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Examining​ ​the​ ​Effects​ ​of​ ​Antibiotics​ ​and​ ​Antibacterials​ ​on​ ​Bacteria Introduction Bacteria​ ​are​ ​prokaryotic​ ​organisms​ ​that​ ​have​ ​one​ ​circular​ ​chromosome​ ​and​ ​can​ ​divide​ ​to​ ​produce a​ ​new​ ​generation​ ​of​ ​bacteria​ ​every​ ​20​ ​minutes​ ​if​ ​they​ ​have​ ​enough​ ​food​ ​and​ ​resources.​ ​ ​Even​ ​though they​ ​reproduce​ ​asexually​ ​and​ ​have​ ​to​ ​rely​ ​mostly​ ​on​ ​mutations​ ​for​ ​new​ ​genetic​ ​combinations,​ ​their​ ​rapid reproductive​ ​rate​ ​allows​ ​for​ ​rapid​ ​adaptation​ ​to​ ​a​ ​changing​ ​environment.​ ​ ​Bacteria​ ​have​ ​traditionally been​ ​divided​ ​into​ ​two​ ​broad​ ​groups​ ​–​ ​gram​ ​positive​ ​and​ ​gram​ ​negative.​ ​ ​This​ ​was​ ​based​ ​on​ ​their reactions​ ​to​ ​different​ ​antibiotics.​ ​ ​Today,​ ​because​ ​natural​ ​selection​ ​has​ ​acted​ ​on​ ​(and​ ​continues​ ​to​ ​act​ ​on) bacteria,​ ​favoring​ ​traits​ ​that​ ​make​ ​bacteria​ ​better​ ​able​ ​to​ ​survive​ ​their​ ​environments​ ​(i.e.​ ​be​ ​resistant​ ​to antibiotics),​ ​these​ ​traditional​ ​groupings​ ​are​ ​no​ ​longer​ ​relevant​ ​as​ ​the​ ​antibiotics​ ​that​ ​were​ ​once​ ​widely used​ ​are​ ​less​ ​and​ ​less​ ​effective. Over-the-counter​ ​medications​ ​and​ ​soaps​ ​advertise​ ​their​ ​ability​ ​to​ ​kill​ ​"germs"​ ​(bacteria).​ ​ ​Most soaps​ ​and​ ​cleaning​ ​agents​ ​today​ ​claim​ ​to​ ​be​ ​"antibacterial."​ ​Technically,​ ​antibacterial​ ​substances​ ​used on​ ​living​ ​organisms​ ​are​ ​called​ ​antiseptics​ ​and​ ​those​ ​used​ ​for​ ​cleaning​ ​surfaces​ ​are​ ​called​ ​disinfectants. Studies​ ​are​ ​showing​ ​that​ ​many​ ​common​ ​bacterial​ ​strains​ ​are​ ​adapting​ ​to​ ​these​ ​over-the-counter​ ​products and​ ​becoming​ ​resistant​ ​to​ ​them. Traditionally​ ​it​ ​made​ ​a​ ​difference​ ​ ​in​ ​how​ ​you​ ​treated​ ​a​ ​bacterial​ ​infection​ ​depending​ ​on​ ​whether bacteria​ ​were​ ​gram​ ​positive​ ​or​ ​gram​ ​negative.​ ​ ​With​ ​widespread​ ​antibiotic​ ​resistance​ ​in​ ​the​ ​bacteria​ ​that cause​ ​disease,​ ​the​ ​distinction​ ​between​ ​gram​ ​positive​ ​and​ ​gram​ ​negative​ ​is​ ​not​ ​as​ ​important​ ​when​ ​we​ ​are forced​ ​to​ ​use​ ​our​ ​strongest​ ​and​ ​most​ ​sophisticated​ ​antibiotics.​ ​ ​That​ ​said,​ ​we​ ​have​ ​strains​ ​of​ ​harmless gram​ ​positive​ ​and​ ​gram​ ​negative​ ​bacteria​ ​that​ ​we​ ​will​ ​use​ ​to​ ​examine​ ​the​ ​effects​ ​of​ ​various​ ​antibiotics and​ ​antibacterial​ ​on​ ​different​ ​bacteria.​ ​ ​E.​ ​coli​ ​is​ ​a​ ​gram​ ​negative​ ​bacteria​ ​normally​ ​found​ ​in​ ​the​ ​large intestine​ ​of​ ​people.​ ​ ​They​ ​help​ ​us​ ​absorb​ ​water​ ​and​ ​secrete​ ​vitamin​ ​K.​ ​B.​ ​cereus​ ​are​ ​gram​ ​positive​ ​soil bacterium. Bacteria​ ​are​ ​cultured​ ​or​ ​grown​ ​on​ ​plates​ ​(in​ ​agar​ ​media)​ ​and​ ​in​ ​culture​ ​tubes​ ​(in​ ​liquid​ ​media)​ ​for study​ ​in​ ​the​ ​laboratory.​ ​ ​Using​ ​sterile​ ​technique,​ ​we​ ​will​ ​culture​ ​our​ ​E.​ ​coli​ ​ ​and​ ​ ​B.​ ​cereus​ ​on​ ​LB​ ​agar plates​ ​and​ ​observe​ ​the​ ​effects​ ​of​ ​antibiotics​ ​and​ ​antibacterials​ ​on​ ​each​ ​kind​ ​of​ ​bacteria.​ ​ ​The​ ​sterile technique​ ​we​ ​will​ ​use​ ​will​ ​help​ ​us​ ​prevent​ ​contaminating​ ​our​ ​bacterial​ ​cultures​ ​and​ ​ourselves. Materials​ ​(per​ ​group) cell​ ​spreader(1) permanent​ ​marker​ ​(1) Bunsen​ ​burner​ ​(1) Sterile,​ ​disposable​ ​1ml​ ​pipette​ ​(4) 10%​ ​bleach​ ​solution waste​ ​beaker LB​ ​agar​ ​plates​ ​(4) various​ ​antibiotic​ ​disks​ ​blank​ ​disks flint various​ ​antibacterials E.​ ​coli​ ​culture B.​ ​cereus​ ​culture Procedure​ ​(Day​ ​1:​ ​ ​Setting​ ​up​ ​the​ ​Plates) Getting​ ​Started 1. Leave​ ​all​ ​of​ ​your​ ​personal​ ​items​ ​on​ ​your​ ​desk.​ ​ ​You​ ​should​ ​NOT​ ​have​ ​any​ ​of​ ​your​ ​personal belongings​ ​(backpacks,​ ​sweatshirts,​ ​etc.)​ ​on​ ​the​ ​laboratory​ ​table​ ​during​ ​ ​this​ ​investigation!! 2. Use​ ​the​ ​spray​ ​bottle​ ​with​ ​10%​ ​bleach​ ​solution​ ​to​ ​wipe​ ​down​ ​your​ ​table. 3. Obtain​ ​stock​ ​culture​ ​bottles​ ​of​ ​E.​ ​Coli​ ​and​ ​B​ ​cereus.​ ​ ​Be​ ​careful​ ​when​ ​handling​ ​these!​ ​Do​ ​not​ ​open them​ ​until​ ​you​ ​are​ ​ready​ ​to​ ​use​ ​them! 4.​ ​ ​Obtain​ ​4​ ​nutrient​ ​agar​ ​plates.​ ​ ​Be​ ​careful​ ​when​ ​handling​ ​these​ ​as​ ​they​ ​are​ ​sterile!!

5. Organize​ ​the​ ​supplies​ ​at​ ​your​ ​table. Labeling​ ​the​ ​Plates 1. Write​ ​your​ ​period​ ​and​ ​lab​ ​group​ ​on​ ​the​ ​ ​bottom​​ ​of​ ​each​ ​of​ ​your​ ​LB​ ​agar​ ​plates. 2.​ ​ ​ ​On​ ​all​ ​of​ ​the​ ​plates,​ ​draw​ ​intersecting​ ​lines​ ​on​ ​the​ ​bottom​ ​of​ ​the​ ​dish​ ​so​ ​that​ ​it​ ​is​ ​divided​ ​into quarters​ ​(see​ ​picture​ ​below):

a,. On​ ​two​ ​of​ ​the​ ​plates,​ ​write​ ​E.​ ​coli.​ ​ ​You​ ​will​ ​spread​ ​E.​ ​coli​ ​cells​ ​onto​ ​this​ ​plate. b. On​ t​ he​ ​other​ ​two​ ​plates,​ ​write​ ​B.​ ​cereus.​ ​ ​You​ ​will​ ​spread​ ​B.​ ​cereus​ ​cells​ ​onto​ ​this​ ​plate. Transferring​ ​your​ ​Cells 1. Light​ ​your​ ​Bunsen​ ​burner​ ​and​ ​adjust​ ​the​ ​valves​ ​on​ ​the​ ​bottom​ ​of​ ​the​ ​burner​ ​as​ ​needed​ ​to​ ​ensure​ ​a good​ ​flame.​ ​ ​If​ ​you​ ​need​ ​help​ ​lighting​ ​your​ ​Bunsen​ ​burner,​ ​ask.​ ​ ​Do​ ​NOT​ ​fool​ ​around​ ​with​ ​the​ ​gas! Any​ ​horseplay​ ​will​ ​result​ ​in​ ​a​ ​zero​ ​on​ ​this​ ​lab,​ ​a​ ​referral,​ ​and​ ​a​ ​trip​ ​to​ ​the​ ​Associate​ ​Principal! 2. You​ ​are​ ​going​ ​to​ ​spread​ ​1​ ​ml​ ​of​ ​cells​ ​from​ ​the​ ​E.​ ​coli​ ​culture​ ​bottle​ ​onto​ ​each​ ​of​ ​the​ ​two​ ​plates​ ​you have​ ​labeled​ ​E.​ ​coli.​ ​ ​Here​ ​is​ ​the​ ​procedure​ ​for​ ​spreading​ ​cells: (1) Remove​ ​the​ ​lid​ ​of​ ​the​ ​E.​ ​coli​ ​culture,​ ​press​ ​the​ ​bulb​ ​on​ ​the​ ​pipette​ ​and​ ​then​ ​insert​ ​the​ ​tip​ ​into the​ ​E.​ ​coli​ ​suspension (2) Release​ ​the​ ​bulb​ ​of​ ​the​ ​pipette​ ​and​ ​withdraw​ ​1​ ​ml​ ​of​ ​the​ ​E.​ ​coli​ ​suspension​ ​(the​ ​line​ ​right​ ​below the​ ​bulb​ ​on​ ​the​ ​pipette) (3) Open​ ​the​ ​plate​ ​marked​ ​"E.​ ​coli"​ ​and​ ​depress​ ​the​ ​bulb​ ​of​ ​the​ ​pipette​ ​to​ ​expel​ ​the​ ​E.​ ​Coli suspension​ ​onto​ ​the​ ​top​ ​of​ ​the​ ​agar. (4) Place​ ​the​ ​pipette​ ​in​ ​the​ ​waste​ ​beaker. (5) Pour​ ​the​ ​ethanol​ ​from​ ​the​ ​culture​ ​tube​ ​into​ ​the​ ​petri​ ​dish​ ​ ​Dip​ ​the​ ​spreader​ ​into​ ​the​ ​petri​ ​dish​ ​with ethanol​ ​and​ ​briefly​ ​pass​ ​it​ ​through​ ​the​ ​Bunsen​ ​burner​ ​flame​ ​to​ ​ignite​ ​the​ ​alcohol.​ ​ ​Allow​ ​the alcohol​ ​to​ ​burn​ ​off​ ​away​ ​from​ ​the​ ​Bunsen​ ​burner​ ​flame.​ ​ ​The​ ​spreader​ ​will​ ​be​ ​too​ ​hot​ ​if​ ​you leave​ ​it​ ​in​ ​the​ ​flame.​ ​ ​Do​ ​not​ ​play​ ​with​ ​the​ ​alcohol​ ​and​ ​flame!! (6)​ ​Lift​ ​the​ ​lid​ ​of​ ​one​ ​of​ ​the​ ​E.​ ​coli​ ​plate​ ​enough​ ​to​ ​allow​ ​spreading;​ ​do​ ​not​ ​place​ ​the​ ​lid​ ​on​ ​the lab​ ​table! (7) Cool​ ​the​ ​spreader​ ​by​ ​gently​ ​rubbing​ ​it​ ​on​ ​the​ ​surface​ ​of​ ​the​ ​agar​ ​away​ ​from​ ​the​ ​cell​ ​suspension (8) Touch​ ​the​ ​spreader​ ​to​ ​the​ ​cell​ ​suspension​ ​and​ ​gently​ ​drag​ ​it​ ​back​ ​and​ ​forth​ ​several​ ​times​ ​across the​ ​surface​ ​of​ ​the​ ​agar.​ ​ ​Rotate​ ​the​ ​plate​ ​one​ ​quarter​ ​turn​ ​and​ ​repeat​ ​the​ ​spreading​ ​motion.​ ​ ​Be careful​ ​not​ ​to​ ​gouge​ ​the​ ​agar. (9) Replace​ ​the​ ​lid.​ ​ ​Return​ ​the​ ​cell​ ​spreader​ ​to​ ​the​ ​ethanol​ ​without​ ​flaming (10)​ ​Set​ ​the​ ​plate​ ​aside​ ​for​ ​now. (11)​ ​Repeat​ ​this​ ​procedure​ ​for​ ​your​ ​second​ ​E.​ ​coli​ ​plate. 3.​ ​Now​ ​take​ ​the​ ​plates​ ​labeled​ ​B.​ ​cereus​ ​and​ ​spread​ ​the​ ​B.​ ​cereus​ ​cells​ ​from​ ​the​ ​B.​ ​cereus​ ​bottle onto​ ​these​ ​plates,​ ​repeating​ ​the​ ​procedure​ ​for​ ​spreading​ ​cells​ ​outlined​ ​in​ ​steps​ ​1-11​ ​above​ ​using​ ​a fresh​ ​1​ ​ml​ ​pipette. Transferring​ ​Antibiotic​ ​Disks 1.​ ​You​ ​are​ ​now​ ​going​ ​to​ ​add​ ​antibiotic​ ​disks​ ​one​ ​of​ ​the​ ​E.​ ​coli​ ​and​ ​one​ ​of​ ​the​ ​B.​ ​cereus​ ​plates​ ​you​ ​just

spread.​ ​ ​You​ ​will​ ​be​ ​transferring​ ​4​ ​disks​ ​to​ ​each​ ​plate​ ​–​ ​one​ ​disk​ ​in​ ​each​ ​quadrant.​ ​ ​ ​ ​You​ ​are​ ​going to​ ​add​ ​the​ ​same​ ​four​ ​antibiotic​ ​disks​ ​to​ ​each​ ​plate​ ​in​ ​the​ ​same​ ​quadrant​ ​(1,​ ​2,​ ​3,​ ​and​ ​4).​ ​ ​Use​ ​the antibiotics​ ​that​ ​are​ ​at​ ​your​ ​table. What​ ​four​ ​antibiotics​ ​are​ ​you​ ​using? Antibiotic​ ​in​ ​quadrant​ ​1: Antibiotic​ ​in​ ​quadrant​ ​2: Antibiotic​ ​in​ ​quadrant​ ​3: Antibiotic​ ​in​ ​quadrant​ ​4:

2. The​ ​sketch​ ​below​ ​gives​ ​you​ ​an​ ​idea​ ​of​ ​where​ ​to​ ​place​ ​each​ ​disk.​ ​ ​Use​ ​the​ ​dispenser​ ​to​ ​carefully place​ ​each​ ​antibiotic​ ​disk​ ​on​ ​the​ ​two​ ​plates.​ ​ ​If​ ​a​ ​disk​ ​does​ ​not​ ​go​ ​on​ ​exactly​ ​the​ ​right​ ​location, sterilize​ ​your​ ​forceps​ ​in​ ​the​ ​Bunsen​ ​burner​ ​and​ ​use​ ​them​ ​to​ ​move​ ​the​ ​disk​ ​to​ ​the​ ​correct​ ​spot​ ​on​ ​the plate.

Transferring​ ​Antibacterials 1. You​ ​are​ ​now​ ​going​ ​to​ ​transfer​ ​four​ ​different​ ​antibacterials​ ​to​ ​your​ ​other​ ​E.​ ​coli​ ​and​ ​B.​ ​cereus​ ​plates. 2. Before​ ​you​ ​start,​ ​record​ ​which​ ​antibacterials​ ​you​ ​are​ ​using​ ​below.​ ​ ​You​ ​will​ ​dip​ ​blank disks​ ​into​ ​each​ ​antibacterial​ ​and​ ​put​ ​the​ ​disk​ ​on​ ​your​ ​plate​ ​in​ ​one​ ​of​ ​the​ ​quadrants​ ​like​ ​you​ ​did​ ​with the​ ​antibiotic​ ​disks.​ ​ ​The​ ​same​ ​antibacterial​ ​needs​ ​to​ ​be​ ​in​ ​the​ ​same​ ​quadrant​ ​on​ ​each​ ​plate. What​ ​four​ ​antibacterials​ ​are​ ​you​ ​using? Antibacterial​ ​in​ ​quadrant​ ​1: Antibacterial​ ​in​ ​quadrant​ ​2: Antibacterial​ ​in​ ​quadrant​ ​3: Antibacterial​ ​in​ ​quadrant​ ​4: 3. Sterilize​ ​your​ ​forceps​ ​by​ ​running​ ​them​ ​through​ ​the​ ​flame,​ ​and​ ​then​ ​use​ ​them​ ​to​ ​pick​ ​up​ ​a​ ​blank disk,​ ​dip​ ​it​ ​into​ ​one​ ​of​ ​your​ ​"antibacterials"​ ​and​ ​then​ ​place​ ​the​ ​disk​ ​in​ ​the​ ​appropriate​ ​quadrant​ ​on your​ ​E.​ ​coli​ ​plate. a. Repeat​ ​the​ ​procedure​ ​for​ ​the​ ​same​ ​antibacterial​ ​onto​ ​your​ ​B.​ ​cereus​ ​plate. b. Repeat​ ​the​ ​procedure​ ​for​ ​the​ ​rest​ ​of​ ​your​ ​antibacterials​ ​and​ ​place​ ​these​ ​disks​ ​onto​ ​the​ ​appropriate quadrants​ ​that​ ​you​ ​identified​ ​in​ ​#2​ ​above. After​ ​the​ ​Transfers 1. The​ ​cells​ ​need​ ​to​ ​soak​ ​in​ ​on​ ​the​ ​plates​ ​for​ ​a​ ​while​ ​so​ ​leave​ ​them,​ ​face​ ​up,​ ​on​ ​your​ ​tables​ ​as​ ​you​ ​clean up​ ​the​ ​rest​ ​of​ ​your​ ​supplies.

2. Turn​ ​off​ ​your​ ​Bunsen​ ​burner,​ ​and​ ​return​ ​your​ ​supplies​ ​to​ ​where​ ​you​ ​got​ ​them.​ ​ ​Carefully​ ​bring​ ​the stock​ ​culture​ ​bottles​ ​of​ ​E.​ ​Coli​ ​ ​and​ ​B.​ ​cereus​ ​ ​up​ ​to​ ​the​ ​teacher​ ​demonstration​ ​table.​ ​ ​Make​ ​sure​ ​you keep​ ​the​ ​lids​ ​on!! 3. Wash​ ​your​ ​hands​ ​with​ ​soap.​ ​ ​After​ ​you​ ​have​ ​put​ ​your​ ​plates​ ​in​ ​the​ ​incubator​ ​(Mr.​ ​Stewart​ ​will​ ​tell you​ ​when​ ​to​ ​do​ ​this),​ ​you​ ​will​ ​need​ ​to​ ​re-bleach​ ​your​ ​table. Procedure​ ​(Day​ ​2:​ ​ ​Observing,​ ​Sharing​ ​Data,​ ​and​ ​Clean​ ​Up) 1.​ ​ ​Obtain​ ​your​ ​plates. 2.​ ​ ​ ​Observe​ ​the​ ​two​ ​plates​ ​that​ ​had​ ​antibiotics​​ ​on​ ​them.​ ​ ​Record​ ​your​ ​results​ ​below​ ​and​ ​add​ ​your results​ ​to​ ​the​ ​class​ ​data​ ​sheet. 3.​ ​ ​ ​Observe​ ​the​ ​two​ ​plates​ ​that​ ​had​ ​antibacterials​ ​on​ ​them.​ ​ ​Record​ ​your​ ​results​ ​below​ ​and​ ​add your​ ​results​ ​to​ ​the​ ​class​ ​data​ ​sheet. 4. Copy​ ​down​ ​the​ ​class​ ​data​ ​as​ ​you​ ​will​ ​need​ ​this​ ​to​ ​answer​ ​the​ ​lab​ ​questions.