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 Lab4–SporeandNegativeStaining,HangingDropSlidePreparation BIOL2730–PathologicMicrobiology

1. Define: a) Sporulate–Processofendosporeformationwithinavegetativecell. b) Negativestaining–Itallowsyoutodeterminethesize,shape,andcellular arrangementofthebacterialcellsthataretoodelicatetoheatfix. c) Motility–thedirectedmovements("runs"and"tumbles")ofmicroorganisms observedunderthemicroscope. d) Brownianmotion–Theappearancethatthecellsaremotilewhen,really,theyare justmovingasthewateraroundthemmoves. PartI–SporeStain 2. Listthebacteriumemployedinthesporestainingexercise. Bacillus megaterium  3. Listtheprocedureforthesporestainingexercise. 1.Usingsteriletechnique,makeasmearofB.megateriumincenterofslide,dilutedwith waterfromsqueezebottle.  2.Airdryandheatfixsmear.  3.Coversmearwithmalachitegreenandplaceslideonhotplatefor5minutes.  4.Removeslidefromhotplate,allowtocoolfor3minutes,andrinsewithwater.  5.Counterstainwithsafraninfor1minute.  6.Rinseslidewithwaterandgentlyblotdrywithpapertowel. PartII–NegativeStain Namethebacteriumstudiedinthenegativestainingexercise. Rhodospirillumrubrum,slide45degreeangleslideacrossslide  4. Listtheprocedureforthenegativestainingexercise. 1. Placeasingledropofnigrosinonacleanmicroscopeslide,adjacenttothefrostededge. 2. Usingaflamedloopandsteriletechnique,removesomeorganismfromyourtubeor plate andmixitintothedropofnigrosin.Besuretherearenolargeclumpsoforganism,buttry

toavoidspreadingthedrop. 3. Placetheendofanothercleanmicroscopeslideatanangletotheendoftheslide containing theorganismandspreadthedropoutintoafilm.Thisisdonebycontactingthedropof nigrosinwiththecleanmicroscopeslideandusingthecapillaryactionofthe dye/microscopeslidetospreadthenigrosinacrossthesmear: 4.Allowthefilmtoairdry.

5.Observetheslideunderthemicroscope,usingpropermicroscopetechnique PartIII–HangingDropSlide 5. Listtheprocedureforthehangingdroppreparationstudy. 1.Applyalightringofpetroleumjellyaroundthewellofadepressionslide 2.Applyadropofwatertoacoverglass,ifusingabrothculturethenomitthewater 3.Asepticallyaddadropofbacteriatothewater.Flameloop 4.Invertthedepressionslidesothedropiscenteredinthewell.Gentlypressuntilthe petroleumjellyhascreatedasealbetweentheslideandcoverglass. 5.Thedropshouldnotbeincontactwiththedepressionslide.Observeunder40xoroil immersion. 6. Statethenamesandcharacteristicmotionsoftheorganismsstudiedinthehanging droppreparations. Pseudomonasaeruginosamotile;darting,spinning,someslowmoversinterspersedwith someveryfast,longdistancemoversthancomeinandoutofthefield. Name Bacillusmegaterium Rhodospirillumrubrum 

Motilitytestresult motil Brownianmotion

7. Statethepointonthehangingdropatwhichfocusingshouldbeinitiated,anddescribe themanneroflightcontrolrequired. Theedgeofthedropshouldbecenteredinthelightpathandfocusedupon.Thelight intensityshouldbekeptlowusingtheirisdiaphragm,lampcontroladjustment,and adjustmentofthesubstagecondenser. ...