Microbio: Lab Exam 2

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Radhika316
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220218
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Microbio: Lab Exam 2
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2013-05-20 00:57:02
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Microbio
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1.Serial Dilutions/Counting Microbes 2. Transformation Lab w/ Handout (✔) 3. Helminths 4. Fungi (✔) 5. Gel Electrophoresis & Recombinant DNA (107&110) 6. Streptococcus and Staphylococcus lab (lab handout) 7. White blood cell lab & blood typing (handout) 8. Antibiotic sensitivity test/Kirby Bauer test (✔) 9. Nitrogen Ex. 8-6 cycle experiment (✔)
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  1. Antiseptics and disinfectants Background:
    --zone of inhibition and different zones
    • -Test if chemical inhibits bacterial
    • growth


    • zone of inhibition: the area of no bacterial growth around an antimicrobial agent in the disk diffusion method
    • --> Resistant zone: Bacteria is not affected by antiseptic.
    • --> Intermediate: ...ehhh.
    • --> Sensitive: larger diffusion, larger diameter, means the bacteria is sensitive to the antimicrobial agent which is GOOD.
  2. Kirby-Bauer Questions
    1. Which antibiotics is effective against all species? Explain.

    2. Which species of bacteria is the most resistant? Explain.
    1. Ciprofloaxin and Gentamycin: All four microbe species are sensitive to them meaning that they have the greatest zone of inhibition.

    2. Pseudomonas aeruginosa is the most resistant to a variety of drugs and have the smallest zone of inhibition.
  3. Transformation lab: Purpose & Background
    --> transformation:
    -->Plasmid used? amp, GFP, araC
    --> what is the drug used and what species is sensitive to it? will it grow?
    Purpose: To transform E. coli by inserting a plasmid (pGLO ) into E. coli

    • Background:
    • ==>Transformation: process of bacteria taking up genes (naked pieces of dna) in a solution by a recipient cell; observed by Griffith in 1928

    • ==> pGLO (plasmid: circular piece of dna) has genes for:
    • 1. amp: ampicillin resistance; if taken up by E. coli then e. coli which was previously sensitive would be able to multiply on ampicillin plates
    • 2. GFP: codes for fluorescent protein
    • 3. araC: codes for a regulatory protein that turns GFP protein on or off. If arabinose is present, the araC protein turns on GFP so that fluorescent protein is produced. But if there is a lack of arabinose, then araC will PREVENT GFP from producing fluorescent proteins.
    • -->araC protein + arabinose sugar =GFP turns ON.
    • -->araC protein alone= GFP STAYS OFF.
    • --> If ENTIRE pGLO is taken up by E. coli will both grown on ampicillin plate AND glow under UV exposure due to arabinose being present.
    • ==>E. coli is sensitive to ampicillin; will not grow.
  4. Transformation lab: Method
    • 1. Use P-1000 micropipette to add 250 microliters of Calcium Chloride (CaCl2) to EACH of the 1.5 microfuge tubes. Place in crushed ice. (to make bacterial cells competent)
    • 2. Then use a sterilized loop to inoculate the “pGLO+” microfuge tube with 2 loops of E. coli; make sure it’s abundant to see on loop. Use the P1000 micropipette to mix the liquid by gently sucking the liquid back up and pumping out to the first stop and several times, until well blended to a milky white with no lumps or air bubbles. Ice the microfuge tube.
    • 3. Then add 10 mL of pGLO plasmid to the pGLO+ tube. Tap to mix and before returning it to the ice to cool down for 15 minutes.
    • 4. Now heat shock the bacterial cells by suspending the “pGLO+” microfuge in a hot water bath (42oC ) for exactly 25 seconds before returning it to ice for 1 minute (this makes the cells fragile and more susceptible to take up the plasmid DNA)
    • 5. Then return the microfuge tube to room temperature, and use a P1000 micropipette to add 250 microliters “LB medium” (from the “L-Tube” ). Mix by tapping.
    • 6. Take already labeled petri plates: “L” (LB medium), “LA” (LB + ampicillin), “LAa” (LB+ ampicillin+ arabinose).
    • 7. In the above listed order; use the P1000 micropipette to transfer 100 microliters of transformed cells from the “pGLO+” microfuge tube to each plate, using new sterilized tips between each transfer. Inoculate in order to prevent spreading ampicillin..
    • 8. Remove foil from the plastic spreader and glide it across the surface of the agar to evenly spread the bacteria on plate, making sure to rotate the plate to completely cover the plate. Spread all three plates in order of inoculation so that arabinose sugar will not be spread in wrong direction. Then dispose into bleach container.
    • 9. Then allow all three plates to sit for about 10 minutes to allow cells to be absorbed into the surface of the agar before incubating upside-down.
  5. Transformation lab Questions:
    1. How should the growth of transformed cells differ on each plate?

    2. Which plate should have fewest colonies?
    1.Transformed cells should be colonies.

    2. L plate because it has no Amp gene to make the cells resistant to the ampicillin and be able to grow normally and freely.
  6. Nitrogen and sulfur cycles Purpose & Method
    Purpose: To observe the products of denitrification/ nitrogen reduction in a soil sample and measure how much nitrate concentration was used to form nitrite and if there was any gas produced.

    • Method:
    • Day One (04.23.13):
    • 1. Obtain two different samples of soil each from different sources; work in pairs and designate a soil sample for each pair. (We chose courtyard soil)
    • 2. Obtain negative control test-tubes and put a pinch of soil sample into it (negative control-label)
    • 3. Then put a pinch of soil sample in a test tube with nitrate broth and a Durham tube (positive control-label)
    • 4. Set both Negative and Positive controls aside to incubate for 7 days

    • Day Two (04.30.13):
    • 1. Record any observable gas bubbles
    • 2. Then transfer .75 ml of broth from each test tube (the positive and negative controls) to a microfuge test tube using a transfer pipette, making sure both microfuge test tubes are equal in volume; label with a plus or minus to indicate which control.
    • 3. Place both microfuge tubes opposite one another in microfuge machine to maintain balance. Spin both tubes in a microfuge for 2 minutes.
    • 4. Then use P1000 to transfer a smaller volume (500 mL; .5 L) from both positive and negative microfuge tubes to individual designated to clean glass test tube; label test tube with either a + or – to indicate control.
    • 5. To both the positive and negative glass test-tubes:
    • a. Reagent 1: add 1 drop and mix 5 seconds.
    • b. Reagent 2: shake the reagent bottle and add 1 drop and mix for one MINUTE after parafilming.
    • 6. Compare the colors in the positive and negative controls to the “freshwater nitrate card” to determine nitrate concentration (mg/L).
  7. Nitrogen and sulfur cycles Analysis & Questions:
    --> negative vs. positive results?
    --> How to calculate Amount of nitrate used
    ==>Did you soil sample contain microbes capable of denitrication& How do you know:
    • Analysis:
    • Negative:
    • -->No bubbles captured: therefore no denitrification occurred because no Nitrogen gas was made
    • -->Red color=160 mg/L of nitrate left in tube: no conversion occurred.

    • Positive:
    • -->Bubble formed: meaning nitrogen gas was produced where nitrate was converted to N2 gas (denitrification process occurred).
    • -->Yellow= 0 mg/L of nitrate in tube. The entire amount of nitrate was converted into gas.

    **Amount of nitrate used: mg/L control – mg/L experimental: 160 mg/L Nitrate.

    • ANSWER: Yes. Because bubbles were formed meaning nitrogen gas was produced from
    • nitrate and the yellow color in comparison means that there was 0 mg/L
    • nitrate left in the test tube because all of it was converted into gas.
  8. Microbial ecology & Biogeochemical cycles
    Microbial ecology: interactions b/w microbes and environment.

    Biogeochemical cycles: the recycling or elements by living things
  9. Nitrogen fixation:
    --bacterial ex's
    -- what enzyme needed?
    • Nitrogen gas converted to ammonia (N2 --> NH3)

    **bacterial examples:
    Rhizobium, Azotobacter, Anabena

    need nitrogenase enzyme.
  10. Ammonification:
    Ammonia (NH3) MADE by microbe decomposition of dead organisms (fungi & protozoa)

    ==>THEN: Ammonia (NH3) becomes Ammonium (NH4)
  11. Nitrification & Denitrification
    Nitrification::[[Opposite of nitrate reduction]]

    Ammonium (NH4+) -->Nitrite (NO2-); Nitrosomonas bacteria

    Nitrite (NO2-) --> Nitrate (NO3-); Nitrobacter bacteria; Nitrate: used by plants as fertilizer to make proteins

    Denitrification/nitrogen reduction: (Nitrate--> makes N2 gas) by Pseudomonas.
  12. Sulfur cycle:
    Sulfate (SO42-) used by plants to make amino acids, eaten by animals; die and return to soil; elemental sulfur is produced

    * Elemental sulfur-->SO42- (by Thiobacillus bacteria: chemolithotrophs, aerobic, sulfur oxidizers)

    *Sulfate (SO42-)-->Hydrogen sulfide (H2S) by Desulfibrio--Sulfur reducers

    *In soil: H2S returns to elemental sulfur (green/purple sulfur bacteria)
  13. Fungi Lab Purpose & Background:
    How to identify Fungi (3 ways; four types of microscopy)
    Purpose: To observe various fungi under a microscope.

    • Background:
    • How to identify Fungi:
    • 1. Detect RNA and DNA
    • 2. Culturing.
    • 3. Microscopy:
    • a. Calcofluorowhite: Fluorescence under UV light, binds chitin in cell walls; appears light blue against dark background
    • b. Negative Staining: nigrosin to stain background
    • c. Gram Staining: yeasts are purple and molds are pink
    • d. Lactophenol Cotton Blue: Phenol kills cells and cotton blue stains chitin.
  14. Rhizopus:
    • bread mold, common in soil, can cause mucormycosis
    • -->sporangium produces sporangiospores=asexual




  15. Penicillium:
    • makes penicillin;
    • conidia/condiospores=sexual
  16. Aspergillus:
    • Found on dying vegetation
    • conidiophores
    • A. oryzae: soy products

     
  17. Coprinus:
    mushroom, sketch gills w/ basidiospores.

  18. Peziza:
    phylum Ascomycota; observe ascus

  19. Saccharomyces cerevisiae:
    • wet mount; baker’s yeast
    • -ascospores

  20. Candida albicans:
    • yeast infection
    • -part of normal flora

  21. Water Plate count: Purpose and Background
    --What should be tested for bacterial contamination:
    --Ways to count bacteria in H20:
    --Coliform characteristics and two species?
    -- Three qualities of a good quality water indicator
    Purpose: To create a serial dilution of E. coli to count the bacteria

    • Background:
    • ==>What should be tested for bacterial contamination: water, food, milk, ocean water…

    • ==>Ways to count bacteria in H20:
    • a) MPN: most probably number and b) standard plate count

    • ==>Coliform: gram negative rods, ferments lactose and produces gas. No Endospores. Facultative.
    • Enterobacter aerogenes: normally found in environment.

    Escherichia coli: found in intestines (humans and some animals)

    • ==>Three qualities of a good quality water indicator:
    • (1) Easy to test for.

    (2) Normal not found in environment.

    (3) Lives little longer than pathogens.
  22. Water Plate count: Method and Results
    1. Designated and label three bottles already filled with 99 mL of water: A, B, and C.

    2. Then Label the bottom half of four petri plates with “B: 1 mL H20”, “B: .10 mL H20”, “C: 1 mL H20”, and “C: .10 mL H20”.

    3. Add 1 mL of E. coli to Bottle “A”. Cap it, and cover it with parafilm and use wrist to flick bottle away from face to mix. Count 25 flicks.

    4. Open Bottle “A” after mixing and from it dispense out 1 mL of water using a new serological pipette

    5. Transfer the 1 mL H20 to Bottle “B”. Cap this bottle, and cover with parafilm. Flick wrist 25 times to mix it.

    6. Use the pipette to dispense 1 mL and .10 mL each from “Bottle B” to the two designated separate Petri plates. Cover plates and swirl gently. Set Aside.

    7. Replace pipette with a new one and dilute “Bottle C” by adding 1 mL from “Bottle B” to it. Cap the bottle, protect it with parafilm and flick/shake it 25 times.

    8. Use the pipette to dispense 1 mL and .10 mL each from “Bottle C” to the two designated separate Petri plates. Cover plates and swirl gently. Set Aside.

    9. Collect four petri plates and to each pour 50oC liquid agar medium until the smaller half of the plate is sufficiently covered.

    10. Gently swirl the plates to mix agar with diluted water and set aside for 10 to 15 minutes to allow it cool before incubating.

    RESULTS::


    • Results:
    • Only Plate C with 1 mL had 103 colonies which is a significant number.
  23. Plaque Assay Experiment: Method
    • METHOD:
    • 1. Place 3 ml of colored beads (diluent) into each of the test tubes labeled A, B, C, and D.

    2. Make a dilution of virus (white beads) by placing 3 ml of white beads into test tube A. Cap and mix by tipping the tube gently. What is the dilution in test tube A? 1 part white beats and 1 part black beads ==> 1:2

    3. Make a 1:2 dilution of virus from test tube A by placing 3 ml of beads from test tube A into test tube B. Cap and mix. What is the total dilution in tube B?_1:4_

    4. Make a 1:2 dilution of virus from test tube B by placing 3 ml of beads from test tube B into test tube C. Cap and mix. What is the total dilution in tube C?_1:8__

    5. Make a 1:2 dilution of virus from test tube C by placing 3 ml of beads from test tube C into test tube D. Cap and mix. What is the total dilution in tube D?_1:16_
  24. Plaque Assay Experiment: Purpose and Background
    --> serial dilutions
    --> Plaque assay
    a. Plaques
    b. Dilution numbers
    --> Analysis:
    Purpose: to determine the amount of the substance (like antibodies, number of microbes) in a sample.

    • BACKGROUND:
    • ==>Serial Dilutions: dilutions of a substance or microbe (serum, virus or bacteria) that are done in a specific sequence so that the substance become more and more dilute.

    • ==> Plaque assay: a test that is used to determine how much virus is present in a sample; virus are spread on pertri dishes that contain medium
    • -Plaques: Clear spots where infected cells of virus will die; these can be counted. "accuracy number": from 30 to 300
    • -Dilutions numbers: Amount of substance being diluted(virus) OVER total amount of substance + Diluent (medium) ex: 1 ml of virus over 9 ml of diluent= 1:10 dilution; for serial dilutions: multiply the dilutions for a final dilutions

    ==> Analysis: calculate number of plague forming units/mL (pfu/mL) by multiplying SIGNIFICANT plaque count number (b/w 30 and 300) with the dilution factor of that dish (second number of dilution)
  25. Standard Food Plate count: Purpose and Backgound
    -->Sources of bacteria
    --> Sources of pathogens?
    --> how to get rid of bacteria on food?
    Purpose: To count bacteria in food (harmless microbes and pathogens)

    • Background
    • ==>Sources of bacteria on food, air, soil,water, animals, people who handle food processing

    • ==>Sources of pathogens:
    • Cattle: E.coli O157:H7
    • Soil Microbes: maybe hard to remove ex: listeria on cantaloupe rinds
    • • Leaky roof in factory that makes PB --> salmonella contamination
    • • Sick worker
    • • Eggs with Salmonella

    ==>How to get rid of bacteria on Food:Cooking, Peeling food (with clean hands) and Washing foods (may help)
  26. Standard Food Plate count Method
    • Method: (03.2213)
    • 1. Chop up strawberries.

    2. Measure on 20 grams using a scale and pour it into the magic bullet container. Then add 180 mL of water to the container. (note: this has a 1:10 Dilution because 20/200)

    3. Screw on cap and twist the container upside down into the magic bullet system and press down to blend. Blend in 20 second intervals until well blended (smoothie-like)

    4. From the bottom of the container (to avoid the floating chunks), use a pipette to extract 1 mL of the mixture and put into Bottle B: 99mL sterile water (note: dilution factor for bottle B is 1:100 since multiply serial dilutions)

    5. Close and parafilm Bottle B and mix 25 times carefully using wrist motions. From Bottle B extract .1 ml and transfer to petri plate (1:100)

    6. Now Dilute Bottle C: Add 1 mL from Bottle B to 99mL of sterile water in Bottle C to make a 1:1000 dilution. Then From Bottle C: transfer .01 mL to one plate (1:10,000) and 1 mL to another plate (1:1,000)

    7. Add warm liquid TSA medium to all three plates and swirl gently. Allow to cool and set before incubation
  27. Water Plate Count...
    -> Dilutions?
    -> Cfu/mL meaning and how to calculate it.
    -> Method:
    • Serial Dilutions: Diluting a bacterial sample over and over again.
    • Colony Forming Unit: Visible Bacterial colonies on solid media
    • ==> Calculate CFU/mL: dilution factor x significant number


    • Bottle A: 1/100
    • Bottle B: 1/10,000
    • Bottle C: 1/1,000,000

    • Method:
    • 1. Designated and label three bottles already filled with 99 mL of water: A, B, and C.
    • 2. Then Label the bottom half of four petri plates with “B: 1 mL H20”, “B: .10 mL H20”, “C: 1 mL H20”, and “C: .10 mL H20”.
    • 3. Add 1 mL of E. coli to Bottle “A”. Cap it, and cover it with parafilm and use wrist to flick bottle away from face to mix. Count 25 flicks.
    • 4. Transfer the 1 mL H20 to Bottle “B”. Cap this bottle, and cover with parafilm. Flick wrist 25 times to mix it.
    • 5. Use the pipette to dispense 1 mL and .10 mL each from “Bottle B” to the two designated separate Petri plates. Cover plates and swirl gently. Set Aside.
    • 6. Replace pipette with a new one and dilute “Bottle C” by adding 1 mL from “Bottle B” to it. Cap the bottle, protect it with parafilm and flick/shake it 25 times.
    • 7. Use the pipette to dispense 1 mL and .10 mL each from “Bottle C” to the two designated separate Petri plates. Cover plates and swirl gently. Set Aside.
    • 8. Collect four petri plates and to each pour 50oC liquid agar medium until the smaller half of the plate is sufficiently covered.
    • 9. Gently swirl the plates to mix agar with diluted water and set aside for 10 to 15 minutes to allow it cool before incubating.
  28. Water Plate Count...
    -> Dilutions?
    -> Cfu/mL meaning and how to calculate it.
    -> Method:
    • Serial Dilutions: Diluting a bacterial sample over and over again.
    • Colony Forming Unit: Visible Bacterial colonies on solid media
    • ==> Calculate CFU/mL: dilution factor x significant number


    • Bottle A: 1/100
    • Bottle B: 1/10,000
    • Bottle C: 1/1,000,000

    • Method:
    • 1. Designated and label three bottles already filled with 99 mL of water: A, B, and C.
    • 2. Then Label the bottom half of four petri plates with “B: 1 mL H20”, “B: .10 mL H20”, “C: 1 mL H20”, and “C: .10 mL H20”.
    • 3. Add 1 mL of E. coli to Bottle “A”. Cap it, and cover it with parafilm and use wrist to flick bottle away from face to mix. Count 25 flicks.
    • 4. Transfer the 1 mL H20 to Bottle “B”. Cap this bottle, and cover with parafilm. Flick wrist 25 times to mix it.
    • 5. Use the pipette to dispense 1 mL and .10 mL each from “Bottle B” to the two designated separate Petri plates. Cover plates and swirl gently. Set Aside.
    • 6. Replace pipette with a new one and dilute “Bottle C” by adding 1 mL from “Bottle B” to it. Cap the bottle, protect it with parafilm and flick/shake it 25 times.
    • 7. Use the pipette to dispense 1 mL and .10 mL each from “Bottle C” to the two designated separate Petri plates. Cover plates and swirl gently. Set Aside.
    • 8. Collect four petri plates and to each pour 50oC liquid agar medium until the smaller half of the plate is sufficiently covered.
    • 9. Gently swirl the plates to mix agar with diluted water and set aside for 10 to 15 minutes to allow it cool before incubating.
  29. Schistosoma mansoni EGG
    --disease?
    --worm type/phylum
    --life cycle
    --signs/symptoms
    • Flatworm (Phylum Platyhelminthes)--Blood FLUKE (Termatodes)
    • -causes schistosomiasis

    -Life cycle: Ova in stool/urine --> larvae (miracidia) reach water--> infect snails (intermediate host) & develop into cercariae--> infects humans in water (definitive host; site of parasite reproduction; travels to intestines thru blood vessels)

    --signs/symptoms: itchy skins, fever, rash, no death
  30. Taenia Pistiformis scolex
    -worm type/ phylum?
    -causes?
    -bodyparts?
    -lifecycle: intermediate and definitive host?


    • Flatworm (Phylum Platyhelminthes)
    • Tapeworm/Cestodes--> causes taeniasis

    • Body parts:
    • 1. Scolex: head w/ suckers
    • 2. Proglottids: tapeworm sections (egg production)
    • 3. cuticle: covering; absorb nutrients
    • 4. Hermaphroditic...

    • LIFE CYCLE:
    • Live in human's small intestines (definitive host) & mate; eggs released into feces--> pigs/cows (enter larval stage; cysts found in muscle)--> undercooked meat infects ppl.
  31. Trichinella spiralis
    -worm type/phylum
    -cause?
    -Life cycle
    -Diagnosis & Prevention
    • ROUND WORM: PHYLUM NEMATODA
    • -Cause trichinosis

    • LIFE CYCLE:
    • Human eats larvae in uncooked meat; grow into adult worm,mate, produce larvae--> encyst in human muscle (now rare in us)

    Diagnosis: serology (look for antibodies; past resistance) & muscle biopsy

    Prevent: cook meat well, avoid pork..
  32. Enterobius vermicularis egg
    -worm type/phylum
    -cause?
    -Life cycle
    -Diagnosis & Prevention
    • ROUND WORM: PHYLUM NEMATODA
    • -causes: enterobiasis; world wide disease

    • Life Cycle:
    • eggs layed in anal area-> fingers -> ingested & hatch in small intestine & adult worms infect large intestine (living there for 2 months)
    • **doesn't need intermediate host**

    • Diagnose: sticky clear tape to collect eggs
    • -drug treatment
    • Prevent: good hygiene/sanitation
  33. Enterobius vermicularis Adult Worm

    -worm type/phylum
    -cause?
    -Life cycle
    -Diagnosis & Prevention
    • ROUND WORM: PHYLUM NEMATODA
    • -causes: enterobiasis; world wide disease

    • Life Cycle:
    • eggs layed in anal area-> fingers -> ingested & hatch in small intestine & adult worms infect large intestine (living there for 2 months)
    • **doesn't need intermediate host**

    Diagnose: sticky clear tape to collect eggs-drug treatment

    Prevent: good hygiene/sanitation
  34. Staphylococcus epidermidis
    • -Gram positive
    • -Facultative anaerobic; on human skin
    • -white..

  35. Staphylococcus aureus
    • -coagulase: causes plaasma to clot (coagulae test)
    • -found on noses, yellow 
    • -causes common infections: skin, toxic shock, and pneumonia
    • -enterotoxin (exotoxin): causes food poisioning
    • -MRSA

  36. Streptococci:
    • gram positive; forms in chains; mouth/throat;
    • --lacks catalase
    • -strict or facultative anaerobes
    • -grouped by hemolysis or lancefield groups (based on detecting carb antigents thru serological methods)
    • a. Streptococcus pyogenes:
    • b. Streptococcus pneumonia:
  37. Streptococcus pyogenes:
    -aka "Group A Strep" or "beta hemolytic strep"

    • -causes mild infections: ear, impetigo, pharyngitis (strep throat--> scarlet fever)
    • -can be carriers; in throat
    • -Invasive GAS: necrotiziing fascitis ("flesh eating bacteria") and strep toxic shock.
    • -Diagnosis: agglutination test

    • --> Related infections:
    • 1. strep throat: fever, painful sore throate, swollen lymph nodes
    • 2. scarlet fever: rash, red/strawberry tongue
    • 3.Rheumatic fever: immune response against strep==> damage heart valves; multiple rounds of strep
    • 4. necrotizing fasciitis: rapid destruction; need skin grafts
    • 5. Strep toxic shock: drop in blood pressure/organ failure
  38. Streptococcus pneumonia:
    • -aka pneumococcus;
    • major cause of pneumonia and bacterial meningitis
    • -virulence: uses capsule
  39. Streptococcus LAB:
    -blood agar...what kind of mediums?
    -alpha vs. beta?
    • -blood agar: TSA + RBC
    • -enrichment medium: good for "picky organisms"
    • -differential medium: different microbes appear on diff mediums
    • -throat swab and use for first streak on an isolation plate

    • --> observations around colonies...
    • a. Alpha: greenish brown; partial lysing of RBC; strep pneumonia and strep mutans

    b. beta: clear areas; complete lysing, strep pyogenes & staph aureus

  40. Staphylococcus aureus LAB:
    -what kinda medium
    -method
    -observations?
    -Pink agar: mannitol (sugar) & salt (75%)

    -selective medium: inhibits growth of some microbes and allows others to grow (staph)

    -Method: swab nose...

    --> observation: if agar REMAINED pink=not staph aureus, some other staph

  41. Gel Electrophoresis
    • Negatively charged DNA travels thru agarose gel toward positive pole when current is applied; smallest pieces travel fastest...
    • -goal: separate DNA segments by size
    • -agarose gel: agarose + buffer (salt/water)
    • -Restriction enzymes: made by bacteria; cut bacteria at specific sequences
  42. Recombinant DNA:
    covalently linking two or more dna pieces to create a new dna molecule.

    -plasmid: small circular dna molecule which has few genes can replicate independently of a bacterial chromosome.

    • --> RELATED APPLICATIONS:
    • 1. Basic research: for study
    • 2. Biotechnology: transform bacteria w/ plasmid containing gene to produce large protein quantities; pharmaceutical
    • 3. Vaccine development: genes placed into a weaker virus to make vaccine; or linked to genes for immune stimulating proteins (Hepatitis B vaccine)

    • --> RECOMBINANT TOOLS:
    • 1. Restriction enzymes: recognize specific dna sequences and cut at them (Eco RI, HindIII, BamHI)
    • 2. Ligase: covalently links DNA pieces
    • 3: Plasmid map: diagram of restriction sites and genes on a plasmid.
  43. WBC's
    • Granulocytes: Neutrophils, Basophils, Eosinophils
    • Monocytes
    • Lymphocytes: T & B cells

    *all Leukocytes*
  44. Neutrophils:
    • Granulocytes;
    • -first responders to any infection
    • -important in inflammatory response
    • -phagocytose antigens and produce inflammatory cytokines

    --> multinucleated lobes

     
  45. Eosinophils
    • Granulocytes;
    • -biloped nucleus
    • -important in fighting parasitic infections;
  46. Basophils:
    • Granulocytes;
    • -pinched nucleus
    • -stains bluish purple
  47. Monocytes:
    • Kidney shaped nucleus, travel in blood.
    • -become macrophages in tissues; phagocytic
    • - important in stimulating specific immune sytem cells.

     
  48. Lymphocytes
    • slightly larger than RBC;
    • round nucleus takes up most of the cells
    • -T lymphocytes: responsible for specific cellular immunity
    • -B Lymphocytes: humoral immunity (antibody protein production)

     
  49. Bloood Typing: A, B, AB, & O
    -antibodies and receives ?
    A: antibodies: Anti-B; Receives: A & O

    B: antibodies: Anti-A  ; Receives:  B & O

    AB: antibodies: none  ; Receives: ALL (universal receiver)

    O: antibodies: Anti-A & Anti B (both)  ; Receives: ONLY O (UNIVERSAL DONOR)
  50. RH Factor..
    -RH Incompatiblility:
    - Hemolytic disease in newborn:
    RH Incompatiblility: RH- mom & RH + baby

    Hemolytic disease in newborn: RH antibodies of mom attack the babies RBC's
  51. Blood Type LAB::

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