar? |
Bromo thymol blue is the pH indicator.
Yellow= Acid, Blue= Basic |
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What are the results for the yellow colony? |
Growth and Yellow. Can grow, Gram Negative. and Yellow > Cant ferment lactose |
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What is the name of this medium? |
MacConkey's Agar |
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What type of medium is this? What does it test for? |
Selective- has crystal violet, which inhibits the growth of Gram+ organisms. Also it is Differential- bright pink- it can ferment lactose |
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What type of media is this? |
Eosin-Methylene Blue Agar- *Selective- eosin+ methylene blye- inhibits Gram + Growth * Differential- Metallic Green- Can ferement Lactose to lots of acid, No black or metallic green colonies, it doesn't ferment lactose to acid |
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Eosin-Methylene Blue Agar- Growth shows Gram -, Black/Metallic Green Postive for lactose fermentations |
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What is a bacteriophage?
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A virus that infects a bacteria |
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What is Phage Typing? Be able to identify an unknown bacterium if given phage typing data |
Using known bacteriophage to ID unknown bacteria |
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What is “pfu”? |
Phage forming units |
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If given plaque data, you should be able to calculate the number of pfu/ml |
Like Serial Dilutions |
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What is burst size? |
How many viruses released by infected cell |
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What is burst time? |
Time from attachment to release
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effects of temperature on microbes in water |
The overall effect of high temperatures (heat) is to denature proteins and stop metabolic activity. If temperatures are high enough, the exposed organisms will die. After one minute of exposure to boiling water, most vegetative cells were killed, but endospore-forming bacteria and some other forms survived. After five minutes of exposure, few if any of the bacteria present remained viable. Exposure time does influence the effects of temperature. |
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interpret the effects of UV light on bacterial growth.o What does UV light do to DNA and cells? |
Lid protects the bacterium from UV Rays, Longer UV =more death, Causes thymine dimmers in DNA, used on lab surfaces/air, waste water treatment plants.. doesn't penetrate well |
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sterilization? |
treatment that removes all living cells, viruses, endospores (Doesn't include prions)
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disinfectant |
used for control of growth of an organism on non living surface ( harms eukaryotic cells) |
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antiseptic |
used for control of growth of an organism on living surfaces. (gentle enough to not harm eukaryotic cells) |
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Which is the only method of sterilization that will kill endospores? How does it work? |
Autoclaving- Uses special test tubes with B. stearothermophilus to test effectiveness. *Uses pressurized steam to get to 121*C for 15-20 mins |
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What type of disinfectant is Gram’s iodine? How does it work? |
Halogen- Powerful oxidizers- charge ions attach to proteins and denature, |
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What type of disinfectant is Lab Disinfectant? How does it work? |
Surfactant- disrupts cell membranes in high concentrations> cell lysis |
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What type of disinfectant is Copper Sulfate? How does it work? |
Metal Ion. Interacts with proteins and inactivates them. Antiseptics and disinfectants. |
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What type of disinfectant is Silver Nitrate? How does it work? |
Metal Ion.Interacts with proteins and inactivates them. Antiseptics and disinfectants. |
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What type of disinfectant is Ethanol? How does it work? |
Alcohol- High concentrations denature proteins > kills most |
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What type of disinfectant is Isopropanol? How does it work? |
Alcohol- High concentrations denature proteins > kills most |
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What type of disinfectant is Carbol Fuschin? How does it work? |
Phenol derivative. dirupts cell membranes- kills cells, Lysol Pine sol- used by Joseph Lister |
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Why are Gram+ bacteria easier to kill (with these compounds OR Antibiotics) than Gram- negative bacteria? |
In general, Gram-negative organisms are less sensitive to chemicals than are Gram-positive organisms, but different chemicals will have variable effects on the organisms being tested.
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Kirby-Bauer disk diffusion techniqueo What is it? |
it is a stamper than puts paper disks of antibiotics onto a petri dish to test for resistance or sensitivity. |
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What is an antibiotic? Where do they come from? |
chemicals of biological origin only effective against bacteria. They come from bacteria, fungi, trees, frogs, hippos. |
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Distinguish between bacteriostatic & bacteriocidal antibiotics |
Bacteriostatic just inhibit growth (refridgeration) where as bacteriocidal antibiotics kill bacteria. |
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How do Antibiotics work? |
They kill bacteria in your body. |
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What parts/pathways of the bacterial cell do antibiotics target? |
. |
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How do you choose which antibiotic to use? |
You want to choose an antibiotic that is perfectly effective towards the infection you are treating, but one that doesnt damage your normal flora or cause any other ill effects. |
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What do “broad spectrum” and “narrow spectrum” mean? |
broad spectrum- controls many different types of bacteria (like gram+ and Gram-), Narrow - controls only one or a few bacteria. |
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Define: Allele |
an alternative sequence of a gene. |
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Define: Dominant |
Only need one copy of dominate to be that phenotype |
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Define: Recessive |
need two recessive alleles
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Define: Heterozygous |
having different alleles
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Define: Homozygous |
having the same alleles
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Know the relative concentrations of leukocytes (“never let monkeys eat bananas”) |
Neutrophils, lymphocytes, monocytes, eosininophils, basophils (Least common) |
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If the A Antigen is present and D antigen , What is blood type? |
A+ |
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Determine the antibodies present in type A Blood |
Anti-B Antibodies |
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Group Project |
A water sample wascollected from Pleasant Grove Creek in Roseville California during arain storm, in an attempt to isolate Escherichia colias an indication of any fecal contamination. We hypothesized thatEscherichia coli would be found. The sample wasstreaked onto differential media designed to highlight the presenceof coliforms, and an isolate was selected and cultured on nutrientagar at 37°C. Morphological tests were conducted utilizing severalstains and physiological tests. Test results were used to compare thebest matches obtained through sequencing the 16s rRNA, that wasobtained through PCR, and comparing the sequence to a database. Themethyl red, Voges-Proskauer, lysine decarboxylase, urease, H2S,indole, motility, and citrate tests were instrumental indifferentiating the coliform isolate from other genera inEnterobacteriaceae, and other species in Enterobacter.Although the isolate did not match any of the possible speciesobtained through sequencing, the combination of sequencing resultsand physiological testing showed that the isolated bacteria is amember of genus Enterobacter, and is possiblyEnterobacter hormaechei, an opportunisticpathogen that is ubiquitous in the environment.
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What type of blood if A Antigen only is present? |
A- |
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What type of blood is no antigens are present? |
O- |
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AB-(Neg) Blood |
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A+ |
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B- |
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O- |
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What is the universal donor Blood Type? |
O |
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What is the universal recipient? |
AB |
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Does pasteurization kill endospores? |
No |
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Ionizing Radiation |
Electromagnetic radiation (gamma rays)- high energy, short wave length. very effective at penetration and no heat/steam |
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Antibodies for Type O Blood |
both A and B Antibodies |
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Antibodies for type AB Blood |
No Antibodies |
