PRACTICAL 2 : EXAMINATION OF STAINED CELLS

Introduction Staining cell is one o t!e tec!ni"ues to en!ance t!e contrast o t!e cell #$ %a&ing t!e %icroorganis%s stand out ro% t!e #ac&ground' Not onl$ t!at( t!is tec!ni"ue also !el) to in*estigate di erent grou) o %icroorganis% #$ e+a%ining t!e structure and c!e%icals di erence in cellular structures' In t!is e+)eri%ent( ,e !ad #een e+)osed to t!e )ro)er tec!ni"ue o o#ser*ing s)eci%en under t!e oil i%%ersion o#-ecti*e lens'

O#-ecti*es .' To stud$ t!e )ro)er tec!ni"ue in using i%%ersion oil o#-ecti*e lens' 2' To stud$ t!e di erence in structure in ani%al and )lant cell ,it! a #acteria cell'

Materials: .' Plant cell 2' Ani%al cell /' Sta)!$lococcus aureus 0' 1acillus su#tilis 2' Sal%onella t$)!i%uriu% Procedure: .' T!e entire slide ,as e+a%ined under t!e i%%ersion oil o#-ecti*es lens' 2' T!e si3e and s!a)e and an$ *isi#le structures o organis% ,as o#ser*ed' /' All t!e o#ser*ation ,as recorded'

O#ser*ations

Zea mays ste% 4%onocot ste%5 4.66X5

Bacillus subtilis 4#acteria cell5 4.66X5

Canine !e)atitis 4ani%al cell5 4.66X5

Discussion Fro% t!e o#ser*ation o Zea mays ste% 4%onocot ste%5 under t!e .66 X oil i%%ersion o#-ecti*e lens( t!e structures seen are corte+( e)ider%is *ascular #undle 4+$le% and )!loe%5' 7!ile or ani%al cell 4canine !e)atitis5 is nucleus' 1ut or #acteria cell( no structure ,ere o#ser*ed due to its si3e ,!ic! is too s%all to #e o#ser*ed under a co%)ound lig!t %icrosco)e e*en under t!e !ig!est o#-ecti*e lens' In t!is e+)eri%ent all t!e slides ,ere o#ser*ed under t!e oil i%%ersion o#-ecti*e lens !ence t!e oil i%%ersion tec!ni"ue ,ere a))lied' T!is tec!ni"ue in*ol*ed t!e use o i%%ersion oil' A #lo# o i%%ersion oil ,as a))lied on t!e to) o t!e slides and t!e o#-ecti*e lens ,as lo,ered to t!e )oint t!at t!e lens and t!e slide are in contact' T!is in return ,ill reduced t!e lost o lig!t to t!e surrounding #$ %ini%i3ing t!e di erence in re lecti*e inde+ #et,een t,o %ediu% 4air and t!e o#-ecti*e lens5' T!e re racti*e inde+ o oil is al%ost t!e sa%e as t!e glass !ence lessen t!e #ending o lig!t entering t!e o#-ecti*e lens' T!at e+)lains ,!$ i a slide ,as o#ser*ed under t!e oil i%%ersion o#-ecti*e #ut ,it!out t!e i%%ersion oil t!e i%age a))ear #lurr$' 1ut( ones !as to re%e%#er t!at oil i%%ersion tec!ni"ue onl$ can #e done ,!en o#ser*ing s)eci%en under i%%ersion oil o#-ecti*e lens and not to under lo,er )o,er o#-ecti*e lens #ecause t!e oil %a$ da%age t!e lens ,!en in contact ,it! t!e lo,er o#-ecti*e lens' As %entioned earlier( t!e #acteria slide ,as o#ser*ed under t!e oil i%%ersion #ut t!e structure ,as ailed to #e identi ied' T!is is #ecause t!e #acteria are too s%all and in order to stud$ t!e structure o t!e #acteria a %ore )o,er ul %icrosco)e suc! as electron %icrosco)e or dar& ield %icrosco)e s!ould #e used' 7!at can #e o#ser*ed is t!e s!a)e o t!e #acteria' Not to %ention( t!e lac& o contrasts o t!e slide also contri#uting t!e #ad i%age )roduced' T!e i%age is also lac& in de inition' Fro% t!e o#ser*ation t!e s!a)e o Bacillus subtilis is rod8 s!a)e' It is a di erent ,a$ around ,!en o#ser*ing )lant and ani%al cell' T!e structured o #ot! cells t!at are ,it!in t!e resolution range o co%)ound lig!t %icrosco)e can #e seen clearl$' For e+a%)le( t!e *ascular #undle o t!e Zea mays and t!e nucleus o t!e canine !e)atitis' Not onl$ t!at( t!e co%)ound lig!t %icrosco)e use lig!t as t!e source also contri#ute to t!e act t!e s%aller organelles suc! as %itoc!ondria cannot #e seen'

Conclusion Fro% t!e e+)eri%ent( t!e structured o#ser*ed under t!e co%)ound lig!t %icrosco)e using t!e oil i%%ersion lens is li%ited to it %agni ication )o,er and resolution'

9uestions .' :o, ,ould a stained )re)aration a))ear under t!e oil i%%ersion o#-ecti*es ,it!out t!e oil #et,een t!e slide and t!e o#-ecti*e lens; T!e stained )re)arations a))ear dull and #lurr$ under t!e oil i%%ersion o#-ecti*es ,it!out t!e oil #et,een t!e slide and t!e o#-ecti*e lens #ecause t!ere is an air ga) #et,een t!e o#-ecti*es and t!e slide' Due to t!e air ga)( t!e lig!t ra$ to %iss t!e o#-ecti*es and t!e i%age a))ear #lurr$' T!is related to t!e act t!at t!e air and t!e glass !as di erent re raction inde+(:ence cause t!e lig!t ra$ to #end'

2' 7!at is t!e structural di erence #et,een t!e ani%al and )lant cell; 1ased ro% %$ o#ser*ation( t!e di erence #et,een t!e ani%al and t!e )lant cell are( )lant cell !as cell ,all ,!ile an ani%al cell does not'

/' Could $ou detect t!e )resence or a#sence o t!e structures in 9uestion 2 in t!e #acteria cell; I could not detect t!e )resence or a#sence o t!e structure in "uestion 2 in t!e #acteria cell #ecause e*en t!oug!t using t!e oil i%%ersion o#-ecti*e ,it! .66 X %agni ication t!e #acteria cell is considera#l$ too s%all to #e o#ser*ed using a lig!t %icrosco)e'

PRACTICAL /: EXAMINATION OF LI<IN= 1ACTERIA

Introduction Li*ing #acteria can #e studied #$ conducting !anging dro) %et!od and ,et %ount tec!ni"ue' 1ot! tec!ni"ues !el) retaining t!e #acteria o*er a )eriod o ti%e !ence t!e %otilit$( s!a)e and si3e relations!i)( and reaction to certain c!e%icals can #e stud$' Not onl$ t!at( #ot! tec!ni"ue can reduced t!e distorted e ect ,!en t!e s)eci%en ,as )re)ared traditionall$ ,!ic! re"uire !ead i+ation' 7!en a s)eci%en ,as !eat i+( !ence t!e %otilit$ o t!e s)eci%en cannot #e studied' T!ere are t,o )ossi#le outco%e o t!is e+)eri%ent( t!e #acteria eit!er e+!i#iting 1ro,nian %o*e%ent or true %otilit$' Di erentiating #ot! %o*e%ent ro% one anot!er is i%)ortant in order to deter%ine t!e %otilit$ o t!e #acteria'

O#-ecti*es .' To stud$ t!e slide )re)aration tec!ni"ue 4,et %ount and !anging loo) tec!ni"ue5 2' To de%onstrate t!e %o#ilit$ o t!e #acteria

Material .' 20 > !our nutrient #rot! cultures o a) Staphylococcus aureus b) E.coli 2' :ollo, ground de)ression slide /' Inoculating loo)s 0' =lass slide and co*er sli)s 2' A))licator stic&s

A' T:E :AN=IN= DROP TEC:NI9?E Procedure .' T!e centre o t!e co*er sli) ,as )laced ,it! a dro) o #acterial #rot! culture using a sterile inoculating loo)' 2' T!e de)ression slide ,as in*erted and lo,ered onto t!e )re)ared co*er sli)' T!e slide ,as gentl$ )ressed do,n' /' T!e slide ,as care ull$ turned o*er( and t!e dro) ,as ensured not to run side,a$s' 0' T!e dro) ,as o#ser*ed using .6 X o#-ecti*es' T!en( t!e c!anged to 06 X o#-ecti*e lens and t!e lig!t intensit$ ,as reduced' 2' T!e t$)e o %o*e%ent ,as o#ser*ed: a5 Dri ting o #acteria across t!e ield o *ie, caused #$ e*a)oration o t!e dro) t!roug! an inco%)lete <aseline seal #5 1ro,nian %otion c5 True %otilit$( a c!aracteristic o lagellated #acteria' T!e #acteria ,ill #e o#ser*ed darting a#out and c!anging direction @' T!e inding ,as recorded'

1' TEMPORARA 7ER MO?NT TEC:NI9?E Procedure .' T!e inoculating loo) ,as la%ed to redness' 2' ?nca))ed t!e #acterial culture tu#e to #e used and t!e %out! o t!e tu#e ,as la%ed' /' A loo) ul o t!e culture ,as re%o*ed and )laced in t!e centre o a clean glass slide' 0' T!e %out! o t!e tu#e ,as la%ed and closed t!e ca)' 2' T!e inoculating loo) ,as sterili3ed' @' A co*er sli) ,as )laced o*er t!e #acterial sus)ension and )ressed do,n gentl$' B' T!e )re)aration ,as e+a%ined under lo, )o,er t!en under !ig! )o,er o#-ecti*es'

Discussions Fro% t!e o#ser*ations( t!e %otilit$ o #ot! Staphylococcus aureus and E.coli ,as studied' Staphylococcus aureus is not a %otile #acteria #ecause it e+!i#iting 1ro,nian %o*e%ent' 1ro,nian %o*e%ent can #e %ista&en #$ true %otilit$ due itCs a))ear D%o*ingE #ut ,!at actuall$ !a))en is( t!e ,ater %olecule is #ouncing around &noc&ing t!e #acteria' :ence( it is a non8directional %o*e%ent' T!e Staphylococcus aureus onl$ *i#rate and sta$ a#out t!e sa%e )osition t!roug!out t!e o#ser*ation' I t!ere is a %o*e%ent( t!e Staphylococcus aureus onl$ %o*e #ac& and ort!' 1ut( or E.coli ( it a#le to Fs,i%C across t!e %icrosco)e !ence( e+!i#iting true %otilit$' True %otilit$ can #e c!aracteri3ed t!e distance co*er Fs,i%%ingC is %uc! greater t!an t!eir non %otile counter)arts' Not onl$ t!at( t!e direction o t!eir %o*e%ent is non directional and irregular' Most o t!e ti%e t!eir direction o Fs,i%%ingC is in luenced #$ c!e%ota+is' Not onl$ t!at( t!e E.coli are assu%ed to #e %otile due to t!e act t!at t!e$ )ossessed a lagella ,!ic! )ro)el E.coli #$ t!e t,isting %otion' It is i%)ossi#le to o#ser*ed lagella under a co%)ound lig!t %icrosco)e( unless t!e sa%)le stained' :ence( t!e outline o t!e lagella can #e %ade *isi#le to #e o#ser*ed under t!e co%)ound lig!t %icrosco)e' In t!is e+)eri%ent( #ot! !anging loo) %et!od and ,et %ound tec!ni"ue ,as conducted' For %e( ,et %ount tec!ni"ue is %uc! easier to #e conducted co%)ared to !anging dro) tec!ni"ue' T!is is #ecause or t!e !anging loo) %et!od t!ere is a need to in*ert t!e co*er sli) and t!e slide' 7it!out e+)erienced( it %a$ lead to dri))ing o t!e %ediu% ,!ic! can #e dangerous #ecause ,e are dealing ,it! %icroorganis%s t!at are in*isi#le or t!e na&ed e$e'

1ot! tec!ni"ue need to #e o#ser*ed i%%ediatel$ a ter #eing )re)ared' Es)eciall$ slide )re)ared #$ ,et %ount tec!ni"ue #ecause it tend de!$drate ra)idl$ t!an t!e slide )re)ared #$ !anging dro) %et!od' T!is is #ecause( in !anging dro) %et!od( t!e %ediu% are sealed ,it!in t!e de)ression o t!e slide' :anging dro) tec!ni"ue is #est used to stud$ t!e %otilit$ o t!e %icroorganis% ,!ile t!e ,et %ount tec!ni"ue is #est to stud$ t!e si3e and s!a)e o t!e %icroorganis%' Not onl$ t!at( due to t!e sa%)le ,as not stained t!e i%age )roduced is lo, in contrast cou)led ,it! t!e si3e o t!e #acteriaG t!e i%age )roduced is al%ost trans)arent' In order to %a&e it easier or t!e o#ser*ation o t!e #acteria( t!e condenser ,as lo,ered do,n' 1ut #$ lo,ering t!e condenser( t!e resolution also #een lo,ered do,n' :ence( t!e #acteria is unresol*ed and t!e ine details cannot #e o#ser*ed'

Conclusion Fro% t!e e+)eri%ent( E.coli e+!i#it true %otilit$ t!us a %otile #acteria ,!ile Staphylococcus aureus e+!i#it t!e 1ro,nian %o*e%ent !ence a non%otile #acteria

9uestion .' 7!ic! one o t!e organis% e+!i#it true %otilit$; 1ased ro% %$ o#ser*ation( E.coli e+!i#it true %otilit$ #ecause it can Fs,i%C across t!e %icrosco)e'

2' 7!ic! one o t!e organis% de%onstrated t!e 1ro,nian %o*e%ent; Staphylococcus aureus e+!i#it 1ro,nian %o*e%ent #ecause it a))ears as i it is *i#rating and %o*ing at t!e sa%e ti%e'

/' 7!ic! o t!e t,o tec!ni"ues is easier to )er or%; 7et %ount tec!ni"ue is easier to )er or% co%)ared to !anging dro) tec!ni"ue'

0' Fro% $our o,n o#ser*ation( di erentiate t!e %o*e%ent o t!e organis% e+!i#iting true %otilit$ 4*ital %o*e%ent5 and 1ro,nian %o*e%ent'

Fro% %$ o#ser*ation( to di erentiate true %otilit$ 4as o#ser*ed in E.coli5 ,it! 1ro,nian %o*e%ent 4as o#ser*ed Staphylococcus aureus5 is true %otilit$ can #e c!aracterise #$ t!e #acteria Fs,i%%ingC across t!e %icrosco)e and t!e distance co*er or #acteria ,it! true %otilit$ is %uc! greater co%)ared to 1ro,nian %o*e%ent #acteria' As or 1ro,nian %o*e%ent( t!e #acteria a))ear as i t!e$ are *i#rating and sta$ at t!e sa%e )osition'

2' Motile #acteria s,i% to,ards nutrients #$ t!e )rocess o c!e%ota+is' E+)lain' C!e%ota+is is a non8rando% %o*e%ent o an organis% to,ards and a,a$ ro% a c!e%ical' T!e #acteriu% ,ill %o*e in t!e direction o attractant #$ reducing t!e re"uenc$ o tu%#le and lengt!en its run i it is a )ositi*e c!e%ota+is' 7!ile( i it is a negati*e c!e%ota+is( it ,ill %o*e a,a$ ro% t!e attractant #$ increasing t!e re"uenc$ o tu%#le and s!orten its run'

@' Can t!e )ossession o lagella #$ #acteria #e utili3ed or classi ication )ur)oses; E+)lain

T!e )ossession o lagella #$ #acteria can #e utili3ed or classi ication traits #ecause it is an i%)ortant )!enot$)ic trait' T!e di erence and c!aracteristic o t!e lagellu% ,ill !el) to classi $ t!e #acteriu% into a))ro)riate categor$'

Re erences 1lac&( H' =' I 1lac&( L' H' 426./5' Microbiology' :o#o&en( N'H': 7ile$'

Mo!d Said( S' S' 426665' microbiology practical booklet (MIC 455)' s!a! ala%' Al)enacc'edu' 26.0' Lab ' JonlineK A*aila#le at: !tt)s:LL,,,'al)enacc'eduL acult$L%ilostan%Lla#@'!t% JAccessed: 2B Mar 26.0K' Anon$%ous' 26.0' Je8#oo&K JAccessed: 2B Mar 26.0K' Cellsali*e'co%' 26.0' !o" Bacteria S"im' JonlineK A*aila#le at: !tt):LL,,,'cellsali*e'co%Lani%a#ug'!t% JAccessed: 2B Mar 26.0K' !tt):LL,,,'!cc l'eduL%ediaL2@M.MML.28e+erciseN26*iii')d ' 26.0' JonlineK A*aila#le at: !tt):LL,,,'!cc l'eduL%ediaL2@M.MML.28e+erciseN26*iii')d JAccessed: 2B Mar 26.0K' Micro#e,orld'org' 26.0' !o" Escherichia coli Mo#e' JonlineK A*aila#le at: !tt):LL,,,'%icro#e,orld'orgLco%)onentL-li#rar$L;*ie,OarticleIidO@MM2 JAccessed: 2B Mar 26.0K' Te+t#oo&o #acteriolog$'net' 26.0' Structure a$% &u$ctio$ o' Bacterial Cells' JonlineK A*aila#le at: !tt):LLte+t#oo&o #acteriolog$'netLstructureP2'!t%l JAccessed: 2B Mar 26.0K'