Katelyn Algas BIO 210-101 Lab February 12, 2014 Wednesday, 8:30am-11:30am Lab 3: Microbial Growth Introduction
!e g"al "# e$am%n%ng t!e gr"&t! "# m%'r""rgan%sms at d%##erent tem(eratures &as t" see t!e m%n%mum, ma$%mum, and "(t%mum gr"&t! tem(eratures "# m%'r""rgan%sms) *r"&t! &as d%re'tly de(endent "n !"& tem(erature %n#luen'ed 'ellular en+ymes) n+yme a't%%ty %n'reased unt%l t!e denaturat%"n "# t!e (r"te%n stru'ture "# t!e en+ymes "''urred, &!%'! st"((ed t!e gr"&t!) n+yme %na't%at%"n "''urred as t!e tem(eratures de'reased t"&ard t!e #ree+%ng ("%nt) !e m%'r""rgan%sms "bsered !ad ar%"us tem(erature re.u%rements) !e gr"&t! "# ar%"us m%'r""rgan%sms &as "bsered "n d%##erent%al and sele't%e med%a) /ele't%e med%a all"&ed 'erta%n "rgan%sms t" gr"& &!%le "t!er "rgan%sms &ere n"t able t") %##erent%al med%a d%d n"t (reent t!e gr"&t! "# "rgan%sms, but %t d%d 'ause s(e'%#%' '"l"n%es t" gr"& d%##erently #r"m "t!er "rgan%sms t!at &ere (resent) By !a%ng m%'r""rgan%sms gr"& "n d%##erent%al, sele't%e, "r d%##erent%al and sele't%e med%a, %t !el(ed %s"late t!e (r"(ert%es "# t!e d%##erent m%'r""rgan%sms and %dent%#y t!e m%'r""rgan%sms, su'! as &!at sele't%e agents %n t!e med%a a##e'ted t!e m%'r""rgan%sms "r &!et!er t!e m%'r""rgan%sms &ere *ram-("s%t%e "r *ramnegat%e) An "$%dase test &as (er#"rmed "n d%##erent m%'r""rgan%sms t" see &!et!er t!ey !ad t!e en+yme 'yt"'!r"me c, &!%'! &as an en+yme t!at ass%sted %n 'ellular res(%rat%"n) A 'atalase 'atalase test &as (er#"rmed "n m%'r""rgan%sms t" see %# t!ey !ad t!e en+yme 'atalase, &!%'! &as made b y all a't%ely gr"&%ng aer"b%' m%'r"bes) !ese tests s!"&ed t!e (r"(ert%es "# d%##erent m%'r""rgan%sms) F"r e$am(le, t!e "$%dase test 'an d%##erent%ate bet&een "$%dase-negat%e gram-
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negat%e enter%' ba'ter%al r"ds and ba'ter%al r"ds t!at bel"nged t" t!e genera Pseudomonas genera Pseudomonas and Aeromonas) Aeromonas) !e 'atalase test 'an d%st%ngu%s! bet&een ba'ter%a &%t! s%m%lar m"r(!"l"g%'al '!ara'ter%st%'s but d%##erent metab"l%' a't%%t%es) Materials and Methods
Escherichia coli, coli, Bacillus stearothermophilus stearothermophilus,, Pseudomonas fluorescens, fluorescens, and Serratia and Serratia marcescens &ere streaed "nt" #"ur ry(t%'ase s"y agar (lates t!at &ere se(arated %nt" .uadrants t" see t!e e##e'ts "# tem(erature "n gr"&t!) a'! (late &as %n'ubated at e%t!er 4, 20, 3, "r 05) Saccharomyces cerevisiae &as als" streaed "nt" #"ur s"y agar (lates, and ea'! (late &as %n'ubated at e%t!er 4, 20, 3, "r 05) All "# t!ese (lates &ere "bsered t!e ne$t day #"r gr"&t!) 6ar%"us 'ultures &ere streaed %n (a%rs "nt" #%e ty(es "# med%a t" "bsere t!e%r gr"&t! "n d%##erent ty(es "# med%a) !e (a%rs &ere Escherichia &ere Escherichia coli and coli and Staphylococcus aureus, aureus, Escherichia coli and coli and Enterobacter Enterobacter aerogenes aerogenes,, and Staphylococcus and Staphylococcus aureus and aureus and Enterococcus Enterococcus faecalis) faecalis) a'! 'ulture &as streaed %n a (%t'!#"r (attern "nt" t!e #%e ty(es "# med%a, &!%'! &ere bl""d agar, 7a'5"ney agar, mann%t"l-salt agar, et"en enter%' agar, and ry(t%'ase s"y agar) All "# t!e (lates &ere %n'ubated at 35 and &ere "bsered t!e ne$t day da y #"r gr"&t!) !e "$%dase test &as (er#"rmed "n Escherichia "n Escherichia coli and Pseudomonas fluorescens) fluorescens) A small am"unt "# ea'! 'ulture &as s(read "n %ts "&n (a(er s.uare, and "ne dr"( "# t!e "$%dase reagent &as (la'ed "n t"( "# ea'! 'ulture) I# t!ere &ere a '"l"r '!ange, t!%s meant t!at t!ere &as "$%dase, 'yt"'!r"me c &as (resent, and aer"b%' res(%rat%"n "''urred) !e 'atalase test &as (er#"rmed "n Escherichia "n Escherichia coli) coli) A small am"unt "# t!e 'ulture &as s(read "n a m%'r"s'"(e sl%de, and "ne dr"( "# t!e 'atalase reagent &as (la'ed "n t"( "# t!e 'ulture) I# t!ere &ere bubbles (resent, t!%s meant t!at t!ere &as 'atalase (resent)
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Results Table 1: ##e'ts "# em(erature "n *r"&t! "# 7%'r""rgan%sms Microorganisms
E. coli
B. stearothermophilus
P. fluorescens fluorescens
S. marcescens
9" gr"&t! L%ttle gr"&t!
9" gr"&t! *r"&t!
9" gr"&t! *r"&t!
L%ttle gr"&t! "ne '"l"ny *r"&t!
*r"&t!
*r"&t! "ne '"l"ny 9" gr"&t!
Temperature 4°C 9" gr"&t! L%ttle !°C gr"&t! A l"t "# 3"°C gr"&t! #$°C 9" gr"&t!
9" gr"&t!
S. cerevisiae 9" gr"&t! *r"&t! *r"&t! 9" gr"&t!
Table : *r"&t! "n /ele't%e and %##erent%al 7ed%a Medium T%pe +acterial (pecies
S. aureus
E. faecalis
&i''erential +lood *gar
*r"&t! •
Beta
•
!em"lys%s 5lear
+"nes ar"und '"l"n%es *r"&t! •
•
E. coli
E. aerogenes aerogenes
*amma !em"lys%s 9" +"nes
ar"und '"l"n%es *r"&t! •
Beta
•
!em"lys%s 5lear
+"nes ar"und '"l"n%es *r"&t! •
*amma !em"lys%s
&i''erential and (electi)e MacCon,e% Mannitol-(alt .e,toen *gar *gar /nteric *gar 9" gr"&t! *r"&t! 9" gr"&t! •
7ann%t"l
•
#ermenter ;ell"&
Tr%pticase (o% *gar *r"&t!
+"nes
La't"se
•
#ermenter <%n
*r"&t! •
7ann%t"l
•
#ermenter ;ell"&
9" gr"&t!
*r"&t!
*r"&t!
*r"&t!
+"nes *r"&t! •
La't"se
•
#ermenter <%n
9" gr"&t!
•
La't"se
•
#ermenter /alm"n (%n '"l"n%es
*r"&t! •
La't"se #ermenter
9" gr"&t!
*r"&t! •
La't"se #ermenter
*r"&t!
4
•
9" +"nes
•
<%n
ar"und '"l"n%es
•
Orange '"l"n%es
Table 3: O$%dase and 5atalase est est =ea't%"ns +acterial (pecies E. coli
P. P. fluorescens fluorescens
0idase Result O$%dase negat%e 'yt"'!r"me c absent O$%dase ("s%t%e 'yt"'!r"me c (resent 'ellular res(%rat%"n
Catalase Result 5atalase ("s%t%e 'atalase (resent
&iscussion
In t!e tem(erature e$(er%ments, all t!e ba'ter%a e$!%b%ted at least s"me gr"&t! at 2>5, but Pseudomonas fluorescens, fluorescens, Serratia marcescens, marcescens, and Saccharomyces cerevisiae gre& t!e best) At 35, Escherichia 35, Escherichia coli e$!%b%ted a great am"unt "# gr"&t!, and Pseudomonas and Pseudomonas fluorescens and fluorescens and Saccharomyces cerevisiae gre& cerevisiae gre& at t!at tem(erature as &ell) I# t!e tem(erature &ere ab"e t!e ma$%mum tem(erature "# 'ellular en+ymes, t!ere &"uld be n" m%'r"b%al gr"&t! be'ause m"st 'ell en+ymes &"uld be destr"yed) !ere &"uld be m%'r"b%al gr"&t! %# t!e tem(erature &ere bel"& t!e ma$%mum tem(erature "# 'ellular en+ymes, &!%'! &"uld de(end "n t!e m%'r""rgan%sm) I# t!e tem(erature &ere ab"e t!e m%n%mum tem(erature "# 'ellular en+ymes, t!ere &"uld be m%'r"b%al gr"&t!, and %# t!e tem(erature &ere bel"& t!e m%n%mum tem(erature, t!en t!ere &"uld be n" m%'r"b%al gr"&t!) 5ells &"uld be %na't%e metab"l%'ally) A (sy'!r"(!%l%' m%'r"be, &!%'! gr"&s bet&een ->5 t" 205, &"u ld m"st l%ely n"t be (at!"gen%' %n &arm bl""ded an%mals be'ause ty(%'al b"dy tem(erature %s 35, and (sy'!r"(!%l%' m%'r"bes gr"& best bel"& t!at tem(erature, s(e'%#%'ally ->5 t" 205) Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Enterobacter and Enterobacter aerogenes all gre& "n t!e bl""d agar) Alt!"ug! ea'! ba'ter%um br"e d"&n t!e !em"gl"b%n %n t!e bl""d agar d%##erently, all "# t!e ba'ter%a !ad !em"lys%ns, &!%'! &ere e$tra'ellular en+ymes
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res("ns%ble #"r brea%ng d"&n t!e !em"gl"b%n) On t!e 7a'5"ney agar,Staphylococcus agar, Staphylococcus aureus d%d n"t e$!%b%t any gr"&t! be'ause be' ause t!%s ba'ter%um &as a *ram-("s%t%e '"''us, &!%'! &as %n!%b%ted by t!e sele't%e agents %n t!e 7a'5"ney agar, &!%'! &ere b%le salts and 'rystal %"let) Enterococcus faecalis, Escherichia coli, coli, and Enterobacter and Enterobacter aerogenes gre& aerogenes gre& "n t!e 7a'5"ney agar be'ause t!ey &ere *ram-negat%e r"ds and 'aused la't"se #ermentat%"n) A '!ange %n ( and la't"se #ermentat%"n 'aused t!e med%um t" '!ange '"l"r)Staphylococcus '"l"r) Staphylococcus aureus and Enterococcus faecalis gre& faecalis gre& "n t!e mann%t"l-salt agar be'ause t!e use "# t!e agar &as t" %s"late sta(!yl"'"''%, as &ell as a #e& %nds "# enter"'"''us ba'ter%a) !ey &ere b"t! able t" sur%e t!e salty '"nd%t%"ns) 7ann%t"l &as als" #ermented, &!%'! 'aused t!e ( t" '!ange, as &ell as t!e '"l"r "# t!e med%um) Escherichia med%um) Escherichia coli and Enterobacter and Enterobacter aerogenes d%d aerogenes d%d n"t gr"& "n t!e mann%t"lsalt agar be'ause t!ey &ere *ram-negat%e, and t!e !%g! salt '"n'entrat%"n (reented t!em #r"m gr"&%ng) Staphylococcus aureus and Enterococcus and Enterococcus faecalis d%d faecalis d%d n"t gr"& "n t!e et"en enter%' agar be'ause t!ey &ere *ram-("s%t%e) !e b%le salts and t!e dyes br"t!mt!ym"l blue and a'%d #u'!s%n %n!%b%ted t!e gr"&t! "# t!ese t&" ba'ter%a) Escherichia ba'ter%a) Escherichia coli and Enterobacter and Enterobacter aerogenes gre& "n t!e et"en enter%' agar be'ause t!ey &ere *ram-negat%e and t!ey #ermented la't"se, &!%'! '!anged t!e '"l"r "# t!e '"l"n%es t" salm"n-(%n "r "range) Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, coli, and Enterobacter and Enterobacter aerogenes all gre& "n t!e ry(t%'ase s"y agar be'ause t!e s"y s" y agar %s ne%t!er a d%##erent%al n"r a sele't%e med%um) Anyt!%ng 'an gr"& "n %t) Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Enterobacter and Enterobacter aerogenes e$!%b%ted !em"lys%s) Staphylococcus aureus and Escherichia and Escherichia coli s!"&ed coli s!"&ed beta !em"lys%s, and Enterococcus and Enterococcus faecalis and Enterobacter and Enterobacter aerogenes s!"&ed aerogenes s!"&ed gamma !em"lys%s) !ese results s!"&ed t!at t!e ba'ter%a t!at e$!%b%ted beta !em"lys%s '"m(letely destr"yed red
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bl""d 'ells %n t!e agar, and t!e ba'ter%a t!at e$!%b%ted gamma !em"lys%s d%d n"t destr"y destr"y any red bl""d 'ells %n t!e agar) Out "# all t!e ba'ter%a t!at &ere streaed "n bl""d agar,Staphylococcus agar, Staphylococcus aureus and Escherichia and Escherichia coli &"uld coli &"uld m"st l%ely be (at!"gens be'ause be'au se t!ey &"uld '"m(letely brea d"&n red bl""d 'ells and %n#e't t!e !"st) Enterococcus !"st) Enterococcus faecalis and Enterobacter and Enterobacter aerogenes, aerogenes, &!%'! s!"&ed gamma !em"lys%s, &"uld n"t be (at!"gens be'ause !em"lys%s &"uld n"t een "''ur) Escherichia coli s!"&ed coli s!"&ed a negat%e result %n t!e "$%dase test) !%s s!"&ed t!at t!e en+yme 'yt"'!r"me c &as absent, &!%'! meant t!at res(%rat%"n d%d n"t "''ur %n t!%s ba'ter%um "r a d%##erent met!"d &as used #"r res(%rat%"n) Pseudomonas res(%rat%"n) Pseudomonas fluorescens s!"&ed fluorescens s!"&ed a ("s%t%e result %n t!e "$%dase test, &!%'! s!"&ed t!at 'yt"'!r"me ' yt"'!r"me c &as (resent and res(%rat%"n %a an ele'tr"n trans("rt '!a%n d%d "''ur %n t!%s ba'ter%um) Escherichia ba'ter%um) Escherichia coli s!"&ed coli s!"&ed a ("s%t%e result %n t!e 'atalase test, &!%'! s!"&ed t!at t!e ba'ter%um d%d g" t!r"ug! res(%rat%"n but by a d%##erent met!"d) Fr"m t!e %n#"rmat%"n g%en, t!%s ne& (at!"gen &as *ram-negat%e) Be'ause t!e ba'ter%um gre& %n t!e !uman gastr"%ntest%nal tra't, %t &"uld gr"& best at b"dy tem(erature, "r 35) Out "# t!e bl""d agar, 7a'5"ney agar, mann%t"l-salt agar, et"en enter%' agar, and ry(t%'ase s"y agar, t!e ba'ter%um &"uld gr"& "n all t!e med%a but t!e mann%t"l-salt agar, &!%'! "nly sta(!yl"'"''% and 'erta%n enter"'"''us ba'ter%a gr"& "n t!ere) !e "t!er med%a &"uld be #a%r game be'ause ea'! d%##erent ty(e "# med%um &"uld be %n'ubated at b "dy tem(erature) !e et"en enter%' agar &"uld be s(e'%#%'ally used be'ause %t %s used #"r #e'al s(e'%mens) Fr"m t!ere, .u%' la't"se-#erment%ng "rgan%sms 'an be #"und, &!%'! s"unde d l%e t!%s (at!"gen) I# t!e "$%dase test &ere d"ne, t!e results &"uld be ("s%t%e be'ause t!%s ba'ter%um s"unded l%e %t m"ed and %n#e'ted .u%'ly, &!%'! &"uld s!"& t!at 'ellular res(%rat%"n "''urred) !e 'atalase
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test &"uld be ("s%t%e as &ell, s%n'e %t &as (red%'ted t!at 'ellular res(%rat%"n "''urred) Oerall, t!%s (at!"gen &"uld need t" be st"((ed .u%'ly)