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Pre - Report
Microbe Smear and Colony Observation on Solid Medium
2016 / 03 / 2Section 1 / 201!10110" / Ra#man $ony %ur
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1. TitleMicrobe Smear and Colony Observation on Solid Medium
2. Purposea& 'earnin( t#e met#od o) preparin( solid
medium&
b& 'earnin( universal microbial cultivation tec#ni*ue
on solid medium&
3. Theory
a& '+ ,'uria +ertani medium
'+ is a .idely used bacterial culture medium& $#e a(ar )orm
o) t#e medium s#ould be
desi(nated ' but it is o)ten re)erred to as '+& lt#ou(#
ori(inally developed )or
bacteriop#a(e studies and S#i(ella (ro.t# '+ subse*uently
became t#e medium o) c#oice
)or (ro.t# o) sc#eric#ia coli and ot#er related enteric
species& '+ #as also been used as a
(eneral-purpose bacterial culture medium )or a variety o)
)acultative or(anisms& n t#e
under(raduate microbiolo(y teac#in( labs '+ is sometimes used as
t#e (ro.t# sur)ace .#en
attemptin( to analye bacterial colony morp#olo(y
a& 'i*uid medium
'i*uid medium are .ater-based solutions t#at do not solidi)y at
temperatures above
)reein( 4 t#at tend to )lo. )reely .#en t#e container is
tilted& $ermed broths, mil5s or
in)usions t#ey are made by dissolvin( various solutes in
distilled .ater& ro.t# occurs
t#rou(#out t#e container 4 can t#en present a dispersed cloudy
or )la5y appearance&
common laboratory medium nutrient broth contains bee) e7tract 4
peptone dissolved in
.ater& Met#ylene blue mil5 4 litmus mil5 are opa*ue li*uids
containin( .#ole mil5 and
dyes& 8luid t#io(lycollate is a sli(#tly viscous brot# used
)or determinin( patterns o)
(ro.t# in o7y(en&
b& Solid medium ,.it# t#e addition o) a(ar
Solid media provide a )irm sur)ace on .#ic# cells can )orm
discrete colonies and are
advanta(eous )or isolatin( and culturin( bacteria and )un(i&
$#e most .idely used and
e))ective o) t#ese a(ents is agar, a polysacc#aride isolated
)rom t#e red Gelidium. $#e
bene)its o) a(ar are numerous& t is solid at room
temperature and it melts at 100℃& Once
li*ue)ied a(ar does not resolidi)y until it cool to 92℃ so it
can be inoculated and poured
in li*uid )orm at 9!℃ to !0℃ t#at .ill not #arm t#e
microbes or t#e #andler ,body
temperature is about 3:℃& (ar is )le7ible moldable and
provides a basic )rame.or5 to
#old moisture and nutrients& not#er use)ul property is t#at
it is not readily di(estible and
t#us not a nutrient )or most microor(anism& ny medium
containin( 1; to !; a(ar usually #as t#e .ord agar in its name&
%utrient
a(ar is a common one& 'i5e nutrient brot# it contains bee)
e7tract and peptone as .ell as
1&!; a(ar by .ei(#t& lt#ou(# (elatin is not nearly as
satis)actory as a(ar it .ill create a
reasonably solid sur)ace in concentrations o) 10; to 1!;&
$#e main dra.bac5 )or (elatin
is t#at it can be di(ested by microbes and .ill melt at room and
.armer temperatures
leavin( a li*uid&
c& Composition o) '+ medium
%aCl 10(/' ,1; ./v
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• $#e strea5in( met#od
is a rapid *ualitative
isolation met#od& t is
essentially a dilution
tec#ni*ue t#at
involves spreadin( a
loop)ul o) culture
over t#e sur)ace o) an
a(ar plate& lt#ou(#
many types o)
procedures are
per)ormed t#e )our-
.ay or *uadrant
strea5 is described& t
is important )or us to
.or5 .it# bacteria t#at are (enetically identical& +y
strea5in( )or sin(le colonies oneisolated colony .ill )orm )rom a
sin(le bacterial cell and t#us t#e colony is (enetically
identical& 8urt#ermore strea5in( #elps to ensure t#at sin(le
colonies can be isolated - 12
#ours later&
• $#e spreadin( tec#ni*ue re*uires t#at a previously diluted
mi7ture o) microor(anisms be
used& =urin( inoculation t#e cells are spread over t#e
sur)ace o) a solid a(ar medium .it# a
sterile '-s#aped bent rod .#ile t#e Petri dis# is spun on a
>lay-Susan? turntable&
4. Experimental Method (Reagent & Apparatus
a& Rea(ent 4 pparatus
1& Eschericia coli & 1% @Cl 1% %aO@
2& 'oop "& Materials )or medium manu)acturin(
3& lco#ol lamp trypton 10(/l
9& Petri dis# yeast e7tract !(/l
!& p@ meter %aCl 10(/l
6& Aei(#in( scale *uantitative spoon (ar 1!(/l
:& utoclave =eionied .ater 1&0l
b& Solid medium manu)acturin(
1& dd )ollo.in( into a 2!0 ml or 300 ml erlenmeyer
)las5&
a& %aCl 1(/100 ml d& =eionied .ater 100ml
b& $ryptone 1(/100ml e& (ar 1&!(/100ml
c&
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%oteD ) t#e bubble #as been (enerated remove t#em *uic5ly usin(
sterilied tips&
& Place t#e lid on eac# plate and allo. t#em to cold do.n to
room temperature )or 10-20
minutes ,until t#e medium (ets solidi)ied and t#en invert t#e
plates& ncubate t#e '+ a(ar
plates at 3:℃ )or overni(#t&
"& 'abel t#e bottom o) t#e plates .it# date& Seal t#e
plates .it# plastic ba(s or para)ilm and
store at 9℃&
!. Strea5in(
1& Sterilie t#e benc# .it# :0; et#anol&
2& Prepare '+ a(ar plates at room temperature&
3& 'abel t#e bottom o) t#e plates .it# t#e strain name and
t#e date&
9& Eeep your table sterilied by .or5in( near a
)lame&
!& et a sin(le colony )rom plate usin( a )lame-sterilied
loop&
%oteD ) you use a .ire loop you can sterilie it by passin( it
t#rou(# a )lame& Fust
be sure to allo. enou(# time to cool t#e loop be)ore
(ettin( a colony&
6& ently strea5 t#e bacteria over a section o) t#e plate as
s#o.n in t#e dia(ram ,2&Strea5in( to create Gone ⓐG&
:& Hsin( t#e )lame-sterilied loop dra( t#rou(# Gone ⓐG and
strea5 t#e bacteria over a
second section o) t#e plate to create Gone ⓑG&
& Repeat t#e procedure to create Gone ⓒG and Gone
ⓓG&
"& ncubate t#e plate )or overni(#t ,12-1 #ours at 3:℃
10& Observe sin(le colonies&
c& Spreadin(
1& Sterilie t#e table .it# :0; et#anol&
2& 'abel t#e petri dis#es&3& pply appropriate amount
o) pre-cultured brot# on t#e '+ a(ar plates usin( a
micropipette and micropipette tips&
9& =ip a (lass spreader into et#anol and t#en pass it
t#rou(# )lame to sterilie t#e
spreader& )ter t#e )lame is e7tin(uis#ed touc# to t#e medium
to ma5e sure it .as cool
do.n&
!& Spread rup t#e spreader smoot#ly on t#e a(ar plate&
,=ra( it bac5 and )ort# rotate&&&&
6& 8lame-sterilie t#e spreader )or t#e ne7t use&
:& ncubate t#e plates GHPS=-=OA%G at 3:℃ )or
overni(#t&
& Observe t#e colony )ormation&
". Re#eren!e1& Fames & Cappuccino %atalie S#ermanI
Microbiolo(y 'aboratory Manual t# ditionI Person
J +enKamin Cummin(sI 200I p13-19
2& Eat#leen Par5 $alaroI 8oundations in Microbiolo(y
:t# ditionI Mcra.-@illI 200"I p60-63
3&
#ttpD//...µbelibrary&or(/component/resource/laboratory-test/3031-luria-brot#-lb-and-
luria-a(ar-la-media-and-t#eir-uses-protocol
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#ttpD//delliss&people&co)c&edu/virtuallabboo5/Strea5Plates/oodStrea5tml
http://www.microbelibrary.org/component/resource/laboratory-test/3031-luria-broth-lb-and-luria-agar-la-media-and-their-uses-protocolhttp://www.microbelibrary.org/component/resource/laboratory-test/3031-luria-broth-lb-and-luria-agar-la-media-and-their-uses-protocolhttp://delliss.people.cofc.edu/virtuallabbook/StreakPlates/GoodStreak.htmlhttp://delliss.people.cofc.edu/virtuallabbook/StreakPlates/GoodStreak.htmlhttp://www.microbelibrary.org/component/resource/laboratory-test/3031-luria-broth-lb-and-luria-agar-la-media-and-their-uses-protocolhttp://www.microbelibrary.org/component/resource/laboratory-test/3031-luria-broth-lb-and-luria-agar-la-media-and-their-uses-protocol
