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Student Handout

Hands-on Activity Winogradsky Columns: Microbial Ecology in the Classroom

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WINDOGRADSKYCOLUMNS:MICROBIALECOLOGYINTHECLASSROOMINTRODUCTION

BacteriaandArchaeaarenotonlythemostancientformsoflife,goingbackatleast3.5billionyears,buttheyarealsothemostdiverseandnumerousorganismsonEarth.Forthefirst2billionyearsofEarth’shistory,theyweretheonlylivingthingsontheplanet.Thesemicroorganismsexhibitastoundingdiversityinmetabolicmechanisms,suchasoxygenicandanoxygenicphotoautotrophy,chemoautotrophy,andphotoheterotrophy,tonameafew.Theyalsoplayanessentialroleincyclingelementsthatmaketheplanethabitableforallothertypesoforganisms.Thediversityofthesesimplelifeformsisevidentinthemyriadofecologicalnichestheyinhabit,fromhydrothermalventstotheacidicliningofyourstomach.

Inthisactivity,youwillobserveasamplingofthediversemicroorganismsthatliveinyourlocalenvironment.YouwillsetupWinogradskycolumns—simpledevicesforconstructingastratifiedecosystem—thatillustratedifferenttypesofmicrobialmetabolisminacolorfulway.

Theactivitytakes6to8weekstocomplete.Itwilltakeacoupleofhourstogetsamplesandsetuptheexperiment,andthenyouwillneedtorecordobservationsforafewminutesaboutonceaweek.

MATERIALS

• 4clear,16-oz.plasticbottles.Largercontainerswillalsowork;adjustthesedimentvolumesaccordingly.

• 1–4disposablecontainers(e.g.,plasticstoragecontainerorplasticbaggie)formixingsediment• 1trowelfordiggingasedimentsample • Permanentmarker• 4smalllabels(1foreachbottle)• Water• 1bucketorcontainerlargeenoughtohold6–10cupsofsediment • Awell-litlocationwherethecolumnscansitundisturbedfor6–8weeks• Digitalcameraorcell-phonecamera• Largemeasuringcuporothercontainerformeasuringsedimentandmixture• Acarbonsource,shreddednewspaper;approximately1cuplooselypacked• 6–10cupsofsediment(mud)• Asulfursource;raweggyolkisbestLargemixingspoon(optional)• 1funnel(optional)Coloredpencils(optional)

PROCEDURE

PlantheActivity

Youaregoingtosetupfour(4)Winogradskycolumns:acontrolcolumnandcolumnsthatcontainaddedcarbon,addedsulfur,andcarbonandsulfur.Pleasebesuretoreadthroughthisentiredocumentfirstsothatyouareawareofwhatisexpected.Youwillneedtogoonashortfieldtriptocollectyoursediment(mud)sample.Onceyouhavethesediment,itwilltakeapproximatelyanhourtosetupthefour(4)columns.Inaddition,setasideafewminutesonceaweekduringthe6-to8-weekexperimentalperiodtomakeobservationsandtakephotosofyourcolumns.

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Student Handout

Hands-on Activity Winogradsky Columns: Microbial Ecology in the Classroom

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Youmaychoosetoletyourcolumnsrunforlongerthan8weeks.Thecolumnscanbeanalyzedforseveralmonthsandmightrevealotherchangesthelongertheyaresetup.

CollecttheSedimentSample

1.Identifyasedimentsourceinyourarea.Anywherewithdirtandwaterisappropriate,suchasastream,creek,marsh,pond,bay,oceanbeach(sand),orevenabackyardpuddle(Figure1).

2.Bringaplasticbucketorothercontainer(withalid,ifavailable),atrowel,andacamera(digitalorphone).

3.Take2or3photographsofyoursamplesitetoillustratethelocationwhereyoursedimentwascollected.

4.Collectapproximately10cupsofsedimentintheplasticbucket.Thesampleshouldbewet.Avoidorpickoutrocks,sticks,andleaves;includesomeadditionalwaterfromthesamplesite.

AssembletheWinogradskyColumns

5.Ifyoudon’thaveafunnel,youcanmakeonebycuttingoffthetopthreeinchesofoneofyourplasticbottles(Figure2).

6.Write“control”ononelabelsticker.Ontheotherthree(3)labels,write“carbon,”“sulfur,”and“carbonandsulfur.”Stickthelabelsonthesidesofthefour16-oz.plasticbottlesnearthebottom. 7.Labelfourdisposablecontainers:control,carbon,sulfur,andcarbon+sulfur.Addapproximately1.5cupsofthesedimentsamplethatyoucollectedtoeachofthedisposablecontainers(i.e.,plasticstoragecontainerorplasticbaggie).Youmayreuseasinglemixingcontainerifyourinseitwithtapwaterbetweenuses.

8.Forthecontrolcontainer,skiptostep10.

9.Fortheotherthree(3)containers,followtheinstructionsbelowforeachcolumn:

a. Forthe“carbon”column:Add½cupofshreddednewspaper(looselypacked)tothesediment(step7above)andmixwithamixingspoonortrowel.(Thenewspapercontainscellulose,asourceofcarbon.)

b. Forthe“sulfur”column:Addtheyolkofanegg(rawisbest)tothesedimentinthecontainerlabeled“sulfur”andmixwithamixingspoonortrowel.Ifusinghard-boiledeggs,crumbletheyolks.(Theeggyolkisasourceofcalciumsulfateinthecolumn.)

c. Forthe“carbonandsulfur”column:Addbothnutrientsasdescribedinstepsaandb.10.Mixeachofthesamplesthoroughly.Makesuretoremoveanylargedebrissuchasleaves,rocks,orsticks.Slowlymixinwater(eitherwaterthatyoucollectedortapwater)untilthemixturehastheconsistencyofamilkshake.

11.Usingalargespoon,slowlyaddapproximately1cupofthemixturetotheappropriatelylabeledplasticcolumn(bottle).Youmaywishtouseafunneloraninverted,cutbottletoptoaddthesedimentmixture(Figure1).Asyouaddthesample,tapthecolumnonacounterortabletoreleaseanytrappedairinthecolumn.Thesediment

Figure2.Funnel

Figure 1. A possible source for collecting sediment.

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Hands-on Activity Winogradsky Columns: Microbial Ecology in the Classroom

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shouldfill½to¾ofthecolumn.

12.Addwaterfromthesedimentcollectionsource(ortapwater)ontopofthesedimentuntilthereisa2-cmlayerofwateronthesurface.(Thereshouldbeairatthetopofthecolumn.)

13.Placethelidoneachcolumnandturnthelida½turn.DONOTtightenthelid!Ifyoucutthebottletop,placeplasticwrapoveryourcolumnandholdthewrapinplacewitharubberband.

14.Placeallfourcolumnsinawell-litspacesuchasawindowsill.

15.Usingacameraorphone,takeaphotographofthecolumnstodocumentweek0oftheexperiment.

MakeandReportObservations

Thefollowingitemsexplaintheobservationanddata-collectionportionsofthisactivity.

A.WeeklyObservations

YoushouldobserveyourWinogradskycolumnsforatleast8weeks--longerisbetter(Figure3).Spendafewminuteseachweekrecordingyourvisualobservationsofthecolumns.Trytomakeobservationsonthesamedayeachweek.Forexample,ifyousetupyourcolumnsonaSunday,checkthemeverySunday.Atableisprovidedforyoutorecordyourobservations,butyoumaychoosetomakeyourowntableonacomputer.

Hint:Considerthesequestionswhenrecordingobservations:

• Hasthecolorofthesedimentchanged?Ifso,notethecolor(s).

• Hasthecolorofthewaterchanged?Ifso,notethecolor(s).• Whatdifferencesbetweenthecolumnsdoyouobserve?• Doesthesedimentappeartohavesettled?Shifted?• Doyouseeanylayersforminginthesediment?Inthewater?• Howdoesthethicknessofthelayerschangefromweektoweek?• Aretheredifferencesbetweenthesidefacingthelightandthesideawayfromthelight?

B.TakeWeeklyPhotographs

Usingadigitalcameraorcellphone,youareencouragedtotakeweeklyphotographsofyourcolumnssidebysidewiththelabelsshowing.Asyousaveyourphotos,werecommendthatthefilenamesincludeyourinitialsandtheweekthephotowastaken.(Forexample,“skn_week1”wouldbeanappropriatephotofilename.)Makesurethatthelabelsonthecolumnsareclearlyvisibleinthephotos.

Hint:Yourcolumnsshouldalwaysfaceinthesamedirectioninrelationtothelightsource.Ifyoumustmovethecolumnstotakethephotographs,besuretoplacethecolumnsbackinthesameorientationrelativetothelight.

C.OnceaMonth,SketchtheColumns

Sketchthecolumns,incolor,3timesduringthecourseofthe8-weekexperiment.Considersketchingonceduringeachcalendarmonthoftheexperiment.Sketchingencouragesdeeperobservationofthecolumns.Blank images on the attached work sheet are for your use.

Figure 3. Examples of columns.

Weekly Observations

Observations During the Week Of: Control Column Carbon Column Sulfur Column

Carbon and Sulfur Column

Week 0

Actual Date: __________

Week 1

Actual Date: __________

Week 2

Actual Date: __________

Week 3

Actual Date: __________

Week 4

Actual Date: __________

Observations During the Week of:

Control Column Carbon Column Sulfur Column Carbon and Sulfur Column

Week 5

Actual Date: __________

Week 6

Actual Date: __________

Week 7

Actual Date: __________

Week 8

Actual Date: __________

Month 1: Date

Control Carbon Sulfur Carbon/Sulfur

Month 2: Date _____________________

Control Carbon Sulfur Carbon/Sulfur

Month 3: Date

Control Carbon Sulfur Carbon/Sulfur

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Student Handout

Hands-on Activity Winogradsky Columns: Microbial Ecology in the Classroom

QUESTIONS During the course of the experiment, spend some time considering the following questions. Some of these questions may require extra research. Feel free to consult a microbiology textbook (e.g., to learn about the sulfur cycle).

1. How do your columns differ? How are they the same? Explain the differences you see.

2. Did you observe changes in the control column? If so, explain why they occurred.

3. Winogradsky columns form oxygen concentration gradients. Predict the distribution of oxygenthroughout the column. (Consider the entire column: the sediment, the water, and the air.)

4. Winogradsky columns form sulfide concentration gradients as well. In the columns that contain egg yolk,predict how sulfide will be distributed throughout the column. (Consider the entire column: thesediment, the water, and the air.)

5. Sulfur reduction is a form of anaerobic respiration. Desulfovibrio are an example of bacteria that reducesulfur as a way of respiring in the absence of oxygen and release sulfide. Where in the columns would youexpect to find them?

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Student Handout

Hands-on Activity Winogradsky Columns: Microbial Ecology in the Classroom

6. Purple sulfur bacteria and green sulfur bacteria are two types of bacteria that use sulfide to support photosynthesis. In general, green sulfur bacteria tolerate higher levels of sulfide than purple sulfur bacteria do. Predict where the green and purple sulfur bacteria would be in relation to each other. Also predict where in the column the purple sulfur bacteria would be in relation to the Desulfovibrio bacteria.

7. If samples were extracted from the various layers of all the columns, where would you find photosynthetic organisms such as cyanobacteria and algae? Explain why.

8. Explain how Winogradsky columns illustrate the diversity of microorganisms found on Earth today in terms of the diversity of niches they occupy.

9. Explain what the Winogradsky columns illustrate about life on early Earth.

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winogradsky-studentWinogradsky4-7Weekly Observations

Winogradsky8-9

Month 1 Date: Month 2 Date: Month 3 Date: Date2: Date1: Date0: Date3: Date4: Date5: Date6: Date7: Date8: Control2: Control1: Control0: Control8: Control7: Control6: Control5: Control4: Control3: Carbon1: Carbon2: Carbon3: Carbon4: Sulfur0: Sulfur1: Sulfur2: Sulfur3: Sulfur4: Carbon0: CarbonSulfur0: CarbonSulfur1: CarbonSulfur2: CarbonSulfur3: CarbonSulfur4: Carbon5: Carbon6: Carbon7: Carbon8: Sulfur5: Sulfur6: Sulfur7: Sulfur8: CarbonSulfur5: CarbonSulfur6: CarbonSulfur7: CarbonSulfur8: Question1: Question2: Question4: Question6: Question7: Question3: Question8: Question9: Question5: