Gram-stainingProcedure

Gram-stainingisafourpartprocedurewhichusescertaindyestomakeabacterialcellstandoutagainstagainstitsbackground.Thespecimenshouldbemountedandfixedonaslidebeforeyouprocedetostainit.Thereagentsyouwillneedtosuccessfullyperformthisoperationare:

• CrystalViolet(thePrimaryStain)
• IodineSolution(theMordant)
• Decolorizer(ethanolisagoodchoice)
• Safranin(theCounterstain)
• Water(preferablyinasquirtbottle)

Beforestarting,makesurethatallreagents,aswellasthesquirt-bottleofwater,areeasilyaccessIBLebecauseyouwon"thavetimetogogetthemduringthestainingprocedure.Also,makesureyouaredoingthisnearasinkbecauseitcangetreallymessy.Wearalabcoat.

STEP1:Placeyourslideonaslideholderorarack.Flood(covercompletely)theentireslidewithcrystalviolet.Letthecrystalvioletstandforabout60seconds.Whenthetimehaselapsed,washyourslidefor5secondswithwater.Thespecimenshouldappearblue-violetwhenobservedwiththenakedeye.

STEP2:Now,floodyourslidewiththeiodinesolution.Letitstandaboutaminuteaswell.Whentimehasexpired,rinsetheslidewithwaterfor5secondsandimmediatelyprocedetostepthree.Atthispoint,thespecimenshouldstillbeblue-violet.

STEP3:Thisstepinvolvesadditionofthedecolorizer,ethanol.Step3issomewhatsubjectivebecauseusingtoomuchdecolorizercouldresultinafalseGram(-)result.Likewise,notusingenoughdecolorizermayyieldafalseGram(+)results.Tobesafe,addtheethanoldropwiseuntiltheblue-violetcolorisnolongeremittedfromyourspecimen.Asintheprevioussteps,rinsewiththewaterfor5seconds.

STEP4:Thefinalstepinvolvesapplyingthecounterstain,safranin.Floodtheslidewiththedyeasyoudidinsteps1and2.Letthisstandforaboutaminutetoallowthebacteriatoincorporatethesaffranin.Grampositivecellswillincorporatelittleornocounterstainandwillremainblue-violetinappearance.Gramnegativebacteria,however,takeonapinkcolorandareeasilydistinguishablefromtheGrampositives.Again,rinsewithwaterfor5secondstoremoveanyexcessofdye.

Afteryouhavecompletedsteps1through4,youshouldblottheslidegentlywithbibulouspaperorallowittoairdrybeforeviewingitunderthemicroscope.DONOTRUBTHESMEAR!