Micro Lab Exam 2 **KEY TERMS**

  1. 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.
  2. 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.
  3. 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.
  4. Nitrogen fixation:
    --bacterial ex's
    -- what enzyme needed?
    • Nitrogen gas converted to ammonia (N2 --> NH3)

    **bacterial examples:
    Rhizobium, Azotobacter, Anabena

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

    ==>THEN: Ammonia (NH3) becomes Ammonium (NH4)
  6. 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.
  7. 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)
  8. Rhizopus:
    • bread mold, common in soil, can cause mucormycosis
    • -->sporangium produces sporangiospores=asexual

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

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

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  12. Peziza:
    phylum Ascomycota; observe ascus

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

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

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  15. Schistosoma mansoni EGG
    --worm type/phylum
    --life cycle
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    • 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
  16. Taenia Pistiformis scolex
    -worm type/ phylum?
    -lifecycle: intermediate and definitive host?
    Image Upload 9

    • 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...

    • 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.
  17. Trichinella spiralis
    -worm type/phylum
    -Life cycle
    -Diagnosis & Prevention
    • Image Upload 10
    • -Cause trichinosis

    • 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..
  18. Enterobius vermicularis egg
    -worm type/phylum
    -Life cycle
    -Diagnosis & Prevention
    • Image Upload 11
    • -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
  19. Enterobius vermicularis Adult Worm

    -worm type/phylum
    -Life cycle
    -Diagnosis & Prevention
    • Image Upload 12
    • -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
  20. Staphylococcus epidermidis
    • -Gram positive
    • -Facultative anaerobic; on human skin
    • -white..

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  21. 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

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  22. 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:
  23. 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
  24. Streptococcus pneumonia:
    • -aka pneumococcus;
    • major cause of pneumonia and bacterial meningitis
    • -virulence: uses capsule
  25. 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

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  26. Staphylococcus aureus LAB:
    -what kinda medium
    -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

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  27. 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
  28. 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.

    • 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)

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

    --> multinucleated lobes

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  30. Monocytes:
    • Kidney shaped nucleus, travel in blood.
    • -become macrophages in tissues; phagocytic
    • - important in stimulating specific immune sytem cells.

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  31. 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)

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  32. 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)
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Micro Lab Exam 2 **KEY TERMS**